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255226410 | pes2o/s2orc | v3-fos-license | Clinical assessment of SARS-CoV-2 infectivity by rapid antigen test compared with virus isolation
Although real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) remains as a golden standard for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, it can not be easily expanded to large-scaled screening during outbreaks, and the positive results do not necessarily correlate with infectious status of the identified subjects. In this study, the performance of Vstrip® RV2 COVID-19 Antigen Rapid Test (RAT) and its correlation with virus infectivity was examined by virus culture using 163 sequential respiratory specimens collected from 26 SARS-CoV-2 infected patients. When the presence of cytopathic effects (CPE) in cell culture was used as a reference method for virus infectivity, the sensitivity, specificity and accuracy of Vstrip® RV2 COVID-19 Antigen Rapid Test was 96.43%, 89.63%, and 90.8%, respectively. The highest Ct value was 27.7 for RdRp gene and 25.79 for E gene within CPE-positive samples, and the highest Ct value was 31.9 for RdRp gene and 29.1 for E gene within RAT positive samples. When the Ct values of specimens were below 25, the CPE and RAT results had high degree of consistency. We concluded that the RAT could be a great alternative method for determining the infectious potential of individuals with high viral load.
Introduction
The Coronavirus disease 2019 (COVID-19) pandemic was caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) [17] . It was mainly transmitted by droplets [8] . Common symptoms included headache, loss of smell and taste, nasal congestion and rhinorrhea, cough, muscle pain, sore throat, fever, diarrhea and difficulty breathing [2] . However, a non-neglectable portion of infected patients are asymptomatic, which would increase the transmission and spreading of SARS-CoV-2 [1] . To control the SARS-CoV-2 pandemic, quarantine is a passive and inevitable measure to block the connection of people but it exhausts a lot of resource. Because of that, a suitable diagnosis method for contagious COVID-19 patients is important to control the spread of SARS-CoV-2.
Rapid antigen test (RAT) is an in-vitro diagnostic device (IVD) which is of low cost and easy to use by general population. Most of the RAT use immunochromatography to detect the interaction between antibody and antigen. For SARS-CoV-2, many RATs use SARS CoV-2 nucleocapsid protein as target antigen due to its abundance on virus particles and sequence conservation during virus evolution [19] . The relatively simple test procedure renders its quick adaption to identify infected individuals during COVID-19 pandemic, and further expand its application as a home-based self-test [15] . Vstrip® RV2 COVID-19 Antigen Rapid Test (Panion & BF Biotech Inc.) is a rapid qualitative detection test for detecting SARS-CoV-2 antigen in human nasopharyngeal specimens. In this study, the correlation of its performance with virus infectivity in the sequential nasopharyngeal specimens from SARS-CoV-2 confirmed cases was evaluated.
Study design and participants
This prospective study was conducted at Taoyuan General Hospital, Ministry of Health and Welfare, Taoyuan, Taiwan between November and December of 2020. Eligible participants were confirmed with SARS-CoV-2 infection by realtime PCR and were ≥ 18 years of age. A standardized case record form was used to collect information on the patients' demographics, comorbidity, treatment history, travel history, and laboratory data. The study was approved by the Research Ethics Committee or institutional review board at Taoyuan General Hospital (registration number TYGH109057). All patients gave written informed consent before enrollment to provide their samples and clinical and laboratory data for research.
A total of 26 patients were recruited for the study. Nasopharyngeal swabs were collected from each participant every day during their stay in the hospital until their real-time RT-PCR results became negative. For comparison, two nasopharyngeal swabs were collected from every participant at each time points. One was stored in universal transport medium (UTM) for real-time RT-PCR by Cobas Z480 and virus infection, the other was stored in lysis buffer and tested by Vstrip® RV2 COVID-19 Antigen Rapid Test (Panion & BF Biotech Inc.). A total of 326 nasopharyngeal swabs were collected for analysis in the study.
Real-time RT-PCR
All samples stored in universal transport medium (UTM) were extracted for viral RNA. Quantitative RT-PCR (qRT-PCR) was performed and analyzed using the Roche Cobas Z480 analyzer (Roche Molecular Systems, Rotkreuz, Switzerland) under the following conditions: 20 min at 50 °C and 20 s at 95 °C, followed by 45 cycles of 3 s at 95 °C and 30 s at 60 °C. Samples were considered SARS-CoV-2 positive if they tested positive for the E or RdRp genes [6] .
Virus infection and isolation
The samples stored in UTM were propagated in Vero or VeroE6 cells in Dulbecco's modified Eagle medium (DMEM) supplemented with 2 g/mL tosylsulfonyl phenylalantyl chloromethyl ketone (TPCK) -trypsin (Sigma -Aldrich). Culture supernatants were harvested when virus-induced cytopathic effects (CPE) were observed in more than 70% of cells. The full -length genomic sequences of the derived clinical isolates from each patient were determined by Sanger sequencing and submitted, along with the patients' travel history and basic information, to the GISAID database. The isolated virus strains were listed in Table 6 .
Rapid antigen test (RAT)
The nasopharyngeal swabs were placed in the lysis buffer and tested by Vstrip® RV2 COVID-19 Antigen Rapid Test (Panion & BF Biotech Inc.). This assay could detect the presence of SARS-CoV-2 nucleocapsid protein (N) by immunochromatography. After the test strip was placed in the lysis buffer for 10-15 min, the result could be interpreted. Samples were considered SARS-CoV-2 positive when both the test lines and control lines could be seen by the naked eye.
Statistical analysis
All subjects' characteristics of demographic data (age, gender, and symptoms etc.) at study entry were listed for all subjects. Frequencies and percentages were reported for all categorical data.
The parameter including sensitivity, specificity, accuracy, positive percent agreement (PPA), negative percent agreement (NPA) and overall percent agreement (OPA) were analyzed according to the test results of CPE and RAT. Categorical variables were compared using chisquare test or Fisher's exact test. Continuous variables were compared using the Kruskal-Wallis one-way analysis of variance or Mann-Whitney U test. Unless otherwise specified, a two-tailed p value < 0.05 was considered statistically significant. All statistical analysis programming was performed using SAS version 9.4 version.
Demographics of study participants
The demographic information of participated COVID-19 patients was shown in Table 1 . The median age of the participants was 32 years old. More than half of the participants were female (61.54%). By travel history, 61.53% of the participants had been to Asia, and among these people, the most had been to Indonesia. Cough (34.61%) was the mostreported symptom from participants, followed by sore throat (23.07%) and congestion or runny nose (23.07%). Besides, the proportion of asymptomatic patients (23.07%) was as same as that of sore throat, congestion or runny nose. The median time for recruitment since disease onset among the participants was 7 days, and median for duration of hospital stay was 37 days. Nevertheless, none of the participants stayed in an intensive care unit (ICU) . During the hospitalization period, respiratory specimens were collected from each subject every day for realtime RT-PCR, virus infection and rapid antigen tests (RAT) until their real-time RT-PCR results became negative (Supplementary Figure 1).
The correlation of rapid antigen test results with virus infectivity
The correlation of rapid antigen test results with virus infectivity was shown in Table 4 . The presence of CPE was used as an indication of virus infectivity in the respiratory specimens. When CPE was used as a reference method, the sensitivity, specificity, positive prediction rate, negative prediction rate and accuracy of the RAT was 96.43% ( Table 5 ). Besides, the area under the curve (AUC) values associated with RAT performance was 0.92 based on the real-time RT-PCR results of RdRp gene and 0.91 based on those of the E gene. ( Fig. 1 ) Further analysis and comparison of the correlation between the results of CPE, RAT and the Ct value of real-time RT-PCR was conducted ( Fig. 2 ). The overall Ct values of the samples with positive RAT result Next, we compared the correlation between the results of CPE and RAT with the amount of viral RNA. For samples with Ct values of RdRp and E genes lower than 20, both CPE and RAT were 100% positive ( Fig. 3 ). The RAT remained 100% positivity when the Ct value was lower than 25 for RdRp gene. In general, when the Ct values of specimens were lower than 25, the CPE and RAT positive rate had high degree of consistency. When the Ct value of samples was lower than 30, the RAT still had over 70% positivity. However, when the Ct value was greater than 30, the positive rate of CPE and RAT dropped significantly, despite of higher positive rate of RAT in RdRp gene. In general, positive RAT results had great correlation with virus infectivity when the Ct value of specimen was lower than 25.
The lineage of viruses isolated from the clinical samples
For those CPE positive specimens, the full-length virus genome sequence PCR-amplified from the culture supernatants and the virus lineage was determined. All viruses isolated in the study had D614G mutation on the S protein, which was the predominant around the world when the clinical specimens were collected. The correlation between results from virus isolation, RAT, and real time RT-PCR across the SARS-CoV-2 lineages was shown in
Discussion
In this study, we evaluated the performance of Vstrip® RV2 COVID-19 Antigen Rapid Test (Panion & BF Biotech Inc.) in correlation with virus infectivity using sequential respiratory specimens collected from SARS-CoV-2 infected individuals and compared the results with those from real-time RT-PCR. The assay showed great performance on confirming virus infectivity by high sensitivity, specificity and accuracy, and the AUC was also greater than 0.9. The study results indicated that Vstrip® RV2 COVID-19 Antigen Rapid Test can serve as a convenient tool to identify the infectivity status of the COVID-19 suspected subjects or confirmed patients and can help to optimize the constrained resource during the pandemic.
In our study, the sensitivity of RAT is lower than the gold-standard method, real-time RT-PCR, with only 41 of 163 real-time RT-PCR positive samples being positive by the RAT. Our results were similar to many previous studies, which had shown that the RAT had a relatively poor sensitivity than that of real-time RT-PCR [10 , 14 , 20] . However, a very high consistence was observed between virus isolation and RAT when the Ct values of specimens were lower than 25. Also, the sensitivity of RAT versus virus isolation results was increased to 96.43%.
Fig. 4. Correlation distribution between virus isolation, rapid antigen test (RAT), and real-time RT-PCR across the SARS-CoV-2 lineages from the study samples.
The samples of participants who had evidence of virus infectivity ( n = 115) were drawn into a dot distribution g raph based on the virus lineage and Ct value. The SARS-CoV-2 lineage for those successfully isolated from the respiratory specimens of recruited participants was shown on the x axis. The Ct values of real-time RT-PCR using (A) RdRP and (B) E gene as targets was shown on the y axis. The white and red dots represented negative and positive results for both cytopathic effect (CPE) and RAT respectively. The green dots represented positive for CPE only, and the blue dots represented positive for RAT only. The two dotted lines represented the highest Ct values for CPE and RAT positive samples, respectively.
Besides assay sensitivity, the possibility of lower sensitivity in RAT and virus isolation versus real-time RT-PCR might be due to viral RNA shedding during SARS-CoV-2 infection. In the clinical course of disease progression, increase of viral RNA in the respiratory specimens could be detected before the symptom onset, and could persist for many weeks even after disappearance of symptoms [11 , 16 , 25 , 27] . It has been reported that viral RNA could be detected in specimens via real-time RT-PCR with high Ct value but failed to isolate SARS-CoV-2 in cell culture [4 , 5 , 9 , 23] . Our study also confirmed that SARS-CoV-2 viruses could not be successfully isolated from the specimens with Ct value above 27.7 of RdRp gene and 25.8 of E gene ( Fig. 2 ). Besides, the collection time point and disease severity are also related to the results of RAT, realtime PCR and virus isolation [3 , 12 , 13 , 24] . It has been reported that the successful rate for virus isolation from specimens collected at later time points after symptom onset would decrease even though the Ct was low [21 , 23 , 27] . Similar results were also observed in our study. Among the four specimens with low Ct values and no development of CPE in virus isolation, two were collected greater than 8 days after symptom onset. Nevertheless, our results demonstrated that RAT has a good sensitivity versus virus isolation in asymptomatic patients. Although only 6 asymptomatic patients were enrolled in our study, the viruses could be detected via RAT in 5 patients' specimens, and their viruses could also be successfully isolated. Therefore, our study indicates that RAT could be used to distinguish contagious people from non-contagious population.
Comparing to real time RT-PCR and virus isolation, RAT had many advantages, including turn-around time, price, and easy-to-operate. It is also easier to be applied to large-scaled screening. However, availability of treatment regimen, capacity of medical support, effectiveness of quarantine strategy and duration of isolation will be affected by the false positive and negative prediction rates of detection method [22] . When wide-spread screening is required, negative predictive value is more important than positive predictive value in the aspect to set up a threshold to rule out infection [26] . Our results demonstrated that RAT had a relatively good negative predictive value (99.18%, Table 5 ) as compared to virus isolation, with only one sample having unexpected results. That sample, with a Ct value of 23.9 for E gene, was positive by virus isolation but negative by RAT. As expected, the negative predictive value of RAT declined to 12.3% when comparing with real time RT-PCR. The infection status, especially the early infection (incubation period) stage, might result in positive real-time RT-PCR but negative RAT. The possibility that the mutations on the epitope of Nucleocapsid (NP) protein would contribute to the false negative results of RAT could not be totally excluded. NP protein is the most abundant protein in coronavirus [19] . Previous studies showed that most of the dominant B cell epitopes are located between amino acid residues 76-82 or 176-206 [18] . This region also harbored the highest prevalence of NP mutations across lineages (Supplementary Figure 2). Two mutations, R203K and G204R, had approximate 100% prevalence in Alpha and Omicron variants. In our study, the sensitivity of different variants of concern (VOC) could not be determined owing to the participates were enrolled in April of 2021, before outbreaks of Alpha and Omicron variants in Taiwan. However, among the 5 specimens used in this study with R203K and G204R mutations on N gene, they could be recognized via RAT, expect lineage B.1.1 ( Fig. 4 ). The results implicated that the recognition of VOC by RAT is similar to the wild type virus. Noteworthy, there are other non-tested high prevalence mutations existing in the VOC (Supplementary Figure 2), and their influences on the sensitivity, positive predictive value and negative predictive value of RAT to VOC need to be further evaluated in the future. The positive predictive value of RAT versus virus isolation was 65.85%. Fourteen specimens were positive by both RAT and realtime PCR, but negative by virus isolation. This phenomenon was likely due to the collection of specimens at later time points or inappropriate storage of specimens.
Overall, Vstrip® RV2 COVID-19 Antigen Rapid Test had good performance to confirm the contagious patients than real-time RT-PCR. The convenient and easy-to-use features of RAT make it the most suitable screening tool to restrict the spreading of asymptomatic infections during the outbreak.
Declaration of Competing Interests
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. | 2022-12-30T14:04:24.090Z | 2022-12-01T00:00:00.000 | {
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3437426 | pes2o/s2orc | v3-fos-license | Avocado fruit maturation and ripening: dynamics of aliphatic acetogenins and lipidomic profiles from mesocarp, idioblasts and seed
Background Avocado fruit contains aliphatic acetogenins (oft-acetylated, odd-chain fatty alcohols) with promising bioactivities for both medical and food industries. However, we have scarce knowledge about their metabolism. The present work aimed to study changes in acetogenin profiles from mesocarp, lipid-containing idioblasts, and seeds from ‘Hass’ cultivar during fruit development, germination, and three harvesting years. An untargeted LC-MS based lipidomic analysis was also conducted to profile the lipidome of avocado fruit in each tissue. Results The targeted analysis showed that acetogenin profiles and contents remained unchanged in avocado mesocarp during maturation and postharvest ripening, germination, and different harvesting years. However, a shift in the acetogenin profile distribution, accompanied with a sharp increase in concentration, was observed in seed during early maturation. Untargeted lipidomics showed that this shift was accompanied with remodeling of glycerolipids: TAGs and DAGs decreased during fruit growing in seed. Remarkably, the majority of the lipidome in mature seed was composed by acetogenins; we suggest that this tissue is able to synthesize them independently from mesocarp. On the other hand, lipid-containing idioblasts accumulated almost the entire acetogenin pool measured in the whole mesocarp, while only having 4% of the total fatty acids. The lipidome of this cell type changed the most when the fruit was ripening after harvesting, TAGs decreased while odd-chain DAGs increased. Notably, idioblast lipidome was more diverse than that from mesocarp. Conclusions Evidence shown here suggests that idioblasts are the main site of acetogenin biosynthesis in avocado mesocarp. This work unveiled the prevalence of aliphatic acetogenins in the avocado fruit lipidome and evidenced TAGs as initial donors of the acetogenin backbones in its biosynthesis. It also sets evidence for acetogenins being included in future works aimed at characterizing the avocado seed, as they are a main component of their lipidome. Electronic supplementary material The online version of this article (10.1186/s12870-017-1103-6) contains supplementary material, which is available to authorized users.
Background
Avocado pulp is an excellent source of macro and micronutrients, characterized for containing highly bioactive lipids that have received increased scientific interest for their potential health-related applications. Although most attention has focused on its contents of monounsaturated oil (15-30% of total fresh weight of the mesocarp [1]), there is a recent interest devoted to a family of fatty acid derivatives called lauraceous acetogenins. Acetogenins, often present as acetylated odd-chain fatty alcohols, have shown relevant bioactivities, such as insecticidal [2][3][4][5], inhibition of acetyl-CoA carboxylase [6] and production of nitric oxide and superoxide in cells [7,8], as well as proapoptotic effects against several cancer cell lines [2,[9][10][11][12], and recently, a promising activity against Acute Myeloid Leukemia cell lines [13], as well as sporostatic and bactericidal properties [14,15].
Avocado fruit development can be easily divided in two different, easily distinguishable processes: fruit maturation, which is the process of growing taking place while in the tree, from 20 to 60 weeks after pollination; and postharvest ripening, comprising the softening of the mesocarp and improvement of organoleptic properties taking place only after the detachment of the fruit [16,17]. Unlike most fruits, in which there is an initial phase of cell division followed by cell growth, in avocado fruit cell division remains active until reaching the last stage of maturation [18]. Being a climacteric fruit, during postharvest ripening an increase in respiration has been observed, coupled with ethylene production, signaling the most drastic physicochemical changes [19]. Notably, it is during fruit maturation, and not during postharvest ripening, that lipids, particularly fatty acids and triacylglycerols (TAGs) are synthesized [20], to the point that oil content is taken as a measure of fruit maturity, but not ripening stage [21].
Apart from the mesocarp, mature avocado fruit contains a single, large seed formed by one embryo surrounded by very thin seed coats; the embryo consists of two particularly large starchy cotyledons with a centrally attached very small embryonic axis [22]. In the early stages of fruit development, the embryo is surrounded by a gelatinous endosperm, which completely disappears at the final stages; thus avocado seeds are considered non-endospermic [23]. It is during these late stages that seed is separated from the vascular network of the fruit [22]. Since avocado is a member of the basal angiosperms, the oldest known flowering plants, branching away before the separation of monocots and dicots [24,25], its seed has no defense mechanisms against dehydration [23,26]. It has to be planted within 9 months of the separation from the mesocarp, and requires 45 days in a high humidity (>70% RH) environment for the radical protrusion to occur [26].
Another peculiarity of avocado fruit is the presence of lipid-containing idioblasts, large (~80 μm) specialized cells, distributed uniformly in the mesocarp which make up to 2 % volume of the edible portion [27] and are characterized by a thick (4 μm) wall consisting of three layers of cellulose, suberin, and lignified cellulose [28,29]. These secretory glands are mainly occupied by a cutinized sac, topologically considered extracellular space, filled with a single, large drop of oil, and surrounded by an electron-dense cytoplasm with partially degraded organelles [27][28][29]. Idioblasts have been hypothesized to play a role in plant defense against herbivores [4] and fungal infections [5] mainly because their high acetogenin content [5].
Little is known about the metabolism of acetogenins in the plant, with most studies focusing on only one derivative, Persin [(Z,Z)-1-acetyloxy-2-hydroxy-12,15heneicosadien-4-one]. Previous works have confirmed incorporation of 14 C-labeled linoleic acid and acetate into Persin [30,31], linking their metabolism to that of long-chain fatty acids. Furthermore, Persin concentrations were found to correlate positively with expression levels of fatty acid editing enzymes, such as Δ9- [31] and Δ12-desaturases [32], and an elongase (avfae1) [33]. On the other hand, a lipoxygenase (LOX) has been shown to degrade Persin in vitro, and the increase in its activity during ripening has been associated with a decrease in Persin contents [34][35][36]. These results suggest a connection between fatty acid and acetogenin metabolism, in both synthesis and degradation, and therefore, studying acetogenin dynamics during the most intense period of fatty acid synthesis, fruit maturation, is expected to yield useful information on this link.
Recently, we fully characterized acetogenin distribution in fruit tissues from 22 avocado cultivars from different genetic backgrounds [37]. We characterized a total of 8 different acetogenin derivatives, sampling the chemical diversity of acetogenins. Apart from generating a chemotaxonomic model, the importance of seed tissue in acetogenin metabolism was documented; and it was also hypothesized that seed and mesocarp are possibly capable of conducting independent biosynthesis of acetogenins. Furthermore, a classification system for lauraceous acetogenins was generated; three groups were proposed based on carbon numbers of their deacetoxylated backbones, which included avocatins, pahuatins and persenins (17,19 or 21 carbons, respectively). Results from work also described in Rodríguez-López et al. favors the hypothesis that each acetogenin group may have a different fatty acid precursor. In addition, this work showed that Persin is not the main component of the acetogenin pool in avocado fruit [37]; thus, more comprehensive studies are needed considering all acetogenins in fruit to start to generating knowledge about their metabolism.
Also, although avocado high oil content has prompted several works in lipidomics characterization, most of the available literature focuses on a single developmental point, using avocado extracts as an example of a recalcitrant matrix. For example Shen et al. used non-polar extracts to test a new Solid Phase Extraction method to enrich glycerophospholipids (by retaining TAGs) and identified a total of 31 putative phospholipids [38]. Similarly, Vaikkinen et al. used avocado mesocarp to test a new ionization technique, Heat-Assisted Laser Ablation (HA-LA-ESI-MS), reaching tentative assignation of 39 features, mainly TAGs, DAGs and phospholipids [39]. Recent work by Horn et al., as a part of a proteomics experiment on avocado lipid bodies, analyzed TAG composition by MALDI-MS Imaging [40] selecting 23 features as putative TAGs, with species ranging from 48:2 to 54:1, most of which were uniformly distributed through the avocado mesocarp [40]. Noticeably, none of these MS studies coupled any chromatographic technique to the MS, and thus, did not report other lipids simultaneously, given that neutral lipids tend to mask the rest of the lipid families by force of concentration [38].
The present work was undertaken with the purpose of studying changes in profiles and concentrations of seven individual acetogenins found in the mesocarp and seed of the commercially relevant 'Hass' avocado cultivar, as affected by growth and postharvest ripening (Additional file 1: Figure S1). The study also aimed to characterize and contrast, for the first time, acetogenin profiles found in specialized, oil-containing idioblasts, with those of mesocarp and seed. Additionally, the effects of seed germination on acetogenin profiles and concentrations, and the effect of environmental changes on acetogenin accumulation in fruit (by comparison of samples from three different harvesting years) are described in this paper. This is also, to the best of our knowledge, the first reported use of MS-based untargeted lipidomics methodology to analyze changes of avocado lipids due to biological effectors, in this case growth and ripening, and the first work using a chromatography-coupled approach. By comparing acetogenin dynamics with the untargeted lipidomics results during different fruit growing and ripening stages, we intend to start unveiling their metabolic cues.
Results
Acetogenin profiles are conserved in mesocarp while seed profiles are dynamic during fruit growth Acetogenin compounds detected in 'Hass' avocado mesocarp and seed are presented in Table 1, and were consistent with those previously reported for the same cultivar [37]. During growth, fruit weight increased considerably from 38 to 343 g, as did the fruit dry weight ranging from 13 to 30%. However, 'Hass' avocado mesocarp acetogenin contents barely changed significantly during fruit growth. There is a slight initial decay in concentrations from very small fruit to mature one (Fig. 1a); however, if acetogenin contents are expressed in fresh weight (FW) there are no significant differences among all fruit development (Additional file 1: Figure S2A). Lipid biosynthesis occurs mainly during fruit maturation rather than during ripening in avocado, and fruit weight gain highly correlated with lipid accumulation (R 2 = 0.978, Kilaru et al. 2015); thus this apparent decrease observed when expressing concentrations in dry weight (DW) is due to the relative concentration with the increasing lipids (Additional file 1: Figure S2C &D). Remarkably, during postharvest ripening there were no differences in acetogenin accumulation (Fig. 1a, Additional file 1: Figure S2A). Acetogenin distribution was also constant in developing mesocarp (inset pie chart of Fig. 1a and Additional file 1: Table S1), always favoring Persenone A accumulation, the main acetogenin found in mesocarp (52.7 ± 4.3%), followed by Persin/ Persenone B (25.8 ± 2.3%).
Contrastingly, seeds from avocados with fresh weights ≤90 g had very small TAC, when compared to seeds bigger than 90 g, with a 4.5-fold increase in concentrations. The same classification was suggested in the Tukey HSD grouping (Fig. 1b); considering the high variability characteristic of the tissue, with the first three stages being significantly different (group 'd') than the group with the highest concentration ('a') and the transition groups ('b' and 'c'). The existence of a large group which is not statistically discernible from the low concentration group is probably due to the high biological variability characteristic of seed tissue [37] as well as the odd distribution of seed dry weight among fruits (when plotted as FW, this early group differentiated from the rest more clearly, Additional file 1: Figure S2B). On this vein, when the acetogenin concentrations in seeds were plotted against seed dry weights (Fig. 1c), a non-linear dependency can be seen ([Acetogenins] = 32.7*Dry Weight 2.246 , R 2 = 0.732).
The distribution of acetogenin species is shown in the inset pie charts of Fig. 1b and listed in Additional file 1: Table S2. While young seeds have a low contribution of Persin/Persenone B to the pool (4.51 ± 2.96%), in mature seeds this relation more than triples (17.4 ± 3.97%); this shift is compensated by UPA, which reduces its contribution roughly two thirds (from 14.6 ± 3.31% to 5.54 ± 3.07%). In a similar fashion, avocatins (short-chain acetogenins, Table 1) contribution is greatly reduced as seed reaches maturity, with a drop on AcO-avocadenyne share from 7.85 ± 1.79% to 4.99 ± 3.49% and for AcOavocadene from 53.7 ± 4.43% to 36.3 ± 4.88%, which was the highest percentual drop (Additional file 1: Table S2). On the other hand, the contents and profiles observed in seeds from mature green fruit remained constant during postharvest ripening (Fig. 1b).
Idioblasts accumulate the majority of acetogenins in avocado mesocarp
Idioblast isolation was conducted on the same samples used for the fruit growth and postharvest ripening experiments. Both mesocarp and idioblast-enriched fractions from avocado fruit were seen under the light microscope, stained with DAPI and Nile Red to easily assure integrity and to observe the lipid droplet. Figure 2a, depicts idioblast cell (top) along with a parenchymatic cell (bottom); as it can be observed, apart from the difference in size (twice bigger than parenchymatic cells), idioblasts were also characterized by their highly dense cytoplasm as previously reported [28,29]. Idioblasts were uniformly dyed with DAPI, unlike parenchymatic cells in which the dye only concentrated in a reduced location. On the other hand, the lipid-specific Nile Red staining, revealed smaller droplets in parenchymatic cells, emitting a high intensity red color, while idioblasts have a much larger droplet, occupying most of the space, glowing on a faint orange-red color. It is important to note that, while the majority of idioblasts show a single, full oil sac, some others appear to have only medium-sized droplets (Additional file 1: Figure S3).
To assess the purity of idioblast-enriched fractions, Nile Red-stained idioblasts preparations were run in a flow cytometer; distribution of cells with idioblast-characteristics was at least 87.9% of the cells in the preparations (Additional file 1: Figure S4 and Table S3). Parallel to mesocarp, when acetogenins were characterized and quantified in idioblasts during fruit growth, no significant differences were found; moreover, these cells had the same acetogenin pool distribution than mesocarp ( Fig. 2b and d, Additional file 1: Table S4). On the other hand, during postharvest ripening, while maintaining the same profile, idioblast TACs steadily increased (Fig. 2c, Additional file 1: Table S4), accumulating, remarkably, almost all (98.1 ± 0.8%) of the acetogenins found in ripe fruits when compared to the corresponding mesocarp replicates Fig. 1a. Fatty acid profiles of idioblasts were also found to be the same as in mesocarp in mature green fruit (Fig. 2d). However, idioblasts accumulated only 4.1% of the saponified fatty acid contents of mesocarp (Table 2). Correspondingly, seed saponified fatty acid profiles differed from those of mesocarp and idioblasts, (Fig. 2d) with seeds having a higher share of stearic, linoleic, and palmitic acid, taking a toll on oleic acid, and had a much lower concentration of total fatty acids: 3.02 ± 0.72 mg/gFW (7.41 ± 1.96 mg/gDW). While idioblasts are reported to be present in all tissues in avocado, including seed, [29,41,42] the methodology used in this work did not allow us to purify idioblasts from this tissue.
Acetogenin profiles do not vary across different harvesting years nor during seed germination
Looking for other hints of metabolic switches for acetogenins, we monitored acetogenin levels during germination, analyzing separately cotyledons, embryonic axis (plumule and radicle) and growing seedlings using the samples collected from the harvest of 2011. Results from these experiments showed no significant trend due to the high variation in cotyledon tissue over a 70 dayperiod on either total acetogenins or each individual one (Fig. 3a). Surprisingly, in embryo's plumule and radicle, or in shoots and roots from seedlings, no measurable amount of acetogenins could be detected (detection limits 0.01 mg/gFW). Fig. 4b and c, respectively) across those years, despite differences expected due environmental stresses, given that 2013 was an atypically dry year in Uruapan, with total rain levels in the month previous to the harvest an order of magnitude less (21 mm) than the average precipitation for that month (270 mm) [43]. These changes are in accordance with a previous study conducted in avocado leaves, which revealed that while there were variations in Persin levels across 21 avocado cultivars, including several 'Hass' ecotypes, there were no changes resulting from leaf age or season, over a two-year period [44].
To gain more insight into the acetogenin and other lipids production in avocado fruit during growing and ripening, acetone extracts from selected samples of fruit growing and ripening were analyzed by HPLC-ESI-TOF. Features were detected from each tissue and analyzed by PCA; then, the most relevant principal component was chosen, and features from it were assigned possible identities by searching the most common adducts on Lipid MAPS® for preliminary identification. By doing this procedure we are presenting for the first time lipidomic profiles from each avocado fruit tissue during growth and ripening. Given that the difference in profiles of smallest fruits overshadows the other differences (PC1, Additional file 1: Figure S5), a PCA was performed on a subset of the samples, excluding the 60 g stage, which successfully separated samples by those that were in the ripening process from the ones that were not (Fig. 4a). Two groups were separated via score plots (top lines, Additional file 1: Figure S6) each with a particular trend, sharing a common characteristic of extremely high variability at the earliest stage, subsequently followed by either an increase in relative intensity (red, abundant in ripening) or a decrease (blue, abundant in mature green), reaching a maximum or minimum, respectively, in the ripe stage (Fig. 4b).
As it can be seen in the cloud plot (Fig. 4c), features present in mature green (top) and ripe fruit (bottom) looked similar, apart from a small section, close to the neutral lipid elution time, unrepresented in ripe fruit. Also, as mentioned above, there were more mature green abundant features (which decrease during ripening) than ripe abundant features, and the latter appeared to have lower Retention Times (RT) and m/z. When analyzing the automatic identity assignations of the features, a difference in the diversity of lipid classes was clear: lipid classes appeared to shift from Glycerolipids and Glycerophospholipids, abundant in mature green ( Fig. 4d), towards Fatty Acyls, the most varied class in the ripe group (Fig. 4e). It is important to note that acetogenins are grouped by the Lipid MAPS® classification in this Fatty Acyl class [45].
Features that were assigned automatically as Glycerolipids were manually curated (Supplementary Table 5) and variations in their intensity through fruit development were plotted into heatmaps ( Fig. 5a and b). It can be clearly observed that TAG diversity drops through fruit development, particularly during ripening: while 100 TAGs decreased, only 30 TAGs, increased (Fig. 5a). Interestingly, distribution of carbon number among TAGs following both trends was fairly conserved (Fig. 5c) while double bonds followed a different tendency, with monounsaturated and disaturated TAGs being the most affected by the ripening process, and saturated and trisaturated TAGs being the most common groups that increased (Fig. 5d). On the other hand, the distribution of DAGs pointed to a remodeling of the DAG pool ( DAGs that decreased during ripening had a higher sidechain carbon number and unsaturations than those that increased ( Fig. 5e and f). Interestingly, the number of DAGs with an even side-chain carbon number in the ripening-decreasing features was the same as those that increased during ripening (21 DAGs); however, DAGs with an odd-carbon number fatty acid are over-all decreasing, with 9 of them reaching a minimum in ripe avocado ( Fig. 5b and e, and Additional file 2: Table S5).
Lipidome changes in idioblasts have differences in the glycerolipid remodeling to that of mesocarp The idioblast-enriched fraction had a more diverse lipidome than mesocarp, with 5333 features detected. The PCA analysis showed the same separation as the PCA from avocado mesocarp: the main component separated the samples belonging to the smallest avocado triplicate, while the second component separated idioblasts extracted from avocados following ripening process from the rest (Fig. 6a). Again, only the second component was taken in consideration, and two populations were extracted from plotting the score values (Additional file 1: Figure S7). Remarkably, the features belonging to these groups followed the same pattern as those from mesocarp, (Fig. 6b). An interesting trend can be observed in the cloud plot ( Fig. 6c) where mature green abundant features, which decreased during ripening, had high m/z values and were concentrated in the chromatographic region corresponding to TAGs. Ripe abundant features, on the other hand, were more disperse and had lower masses and retention times, hinting at a degradation of the aforementioned TAGs, and an increase in diversity of slightly more polar lipids. The automatic classification of the features also points to the same shift as mesocarp, with Glycerolipids and Glycerophospholipids being the most abundant classification in mature green (Fig. 6d); and, Fatty Acyls (with acetogenins in them) were the most abundant in the ripe group (Fig. 6e), confirming our observation from the targeted analysis. When comparing lipidomic profiles, it was evident that the region of acetogenins was prevalent in idioblasts extracts while mesocarp accumulated mostly neutral lipids, confirming again the characterization made by our targeted analysis. Also, unlike mesocarp, the chemical nature of the varying glycerolipids in idioblasts differed greatly (Additional file 2: Table S6) and had encountered trends: while no trend dominated the manually curated TAGs (Fig. 7a), the majority of the DAGs decreased as fruit ripened (Fig. 7b). TAGs that decreased during ripening had higher carbon number (Fig. 7c) and unsaturations (Fig. 7d) than those that increased. DAGs, tended to have lower unsaturations as fruit ripen (Fig. 7f) and, interestingly, higher carbon number than those that decreased (Fig. 7e). Thus, they might be product of TAG degradation. Another interesting phenomenon occurs in this DAG remodeling in idioblasts, especially considering that acetogenins are odd-chain lipids: DAGs containing odd-chain FA were the most common features that increased during ripening, contrary to mesocarp (Fig. 7e). Particularly, the most abundant odd-chain category of decreasing mesocarp DAGs, it had a 39-carbon sidechain (Fig. 5e, Additional file 2: Table S5), the same as the most abundant category of increasing DAGs in idioblasts (Fig. 7e, Additional file 2: Table S6); both were also highly unsaturated (up to 8 unsaturations in mesocarp, Fig. 5f, and up to 7 in idioblast oddchain DAGs, Fig. 7f ).
Lipidomic profiles from seeds show glycerolipid consumption and acetogenin accumulation as main cause of changes in lipids from this tissue Most of the variation found in the 3648 features detected in seed was due to features from PC1 (41%, Fig. 8a), which separated samples in an almost exact fashion as the acetogenins did (Fig. 1c). In fact, the acetogenin contents per dry weight (TAC-DW) could be effectively predicted just by the value of the main component in the sample (R 2 = 0.91, Additional file 1: Figure S8A). This suggests that the most relevant changes occurring in the seed lipidome during fruit growth are due to accumulation of acetogenins, or acetogenin-related metabolites, as seed dry weight increases.
The two main groups responsible of the separation by TAC-DW differed greatly in mass, with most of the scores relating positively to TAC-DW having low m/z, and features with high-intermediate m/z being on the negative side of the scores plot (Additional file 1: Figure S8B). TAC-related features (Fig. 8b, red lines) presented a clear common trend when plotted by stage (Additional file 1: Figure S8C). This trend showed an analog behavior to that of the barplots in Fig. 1b, increasing rapidly in the first stages and reaching a plateau at medium-sized avocadoes; similarly when plotted against DW, with the plateau being reached at about 30% dry weight (Fig. 8b). On the other hand, the trend of the features with high molecular weight (Fig. 8b, blue lines) follows a decline as fruit grows, which, when plotted against dry weight, reaches a minimum at 45% dry weight (Fig. 8b). The cloud plot in Fig. 8c shows the typical lipidome profile from small seeds (bottom) and mature seeds (top). Features in the region of neutral lipids (high molecular weight, longer RT) were more abundant in seeds from smaller avocados, and features in the acetogenin region (low m/z and short RT) were more abundant in mature seeds (Fig. 8c). On the other hand, there were few metabolites in the region of polar lipids of high molecular weight that were more abundant in mature seeds. When analyzing Lipid MAPS® assignations, these assumptions were further supported, given that the most common putative identity of the features belonging to the high molecular weight group (Fig. 8d) was the Glycerolipids class. In contrast, the most common assignation, by far, of the TAC-related features (Fig. 8e) was as Fatty Acyls.
A further manual curation of the assignations was performed, to better characterize the metabolites that had the most relevant changes (Additional file 2: Table S7). Heatmaps of these curated assignations evidenced the decline in TAGs and DAGs as the seed develops, this in complete contrast with the increase in acetogenins (Additional file 1: Figure S9). In total, 139 features assigned as Glycerolipid decreased as seed matures (Additional file 2: Table S7); the most abundant group was TAGs, with 71 putative identity assignations; then DAGs, with 51; MAGs with 3; and 14 assigned as other glycerolipids. Most of these glycerolipids were polyunsaturated, and interestingly, more than one third of the putative TAGs and DAGs had either one or three odd carbon-number fatty acid radicals (Additional file 2: Table S7), although since this method cannot differentiate glycerolipids with a pair of odd carbon-number fatty acids, this number may be greater. A targeted analysis of adducts and fragments of the TAC-related features revealed a group of features sharing signature characteristics with the acetogenin NMR-confirmed standards, such as high oxygen count (O ≥ 4), odd number of carbon atoms, and loss of the acetoxy group, along with the fact that their scores on the PC1 were high enough to be selected (Additional file 1: Table S4). A total of 163 features were assembled in 35 groups that were assigned putative identities as acetogenins, 30 of which were new to us (Additional file 2: Table S7). Some of these putative acetogenins would have characteristics that have not been previously reported, such as completely saturated Persenins, triple acetoxylations, and highly polar acetogenins with short retention times (Additional file 1: Table S4). Therefore, it is reasonable to claim that as seed increases in dry weight, glycerolipid contents, particularly TAGs and DAGs, decrease, while acetogenins and acetogeninrelated metabolites increase. This observation is further supported with correlation analyses (Fig. 9), in which it is very clear the very high negative correlation of acetogenins with both TAGs and DAGs. Interestingly, when analyzing the highest correlations between TAGs, DAGs, and acetogenins, there is a cluster of the former group that has the highest correlation with acetogenins, rather than DAGs.
Discussion
Little is known about aliphatic acetogenin metabolism in avocado. Apart from its response to pathogens [34], elicitation with ethylene and discovery of linoleic acid as one precursor of Persin [30,31]; there have been no further studies about the production and distribution of these metabolites in avocado fruit. As a first approach we fully characterized acetogenins produced in 21 different avocado accessions [37], showing that this is a very conserved metabolism in Persea americana. The current study was undertaken to further characterize acetogenins in the most produced cultivar worldwide, 'Hass'. We followed acetogenin and lipid production in mesocarp, idioblasts and seeds from avocado fruit during maturation, ripening, seed germination and harvesting years looking for indication of possible metabolic Features characteristic of small seeds and (e) seeds from mature fruits ripening fruit by Lipid MAPS® Classification [45] switches that can serve as a system to further characterize acetogenin biosynthesis. In doing this we have produced for the first time full acetogenin profiles during fruit growth and, moreover, to the best of our knowledge we have generated the first LC-MS based lipidomic profiles of tissues of this fruit mostly known for its oil accumulation.
Mesocarp and idioblasts lipid metabolism differs greatly during avocado fruit ripening
The growth of avocado fruit was not a clear differential factor for acetogenin contents in mesocarp and idioblasts. Early works followed only two acetogenins during fruit maturation from the cultivar 'Fuerte' , Persin [5,34] and AcO-avocadene [5], which represent respectively 27 and 12% of the total acetogenin pool in mature green 'Hass' cultivar fruits [37]. AcO-avocadene did not have a significant change in 'Fuerte' mesocarp as the fruit expanded (up to 5-fold increase in fresh weight) [5], which is in accordance to our findings (Fig. 1a). On the other hand, it was reported that Persin contents decreased as fruit expanded [5] and during ripening [36]. The co-elution of Persin/Persenone B did not significantly change during development in avocado mesocarp. However, since Persenone B has an absorption maxima closer to 220 nm than Persin [37] it is possible that it is masking changes in Persin concentration during development.
The main features that changed in mesocarp as fruit ripens were TAGs and DAGs. Interestingly, the level of polyunsaturation decreased in TAGs during ripening, with DAGs having more hits with two and four desaturations following the opposite trend, increasing during ripening. There is only one report about changes in avocado lipids during ripening: the mesocarp of 'Hass' avocados showed that the unsaturated linolenic, linoleic and palmitoleic acids slightly increased during eight days after harvesting, while the main avocado FA, oleic acid, remained constant [21]. It its possible then that the increase in polyunsaturated DAGs is related to this observed trend, probably as intermediates in the catabolism of TAG reserves and not de novo synthesis, as it has been previously suggested that lipid accumulation stops as fruit is harvested [46,47]. On the other hand, and very interestingly, in idioblasts cells there appears to be an increase in chemical diversity as fruit ripens, as the features that increase during ripening more than double those that decrease. Remarkably, we found that almost all total acetogenins in the ripe fruit were stored in these specialized cells, which only account for 2% of the mesocarp volume [27], this suggests that idioblasts are the main site of acetogenin synthesis within mesocarp.
It has been shown that isolated idioblasts are able to synthesize Persin when elicited with ethylene [30] and previously it was roughly estimated that 70-90% of the Persin was accumulated in idioblast from mature avocado mesocarp [5]. In addition, formation and maturation of lipid-containing idioblasts seem to be independent from the fruit developmental stage (idioblasts from all stages in their development have been detected from small ovularies to unripe immature, unripe mature and ripe fruits) [28,29,41]. Therefore, the measurement of whole mesocarp fatty acid contents would mask the contribution of the small amount of lipids present within idioblasts for acetogenin biosynthesis; this is why we did not find a correlation between oil accumulation and acetogenins during fruit growing and only when normalizing by dry weight did acetogenins seem to slightly decrease in mesocarp (Fig. 1a). Nevertheless, it could be argued that idioblasts can be a place for acetogenin storage rather than the place of biosynthesis, we consider this possibility as low for the following reasons. In young fruits, protoplasts of idioblasts react immediately with colored stains, while in mature fruits idioblasts generally remain impervious, probably because of suberin deposition [29,41]. Also, idioblasts do not lose integrity during ripening because the suberin layer is not degraded by neighboring enzymes [41]. It is probable thus, that in immature fruit idioblasts can interchange molecules with parenchymatic cells. However, we have found more acetogenins in idioblasts from ripe fruit when they are closed and would not be able to translocate acetogenins (Figs. 2 and 3). We cannot rule out that parenchyma cells could be another -minor-place of acetogenin biosynthesis, but evidence shown here indicates the majority of acetogenin biosynthesis happens in these specialized cells in avocado mesocarp.
Acetogenins are likely produced independently in avocado seeds and their accumulation is triggered during early fruit growth Untargeted lipidomic analysis of seed tissue set the importance of acetogenins in the developing seed, revealing them as one of the main lipid classes by placing this oftignored compounds in the context of the rest of the lipidome. Lipids have been characterized in mature 'Hass' avocado seeds previously [48], and the total lipid extraction reported to be 11 ± 1.5 mg(g FW) −1 , with a composition mainly consisting of neutral lipids (80.3%) followed by glycolipids (12.3%) and phospholipids (7.4%); the latter distribution calculated as percentage of the total eluate from a silicic acid column. The same study also declares TAGs as the main component (around 75%) of the neutral lipids, followed by DAGs (1 0%) and MAGs (~5%) [48]; which resembles the distribution of the features found here to be relevant in seed development. However, here we report acetogenins as one of the main components of 'Hass' avocado seed lipids, with an average composition in mature green fruits (4.99 ± 1.61 mg[g FW] −1 ) in the same order of magnitude as the total saponifiable fatty acids (3.02 ± 0.72 mg[g FW] −1 ), which would make a significant fraction of the total lipid extract reported. A visual analysis of the chromatograms presented here of seed lipid extracts revealed acetogenins as one of the main contributors to the seed lipidome, particularly in seeds coming from mature green avocados (Fig. 8c, bottom chromatogram). This was further supported by the unsupervised analysis, that revealed that acetogenins (tagged as fatty acyls) were responsible for the main variations in seed during dry weight accumulation, as they increased as seeds reached maturity, while glycerolipids, the main peaks in immature seeds, decreased. In fact, very small seeds presented a visually distinct lipidomic profile than those from fully-grown fruits, coinciding with their very distinct anatomy: here we are profiling lipids from seed coats, endosperm and small embryo while in mature seeds we are profiling lipids mostly from embryo's cotyledons, the tissue that grows the most during avocado seed development, mature seeds consist more than 99% cotyledon tissue. Other studies could not assess this as seeds lipids have been characterized only by targeted analyses and just in one sample of purchased mature fruit [38,48,49].
We found that the main groups of seed lipids that changed through development corresponded to Glycerolipids (neutral lipids + glycolipids) and Glycerophospholipids, which decreased as seed embryo increased in dry weight, and fatty acyls, mainly acetogenins, which were by far the main lipid class enclosed in features that reached a maxima in mature seeds. The latter confirms the observation of the targeted approach, and indicates that the acetogenin metabolism is quite dynamic in this tissue, and probably influenced by substrate, given they present a dry weight dependent accumulation. Moreover, avocatins (C19) are highly concentrated in seed when compared with mesocarp and idioblasts (Fig. 1b, inset, [37]), which coincides with the fatty acid distribution with a higher contribution of palmitoleic acid (C16) in seed than in mesocarp and idioblasts. Interestingly, more than one third of the Glycerolipids that decrease through development have side-chains consisting of either one or three odd-chain fatty acids. It has been reported that, in ripe fruits, odd-chain fatty acids are present in avocado seed oil extracts in a higher percentage than in mesocarp, although in trace quantities [50]. It is therefore expected that in immature seeds, these fatty acids will be present in biologically relevant quantities. Also, since they decreased as acetogenins increased in concentration, the former are the main candidates to being precursors of the latter.
In most avocado cultivars, mature seeds generally accumulate more acetogenins than mesocarp [37]. We have previously hypothesized that this tissue might be capable of an independent synthesis, since it has distinct profile of both acetogenins [37] and fatty acids [50] than mesocarp. It was then very interesting to find here that seeds from very young fruit accumulated acetogenins in minute amounts and with different profiles than seeds from more developed fruits. As previously mentioned, seeds from the smaller fruit samples presented the characteristics of early-immature avocado seeds, which included a predominant gelatinous endosperm tissue covering the embryo and two thick and fleshy seed coats [22]. Conversely, in more mature seeds, the embryo grows steadily while the endosperm tissue disappears and the seed coats shrivel and darken until dry; thus, hindering the translocation of molecules between embryo and pericarp [22]. It was then (at weights >90 g) when acetogenins began to accumulate significantly in our samples; thus, acetogenin accumulation in seeds correlated negatively with endosperm abundance and vascular continuity and positively with the embryo growth. Then, it is tempting to speculate that acetogenin accumulation in seeds may be either triggered to relocation of resources during seed development, or its accumulation would be exclusive to embryo, particularly cotyledons (embryonic axis showed no detectable acetogenin accumulation), which displace the endosperm in later stages of maturity. Correspondingly, because acetogenins accumulated the most when the transfer of molecules seemed to be obstructed by the drying of seed coat vascular system, it is highly probable that cotyledons are capable of independent biosynthesis. Furthermore, it has been recently reported that genes involved in linoleic acid biosynthesis show higher expression levels in mature avocado seed tissue than in fruit [47]; however, mature seeds accumulate minute amounts of this (and every other) fatty acid. Particularly, Fatty acyl ACP thioesterase (FatB) important for long-chain fatty acids export from the plastid, Ketoacyl-CoA synthase (KCS1), in charge of activating and making them available for enzymatic reactions, and Oleate Desaturase (FAD2), which forms C18:2, were expressed more than 4, 3 and 2fold, respectively, in seed than in avocado mesocarp [47]. Given that linoleic and linolenic acids, are orders of magnitude less accumulated in mature seed than in pulp (17 and 26-fold less, Table 2), we speculate that such overexpression of long-chain fatty acid biosynthesis genes is directly related to acetogenin production, as we have shown the relevance of these metabolites in seed tissue. Evidence presented in this work, along with this previously reported information, strongly supports our hypothesis of seeds -particularly cotyledons-being able to independently synthesize these metabolites.
The trigger of acetogenin accumulation in seeds is the clearest indication of a possible switch that we have found in our studies, as acetogenin profiles are quite constant among avocado cultivars [37], changed little during growth and ripening in mesocarp, and environment seemed not to significantly affect them either. On the other hand, during germination, acetogenins from cotyledons did not change while embryonic axis and seedlings were not able to produce detectable amounts of these metabolites. As acetogenins are present in avocado leaves [44], it is highly probable that a similar switch exists also in this tissue, probably coinciding with the shift from auxo-to autotrophy. Therefore, this tissue offers an attractive model to study acetogenin biosynthesis as it seems that at one point during maturation, most of the seed lipid reserves are used to produce them.
Acetogenin building blocks are likely to come from TAG editing and catabolism in avocado idioblasts and seeds
It has been shown that linoleic acid is incorporated to Persin [30,31] and that the expression of some FA editing enzymes positively correlates with this single acetogenin [31,32]. This evidences FAs as precursors, however, until now we can point the metabolic process in which acetogenin biosynthesis might begin. Here we showed that acetogenin contents increased during fruit ripening in idioblasts; in the same manner, the most common DAGs that also increased during ripening, had odd-carbon, highly unsaturated fatty acid chains, while long-chain highly processed TAGs decreased. Seed lipidome showed the same trends ever more clearly, as it seemed that TAGs and DAGs pools were committed to acetogenin synthesis during seed development.
TAG biosynthesis and FA β-oxidation processes have been fully described in plants; however, the steps in TAG catabolism remain elusive. Forward and reverse genetic studies in Arabidopsis have shown that TAG catabolism is triggered by SUGAR-DEPENDENT1 (SDP1) and SDP1-LIKE lipases [51,52]. In fact, sdp1 transcript abundance increases at the beginning of seed maturation and it is expressed the most in late development; this coincides with the decline in TAG that we observe here in avocado seeds. Other lipases that must act on DAGs and MAGs to produce free FA and glycerol are yet to be characterized. The free FAs need to be activated to an acyl-CoA and translocated to the peroxisome for βoxidation [53]. Evidence produced by this work suggests that the biosynthesis of acetogenins happens precisely in between these two processes. Moreover, very interestingly, it has been reported that besides SDP1 lipase, there is a lipoxygenase-dependent degradation of storage lipids in some plants [54]. LOXs (linoleate:oxygen oxidoreductase), in specific 13-LOX, can prime the hydrolysis of polyunsaturated TAGs by oxygenating their esterified FA residues [54,55]. These LOXs produce a wide array of oxygen-rich lipids, to the point where acetogenins may fall within the chemical diversity of their products. Some of the LOX-dependent reactions which may be taking place are side-chain hydroxylations, as well as production of α and γ-ketols [56], also, degradation of LOX-derived oxylipins leads to β-ketols [57] similar to the polar head of acetogenins (Table 1), and LOXs produce keto-PUFAs [54], which have a cis double bond adjacent to the keto group, the same as Persin and other acetogenins. In this paper we report several putative acetogenins containing high number of oxygens (Additional file 2: Table S7), which may correspond to intermediary products with side-chain hydroxylations. Unfortunately, our current approach is database-dependent; therefore, unreported molecules are virtually impossible to identify. Even though, we dare to speculate that acetogenins, −oxygen-rich aliphatic molecules-could start to be produced by editing enzymes acting directly on TAGs and maybe DAGs, rather than on free fatty acids or other fatty acylester moieties in avocado.
Conclusions
For the first time five different acetogenins, plus two tentative aliphatic acetogenins were simultaneously analyzed in avocado mesocarp, seeds, and specialized idioblast cells, during fruit growth, postharvest ripening, germination, and three harvesting years. This is also the first record of a chromatography-coupled, untargeted lipidomics approach at analyzing avocado fruit.
By contextualizing acetogenins in the seed lipidome, our results reveal their importance as a major lipid family in seeds, not only by being one of the most accumulated lipids by weight in mature seeds, but also responsible for the main variations in lipid metabolites as seed matures. This dry weight dependent increase in acetogenins coupled with a decrease in TAGs and DAGs (a third of which have odd-chain fatty acids), along with previously reported high expression of fatty acid synthesis genes in this tissue, supports our claim that seed is independently able to synthetize acetogenins in cotyledons and places their synthesis during oil catabolism. The 35 putative acetogenins found in seeds by the lipidomic analyses shows the high diversity of these compounds and evidences the ramifications of this metabolism in avocado. In addition, the prevalence of acetogenins in the avocado lipidome hints to an important yet to be discovered role of these metabolites in the physiology of the plant.
Lipidomic profiles from mesocarp and idioblasts showed the complexity of the idioblast lipidome, and corroborated the observations from our targeted analysis. In addition, it unveiled the dynamics of lipids during fruit growing and ripening showing that mesocarp and idioblasts, although following the same general tendency, differed on trends within specific subfamilies, such as TAGs and DAGs. Idioblasts were shown to accumulate most of the acetogenins in mesocarp, while only accounting for a minimal fraction of the total fatty acids. They thus become the main candidate for being the place of acetogenin biosynthesis in mesocarp. Characterization of this cell-type will definitely help to describe acetogenin metabolism in avocado.
All the evidence presented here will serve to continue to study acetogenin metabolism focusing on idioblast and seeds and also shows for the first time lipidomic profiles from avocado tissues that evidence the prevalence of these compounds in avocado. Also, this work sets strong evidence for acetogenins being included in all future work aimed at characterizing the avocado seed lipidome, as they are a paramount component of the lipid fraction.
Plant material
Avocado fruits from the 'Hass' cultivar were harvested from orchards located in Uruapan, Michoacán, México (19°25 N, 102°03 W; 1620 m AMSL), which is the major commercial avocado producing region in the country. Samples for the different experiments were collected and studied between the years of 2011 to 2014. After collection, avocados were kept at ambient conditions in perforated bags overnight and shipped in closed containers with activated carbon. Samples for the first studies, described below, were collected in 2013 and upon arrival to the Centro de Biotecnología FEMSA, avocados were divided in three subsets for the fruit growth (Study I), postharvest ripening (Study II), and idioblast isolation studies (Study III). As it is described in the following sections, other avocado samples were also analyzed in Studies IV and V to study the effects of seed germination and harvesting season on acetogenin profiles, respectively.
Study I. Fruit growth -To characterize changes during fruit growth (Additional file 1: Figure S1, first part) avocado fruits were selected and separated directly at the Uruapan orchard by weight. Samples were grouped by two methods: by fruit fresh weight, to assess changes related to growth, into 10 categories (45,60,90,120,150,180,210,240,270 and 300 g) containing three replicates each; and by dry matter, to investigate relation to oil content. Upon arrival at the laboratory, the selected avocados were stored at 4°C for 48 h, followed by freezing at −20°C for other 2 days, and final storage at −80°C. Study II Postharvest ripening -Fully developed avocados (300 g), harvested at the same time than those for Study I were placed under a temperature controlled, ventilated environment until reaching one of the three sought ripening stages, in order to assess postharvest evolution of lipids (Additional file 1: Figure S1, second part). Fruits were separated by hedonic scale in Unripe (one week after detachment; peel still green); Breaker (two weeks after detachment; peel half black, half green, mesocarp still hard but softening) and Ripe (two and a half weeks after detachment; peel black and mesocarp soft, ready to eat); each stage with three replicates. When samples reached each stage, freezing and storage was followed as described in Study I.
Study III. Idioblast characterization-Idioblast isolation was also conducted on the same samples used for Study I at every other stage during growth, and from Study II at all postharvest ripening stages, to characterize acetogenin contents and distribution; and finally from 5 mature green (300 g) avocados to simultaneously assess acetogenin and fatty acid profiles. To avoid compromising cell integrity, idioblast extraction was performed as soon as avocados were at the desired stage. Samples were processed into slices, digested and fractionated to isolate idioblasts as described below; and the remaining tissue was frozen at −20°C until further analysis. Thus, each idioblast replicate has a corresponding mesocarp and seed counterpart from Studies I and II.
Study IV. Germination studies-A set of samples collected on October 2011, were used in seed germination studies. Germination was conducted as reported [26] with minor adjustments. Briefly, avocado seeds from fully ripe black fruit were washed with soap and stored for three days at 4°C in closed plastic bags filled with sterilized peat moss, to avoid dehydration. Prior to germination experiments, seeds were rinsed, decorticated (to remove seed coats) and a horizontal cut was made at the base of the embryo, without damaging the embryonic axis. Cut seeds were placed in a container filled with water covering one quarter of the seed. Samples were changed to individual Magenta™ vessels as they grew, and germinated inside a growth chamber (23°C, 18:6 light/dark cycle). Embryonic axis and cotyledon sub-samples were collected in triplicate at the beginning of the experiment (day 0), twice a week after germination during a 3-week period (days 7 to 24), and after 10 weeks of imbibition (day 70). Embryonic axis (plumule and radicle) and cotyledon sub-samples were flash-frozen in liquid nitrogen, and stored at −80°C until further analysis.
Study V. Seasonal effects-To investigate possible changes in acetogenin profiles throughout the harvesting years, sampling was conducted on October 2011, June 2013 and April 2014. Avocado fruits used for the study were collected at a mature green stage and stored as described above. Studies I, II, and V included acetogenin determination for both fruit mesocarp and seed tissues.
Determination of moisture content
Dry weight was determined by weighting 5 g of material (seed or mesocarp), cutting it in thin slices (mesocarp) or small cubes (seed) and incubating at 105°C until constant weight was achieved (typically 5-6 h) [58].
Idioblast fractionation
Idioblasts were fractionated as described elsewhere [29,59], with modifications. Briefly, 5 g of avocado mesocarp, in a slice, were cut to small pieces and briefly homogenized at 11000 rpm in 10 mL of a buffer containing 10 mM MES, 100 mM sorbitol, 1 mM CaCl 2 , 0.2% BSA and 0.2% DTT, at pH 5.5, and lytic enzymes (Cellulase Onozuka RS, 165 units/mL; and Macerozyme R10 mix, 15 units/mL, final concentration; Phytotechnology Laboratories). Oxygen was removed from the headspace with Nitrogen and the homogenate was incubated for 2 h in the dark at 150 rpm and room temperature. Afterwards, the mix was briefly homogenized again, and then filtered through nylon mesh filters of pore size 140 and 61 μm, and washed in the filter with buffer without enzymes. Fraction F140 contained mainly vascular tissue and undigested tissue, and fraction F60 was the idioblast-enriched fraction. Cell integrity and purity (absence of parenchymatic cells) were checked by microscopy, which clearly differentiates intact from burst idioblast cells and also lipid-containing idioblast from parenchymatic cells (described below).
Acetogenin extraction
Extractions were made as described previously [37], namely, tissues were separated, mesocarp (2 g) was cut in even, longitudinal slices, and cotyledons (1 g) were macerated while frozen. On the other hand, idioblast enriched fractions (recovered from 5 g of mesocarp) were analyzed directly. Extraction was achieved by addition of 15 mL of acetone, where samples were homogenized with the aid of a Polytron homogenizer (Ultra-Turrax T25, IKA-Werke, Germany) for 3 min, sonicated for 1 min and clarified by centrifugation at 10000 g at 25°C for 10 min. A 1 mL aliquot was then taken and dried under nitrogen, redissolved in 2 mL of water and added 2 mL of dichloromethane, and the organic phase was recovered, dried, resuspended in 1 mL isopropanol and filtered through a 0.2 μm PTFE filter, for HPLC injection. The extraction was made under dim light and for every step following homogenization, air was displaced from the headspace using nitrogen gas.
Extracts were separated with the aid of a C18 column (Zorbax Extend-C18, 3x100mm, 3.5 μm; Agilent, CA, USA) using a HPLC-VWD (Series 1100; HP, CA, USA) system and a gradient elution program using water (A) and methanol (B) as mobile phases, as stated in [37] only with a minor modification to column temperature, set to 35°C. Chromatographic profiles were obtained by measuring absorbance at 220 nm and identities were assigned by comparing the retention times to those with NMRconfirmed, purified peaks by Rodríguez-Sánchez et al. [60]. Calibration curves were generated for every purified compound based on weight, except for Persenone A, for which an extinction coefficient is available. Only peak (3), an Unknown Putative Acetogenin (UPA), was quantified in Persenone A equivalents. Since Persin coeluted with Persenone B [37] the chromatographic peak was considered as both Persin and Persenone B, and quantified with a Persenone B calibration curve, as it is the moiety that absorbs the most at 220 nm (Persin absorption maximum is at 208 nm [37]).
Fatty acid extraction
For lipid extraction, a modified Folch method was used [61] in which the tissue or an idioblast fraction (0.5 g) was homogenized in a 2:1 solution of dichloromethane:methanol (10 mL) for 3 min, sonicated for 5 min, and left at room temperature for at least 10 min before centrifugation (10,000 g) at room temperature for 5 min. Clarified phase was then vigorously mixed with a NaCl solution (0.9%, 2 mL), then centrifuged (5000 g, 2 min) and the recovered organic phase was evaporated. The remaining oil was then resuspended in a KOH solution (4 mL, 1 M in 96% ethanol) and left overnight at room temperature, under a nitrogen atmosphere, for saponification. The solution was then mixed with water (10 mL), and extracted 3 times with hexane-diethyl ether (1:1, 10 mL). Organic extract was further washed with water (10 mL), which was then mixed with the previous aqueous phase and acidified with HCl to a pH of 3. Fatty acids are recovered from acidified phase with subsequent extractions (10 mL, 3 times) with hexane-diethyl ether (1:1). Organic extracts were evaporated to dryness, resuspended in isopropanol and passed through a PTFE filter (0.2 μm) prior to injection.
Separation and detection were made by HPLC-ELSD (1200 Series; Agilent) with the aid of a Luna C8(2) column (2.6x75mm, 3.5 μm; Phenomenex) using the vendor application No. 1258 [62] with slight modifications. Solvent gradient was programed to change from 70% acetonitrile in water, to 90% acetonitrile during the first 10 min, followed by a change to 100% acetonitrile by minute 11, and kept for 4 extra minutes, before returning (at minute 15) to the initial conditions for 5 min before the next injection, all at a flow rate of 0.3 mL/min. Detector was set to a temperature of 40°C, with a gain of 4, with no offset, and a sampling rate of 0.1 s with a gas pressure of 3.3 bars; quantification was made by comparing areas to a curve made with certified standards for each fatty acid (Palmitic, Palmitoleic, Stearic, Oleic, Linoleic, and Linolenic acids), which were purchased from Sigma-Aldrich (St. Louis, MO, United States).
Staining experiments
For staining experiments, nucleic acids were stained using 4′,6-diamidino-2-phenylindole (DAPI, ThermoFisher, USA), and a lipid specific dye, Nile Red (Sigma-Aldrich, USA), was used for oil staining following vendor instructions. Idioblast cell integrity was visualized in an AXIO Imager.A2 Microscope (Carl Zeiss, Oberkochen, Germany) with a HXP 120C UV source (OSRAM, Munich, Germany) equipped with a mercury lamp. Cytometric measurements on stained samples of pulp homogenate, idioblast enriched and permeated fractions were performed on a BD FACSCanto II flow cytometer (BD, San Jose, Calif., U.S.A.). Data was acquired from a total of 10,000 events per sample, collected at low flow rate through channels PerCP (670 LP nm band-pass filter) and FITC (530/30 nm band-pass filter), in forward and side scatter. Group discrimination and purity assessment was performed in R, as stated in the Data Analysis section.
Lipidomic analysis
All acetone extracts from Study III (Idioblast characterization) were selected to follow the lipidomics pipeline, along with their corresponding extracts from mesocarp and seed in Study I and II, with the exception of the smallest stage (45 g) for which there was not enough sample and was substituted by the next stage (60 g) in mesocarp and seed. Acetone was evaporated in the dark, under vacuum, at 45°C, until dryness, and resuspended in Isopropanol. Resuspension volume was calculated as to inject a constant amount of dry weight for each tissue.
Chromatographic separation
Extracts were separated with a Luna C18(2) column (150x2mm, 3 μm; Phenomenex, CA, USA) using a HPLC (Series 1100; Agilent, CA, USA) coupled via ESI to a TOF MS Detector (G1969A; Agilent, CA, USA) system and a gradient elution program that included a water:Acetonitrile mix (4:1 v/v; phase A) and an Isopropanol:Acetonitrile mix (9:1 v/v; phase B) as mobile phases, both modified with 10 mM Ammonium Acetate and 0.1% Formic Acid. Samples were separated at 55°C and the elution gradient had a constant flow of 0.2 mL/ min. The 65-min gradient consisted of linear ramps from 40% to 43% B (6 min); jumping to 50% B at minute 6, and ramping linearly to 54% B until minute 36; then changing immediately to 70% B and linearly increasing to reach 99% B by minute 54. This condition was kept until minute 55, when column returned initial conditions (40% B) where it equilibrated (10 min). ESI drying gas (nitrogen) was set to 13 L/h, at 350°C, with a nebulizer pressure of 35 psig; capillary voltage was set to 4.5 kV to favor fragmentation, and the optical parameters were set to 250, 225, and 60 V for the octopole radio frequency voltage (Oct RFV), fragmentor and skimmer, respectively. Runs were performed to acquire mass spectra in positive mode, and files were saved in profile mode, with an m/z range from 150 to 1500 m/z, and reading at 0.94 cycles per second, with a total of 10,000 transients per scan. Samples were injected in a random manner, and began with a set of 5 'dummy' runs, where the same amount of a mix of all samples was injected.
Feature detection
Raw files were converted to CDF using Agilent's Translator Utility (Agilent, CA, USA), and processing was done in the MZmine 2.15 platform. [63]. GridMass algorithm [64] was used for peak detection and base line correction was performed using an in-house implementation of a 2D-baseline correction method as a module for MZmine, which is available in http://bioinformatica.mty.itesm.mx/baseline2d. The baseline algorithm works by considering a range of time points from a window in m/z to reduce the background, which is estimated by a percentage of observed data within the window. This algorithm was run, considering an m/z window of 0.01, a retention time (RT) window of 1.5 min, and a 40% quantile; and peak detection via GridMass using a minimum height of 1000 counts, an m/z tolerance of 0.05, a RT window between 0.1 and 2.5 min, a smoothing time of 0.1 min and an intensity similarity ratio of 0.5. After feature detection, isotopic peaks grouping was performed using an m/z tolerance of 0.001 or 10 ppm, and a RT tolerance of 0.25, assuming a monotonic shape and a maximum charge of 2, with the lowest m/z as the most representative isotope. Alignment of features was achieved by the RANSAC algorithm with an m/z tolerance of 0.025 m/z, or 50 ppm, a RT tolerance of 2 min before and 1.5 min after RT correction, a minimum of 25% points matching the non-linear model below a threshold of 1 min. Finally, gap filling was performed using our own algorithm in R integrating the intensity over non-detected peaks in the RT window predicted using the detected peaks which better predicted the RT window of the detected features in the sample, with an m/z tolerance of 0.025 or 50 ppm. Further analysis of the resulting feature tables was performed in R, as stated in the Data Analysis section.
Grouping
Given the nature of the data, in which molecules may be confounded with isotopes of members of the same family, which is also rich on isomers, many of the detected features included isotopes and artifacts of the feature detection. Also, different adducts of the same molecule may be present, and, given the high voltage selected, molecules may be subject to fragmentation, which can yield information on their structure. Therefore, after processing the raw data with MZmine, an automated grouping procedure was performed in R. On the selected list of features, an in-house built algorithm was used to extract from the raw files information of the peaks in the samples, such as m/z, retention time, and intensity values from each measurement between the full width at quarter maximum (FWQM) of the chromatographic peak. Second, features were compared among each other, and if the retention times overlapped in some point, they were considered as "candidates" to belong to the same compound. Later, this candidate list was further trimmed based on the correlation of the respective intensities of the peaks at each time point, and were considered to belong to the same molecule only if their correlation was above 0.9. Since correlation rapidly degenerates as RT shifts, this threshold is equivalent to a shift of one scan (<1 s) even if the peak follows the exact same peak shape. An example of this grouping is shown and explained in Additional file 1: Figure S10. Selected peaks that belonged in the same group were considered as probably belonging to the same molecule, and were manually cleaned from isotopes by visually assessing the experimental mass spectra, and curated in search for adducts or fragments in the selected and automatically identified features.
Assignation of identity
Using the same information extraction algorithm from the grouping method (2.7.3) each peak was assigned a mean m/z measurement and its standard deviation, and was assigned a charge based on the first isotope. Monoisotopic masses of the most common adducts in positive mode for single ( [65] to access the Lipid MAPS® structure database [66]. The m/z window for the search was taken as 3 standard deviations of the m/z measurement, plus 25 ppm, and assignations were further cleaned by comparing the theoretical isotopic pattern of the molecular formula (including the adduct) with the experimental intensities, using as an allowed window the standard deviation of the intensity divided by the square of the correlation to the monoisotopic feature, plus 5% of the intensity value. Information retrieved included Molecular Formula, Name and Lipid MAPS® classification [45].
Therefore, a single feature could be assigned more than one putative molecular formula, each of which could in turn represent one or more known compounds. However, a set of different compounds that differ in identity, may belong to the same category in the Lipid MAPS® Lipid Classification System, having a similar biological role. Thus, using the main and secondary lipid classes, we estimated an "average" composition of each sample by estimating the proportion of 'fixed' and 'approximate' composition of all possible annotations for each mass. The 'fixed' composition represents the annotations that are independent of the choice of the assigned compound either because it contains only one compound or because all compounds are annotated as belonging to the same family. The 'approximate' composition represents a weighted average composition from all possible compound annotations. For instance, the single-charged feature 421.
Data analysis
All algorithms and statistical procedures, such as Analysis of Variance (ANOVA), t-tests, and Principal Component Analysis (PCA) were made using the R platform [67] with the stats library, unless otherwise stated. Grouping by Tukey's Honestly Significant Difference (HSD) was done with the aid of the agricolae package [68]. For a result to be considered significant, a p-value threshold of 0.05 was set for the ANOVAs and t-tests; similarly, an α value of 0.05 was used for Tukey's HSD. While linear model regressions were estimated using the R stats library, non-linear regression parameters were calculated using Microsoft Excel® (Microsoft Office Professional Plus 2013) "Add Trendline" function. Contents mentioned in the text are shown as mean and standard deviation unless otherwise stated. Flow cytometry data was accessed by use of the flowCore library [69], and purity assessed by predictive Linear Discriminant Analysis (pLDA) with the DiscriMiner library [70]. Direct access to MS files was achieved through ncdf package [71], and generation of theoretical exact mass and isotopic pattern calculations, using Rdisop library [72]. Previous to lipidomics analyses, the matrix with the raw intensities was quantile-normalized and log-transformed; then, centered and scaled feature-wise. All images here presented are of our own creation, using a combination of R, Microsoft Excel® and PowerPoint® (Microsoft Office Professional Plus 2013), and ACD/ChemSketch (ACD/Labs version 12.01, 2010) for chemical structures.
Additional files
Additional file 1: Figures S1 to S10; Table S1 to S4. This file contains a schematic explaining the terminology used in the article to refer to developmental stages (Additional file 1: Figure S1); information regarding acetogenin contents in mesocarp and seed of 'Hass' avocado fruit in a fresh weight basis (Additional file 1: Figure. S2, Tables 1 and 2); and micrographies of dyed idioblasts (Additional file 1: Figure S3), results from the assessment of extract purity (Additional file 1: Figure S4, Table S3), and acetogenin contents (Additional file 1: Table S4). Also, it contains complementary information on the metabolomics analysis, such as PCA of the mesocarp features (Additional file 1: Figure S5) and score plots of the mesocarp (Additional file 1: Figure S6) and idioblast (Additional file 1: Figure S7) samples. The corresponding part in seed tissue (Additional file 1: Figure S8) is included, along with the heatmaps of the manually curated TAGs, DAGs, and Acetogenins data (Additional file 1: Figure S9). Finally it contains an example of the grouping algorithm mentioned in the Methods section (Additional file 1: Figure S10 Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions CERL and RIDG conceived the study and wrote the manuscript; CERL conducted all experiments; CERL and VTA analyzed lipidomic data. CERL, CHB, VTA and RIDG carried out the interpretation of results and critical revision of the manuscript. All authors gave final approval of the published version.
Ethics approval and consent to participate Avocado fruits used in Studies I, II and III were harvested with the permission of the owner of the orchard, Martha de la Peña; and commercial fruits used in Studies IV and V were sampled with the permission of the owners of the fruits that were Rafael Morán, from H.E.B. México, and Rodrigo Treviño, from Frupack, México. All of the above mentioned individuals donated the fruit for the studies and gave their consent to publish the results from the scientific experiments and analyses performed with them.
Consent for publication
Not applicable.
Competing interests
Some of the uses of acetogenins in the food industry are protected under patent application WO/2012/042404 [14].
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | 2017-10-03T08:44:12.133Z | 2017-09-29T00:00:00.000 | {
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57574343 | pes2o/s2orc | v3-fos-license | The Combination of APRI and ALBI Facilitates Preoperative Risk Stratification for Patients Undergoing Liver Surgery After Neoadjuvant Chemotherapy
Background Neoadjuvant chemotherapy (NeoCTx) is performed for most patients with colorectal cancer liver metastases (CRCLM). However, chemotherapy-associated liver injury (CALI) has been associated with poor postoperative outcome. To date, however, no clinically applicable and noninvasive tool exists to assess CALI before liver resection. Methods Routine blood parameters were assessed in 339 patients before and after completion of NeoCTx and before surgery. The study assessed the prognostic potential of the aspartate aminotransferase (AST)-to-platelet ratio index (APRI), the albumin-bilirubin grade (ALBI), and their combinations. Furthermore, an independent multi-center validation cohort (n = 161) was included to confirm the findings concerning the prediction of postoperative outcome. Results Higher ALBI, APRI, and APRI + ALBI were found in patients with postoperative morbidity (P = 0.001, P = 0.064, P = 0.001, respectively), liver dysfunction (LD) (P = 0.009, P = 0.012, P < 0.001), or mortality (P = 0.037, P = 0.045, P = 0.016), and APRI + ALBI had the highest predictive potential for LD (area under the curve [AUC], 0.695). An increase in APRI + ALBI was observed during NeoCTx (P < 0.001). Patients with longer periods between NeoCTx and surgery showed a greater decrease in APRI + ALBI (P = 0.006) and a trend for decreased CALI at surgery. A cutoff for APRI + ALBI at − 2.46 before surgery was found to identify patients with CALI (P = 0.002) and patients at risk for a prolonged hospital stay (P = 0.001), intensive care (P < 0.001), morbidity (P < 0.001), LD (P < 0.001), and mortality (P = 0.021). Importantly, the study was able to confirm the predictive potential of APRI + ALBI for postoperative LD and mortality in a multicenter validation cohort. Conclusion Determination of APRI + ALBI before surgery enables identification of high-risk patients for liver resection. The combined score seems to dynamically reflect CALI. Thus, APRI + ALBI could be a clinically relevant tool for optimizing timing of surgery in CRCLM patients after NeoCTx. Electronic supplementary material The online version of this article (10.1245/s10434-018-07125-6) contains supplementary material, which is available to authorized users.
(LD) (P = 0.009, P = 0.012, P \ 0.001), or mortality (P = 0.037, P = 0.045, P = 0.016), and APRI ? ALBI had the highest predictive potential for LD (area under the curve [AUC], 0.695). An increase in APRI ? ALBI was observed during NeoCTx (P \ 0.001). Patients with longer periods between NeoCTx and surgery showed a greater decrease in APRI ? ALBI (P = 0.006) and a trend for decreased CALI at surgery. A cutoff for APRI ? ALBI at -2.46 before surgery was found to identify patients with CALI (P = 0.002) and patients at risk for a prolonged hospital stay (P = 0.001), intensive care (P \ 0.001), morbidity (P \ 0.001), LD (P \ 0.001), and mortality (P = 0.021). Importantly, the study was able to confirm the predictive potential of APRI ? ALBI for postoperative LD and mortality in a multicenter validation cohort. Conclusion. Determination of APRI ? ALBI before surgery enables identification of high-risk patients for liver resection. The combined score seems to dynamically reflect CALI. Thus, APRI ? ALBI could be a clinically relevant tool for optimizing timing of surgery in CRCLM patients after NeoCTx.
Throughout the past decade, multimodal management of patients with liver metastases of colorectal carcinoma (CRCLM) has improved, leading to a significant advancement in oncologic outcome. 1,2 Liver resection (LR) is considered the only potentially curative treatment of colorectal liver metastases if complete resection can be achieved. In this context, neoadjuvant chemotherapy (NeoCTx) has proved to be efficient as a method to downsize liver metastases, which makes curative LR feasible for a greater number of patients. [3][4][5] Moreover, in addition, findings have shown that patients with primary resectable CRCLM benefit from NeoCTx. [6][7][8] However, preoperative treatment with oxaliplatin or irinotecan, the most commonly used chemotherapeutic agents for patients with CRCLM, is known to cause chemotherapy-associated liver injury (CALI), 9 ranging from steatosis to more severe liver damage such as sinusoidal obstruction syndrome (SOS) and chemotherapy-associated steatohepatitis (CASH). 10 Importantly, the development of postoperative liver dysfunction (LD) has been associated with a higher incidence of morbidity and mortality as well as the presence of CASH and SOS. [11][12][13][14][15] Thus, the amount of NeoCTx has to be optimally balanced between best oncologic efficacy and least induced liver damage. 16,17 Unfortunately, assessment and staging of CALI remains challenging, and a clinically applicable tool for noninvasive evaluation still is missing.
Recently, a number of noninvasive scoring systems for staging chronic liver disease have been established based on routine laboratory parameters. Among these, the aspartate-to-platelet-ratio index (APRI) and the albuminbilirubin grade (ALBI) have been shown to allow estimation of liver function, as reflected by the Child-Pugh score and indocyanine green (ICG) clearance. [18][19][20][21][22][23][24][25] Although APRI was first introduced as a noninvasive marker for hepatitis C-related liver fibrosis, 26,27 it evolved as a general marker for liver function in fibrotic and cirrhotic patients. 28 In addition, findings have shown APRI to be associated with SOS in patients after oxaliplatin-based chemotherapy. 29,30 Similarly, ALBI has been established as a grading system for hepatic function, especially in patients with hepatocellular carcinoma (HCC). 31,32 Interestingly, studies have found both APRI and ALBI to be predictors of postoperative outcome for patients undergoing liver surgery. [33][34][35][36] In particular, studies have shown APRI to be predictive for the development of postoperative LD in patients after major LR, and an association with the presence of SOS has been suggested. 34,35 In parallel, studies have shown ALBI to be a predictive marker for postoperative liver failure in patients undergoing LR for HCC. 37,38 This study aimed to assess the potential of APRI and ALBI to predict a poor postoperative outcome for a homogeneous cohort of CRCLM patients undergoing LR. In addition, the study aimed to assess the potential predictive benefit of APRI and ALBI combined and to elucidate the relation of both markers to NeoCTx.
Patients
The study enrolled patients undergoing LR between 2001 and 2014 at the Medical University of Vienna who had CRCLM. Data including routine blood parameters were prospectively collected. Information on NeoCTx was documented, and if applicable, blood parameters were assessed before chemotherapy (preNeoCTx) and after completion of chemotherapy (postNeoCTx). Blood was taken from all patients before LR (preOP). Both APRI and ALBI were calculated as specified in Fig. S1.
The current study aimed to assess CALI by a rigorous pathologic examination of 170 patients. In addition, the study included a validation cohort recruited at four different institutions (Medical University of Vienna, Rudolfstiftung Hospital Vienna, State Hospital Wiener Neustadt, Inselspital, and University Hospital Bern). Based on data from the exploration cohort, a sample size calculation was performed, and 158 patients were found to be suitable for validation, based on the proportion of patients with LD in the defined risk groups (a = 5%, b = 80%).
All the patients gave written informed consent. The study was conducted in accordance with the Declaration of Helsinki and approved by the institutional ethics committee (#424/2010; #2032/2013).
Statistical Analysis
Statistical analyses, based on nonparametric tests, were performed using SPSS (version 23; IBM Corp, Armonk, NY, USA). A P value lower than 0.05 was considered statistically significant. For more information on statistical methods refer to the supplementary material.
Subsequently, we aimed to compare mathematical combinations of APRI and ALBI and to assess the differences between patients with and without morbidity, LD, or mortality. According to our hypothesis, a summative combination of APRI and ALBI should be the most effective variant because this combination should theoretically increase the discriminatory capacity of each individual marker. Indeed, the patients with postoperative morbidity showed higher values of APRI ? ALBI already before the operation (median no morbidity, -2.48; median morbidity, -2.27; P = 0.001; Fig. 1g). This also was found for patients who would experience LD (median no LD, -2.44; median LD, -2.16; P \ 0.001; Fig. 1h) and for patients who did not survive for 90 postoperative days (median no mortality, -2.41; median mortality, -1.87; P = 0.016; Fig. 1i). Notably, other mathematical combinations were assessed but ultimately found to be of less or no statistical significance, as illustrated in Fig. S2.
APRI and ALBI Combined Improves the Prognostic Potential for Prediction of Postoperative LD
To compare the potential of APRI and ALBI alone with that of both variables combined to discriminate between patients with and without LD, receiver operating characteristic (ROC) curve analysis was performed. Notably, Fig. 2d).
Interestingly, only the patients in the APRI ? ALBI high group experienced clinically relevant postoperative LD graded according to the International Study Group of Liver Surgery (ISGLS) criteria (grades B and C), as visualized in Fig. S3a. Similarly, severe postoperative complications classified higher than Dindo grade 4 were exclusively observed in high-risk patients according to APRI ? ALBI (Fig. S3b). Ultimately, only patients in the high-risk group died within 90 postoperative days (P = 0.021; Fig. 2e). Notably, the study was able to confirm the prediction of a poor outcome for both minor and major LR (Fig. S4).
Prediction of Postoperative LD Using APRI ? ALBI is Independent of Other Variables and Confounders
To test for the independence of the proposed cutoff from other predictors of LD and to identify potential confounding factors, multivariable analysis was performed (Table S2). However, after the proposed cutoff for APRI ? ALBI, the extent of resection, the alkaline phosphatase (AP) level, the gamma-glutamyl transferase (GGT) level, and the retention rate after 15 min for ICG clearance were entered into the multivariable model, only the proposed cutoff (P = 0.002; odds ratio [OR], 5.501; 95% CI, 1.822-16.606) and preoperative gGT (P \ 0.001; OR, 1.006; 95% CI, 1.003-1.010) remained as significant independent variables after stepwise forward selection.
Patients Treated with Neoadjuvant Chemotherapy Display Higher Levels of Preoperative APRI ? ALBI
Because APRI ? ALBI were associated with postoperative outcome, we aimed to investigate the underlying etiology behind elevated levels of APRI ? ALBI. Increased levels of APRI ? ALBI were observed in patients treated with NeoCTx (median no NeoCTx, -2.53; median NeoCTx, -2.39; P = 0.028; Fig. 3a). Intriguingly, patients treated with chemotherapeutic agents with lower hepatotoxicity also showed significantly lower scores than the patients treated with commonly used regimens (i.e., based on oxaliplatin, irinotecan, or a
Levels of APRI ? ALBI Dynamically Change During Neoadjuvant Chemotherapy and Allow Quantification of Liver Function Recovery
To investigate a potential dynamic change of APRI ? ALBI during NeoCTx, the score was assessed before NeoCTx and directly after completion of the last cycle. Indeed, levels of APRI ? ALBI were found to increase significantly after NeoCTx (median preNeoCTx, -2.60; median postNeoCTx, -2.27; P \ 0.001; Fig. 4a). This increase persisted until the time of surgery (median pre-NeoCTx, -2.60; median preOP, -2.40; P \ 0.001; Fig. 4a). Importantly, a continuous decrease in APRI ?
DISCUSSION
For a large fraction of patients with CRCLM, NeoCTx is routinely used. 3,6,7 Indeed, its beneficial effects have been shown for both resectable and primarily unresectable CRCLM. 3,7,41 However, the development of CALI and a concomitantly increased risk for postoperative complications and LD remain drawbacks for NeoCTx. 16 More importantly, reliable diagnosis and staging of CALI remain a challenging task for clinicians. Indeed, radiologic studies do not provide satisfactory information. 42 Furthermore, proposed methods for identifying patients with CALI, such as ICG clearance, 43,44 are expensive and have not found their way into clinical routine. Thus, liver biopsy often is the examination of choice, but due to the high degree of invasiveness, it is reserved for high-risk patients with suspicion of severe liver damage. This leaves the majority of patients undergoing LR after NeoCTx without adequate preoperative assessment for CALI.
Intriguingly, liver biopsy and histopathologic examination have been shown to have low sensitivity for most types of CALI. 45 In a prospective study, Viganò et al. 45 aimed to assess the sensitivity and accuracy of preoperative liver biopsies versus examination of resected liver parenchyma for CALI detection. Notably, only steatosis could be reliably detected, with a sensitivity of 88.9% and an accuracy of 93%. Importantly, diagnosis of SOS had an accuracy of only 63% and a sensitivity of only 21.1%. Similarly, steatohepatitis was diagnosed correctly in only 78% of cases and with a sensitivity of only 21.1%. This suggests that percutaneous liver biopsy does not suitably reflect the actual pathophysiologic degree of liver damage.
Strikingly, using the combination of APRI ? ALBI, we were able to identify CALI (as evaluated in the surgical specimen) with a much higher sensitivity. Whereas severe steatosis was detected with a sensitivity of 90%, the cutoff was able to identify SOS with a sensitivity of 77.8%, and detection of CASH showed sensitivities of 86.7% (NAS C 4) and 92.3% (Brunt = 3). This favors the use of APRI ? ALBI as a noninvasive tool for the sensitive identification of CALI before surgery.
Interestingly, the patients in the high-risk group had a significantly higher prevalence of severe steatosis and CASH and a tendency toward an increased prevalence of SOS. The was paralleled by a substantially higher incidence of adverse clinical outcomes. Importantly, the gathered data could be confirmed in an independent multi- Most importantly, the current study introduced APRI ? ALBI as a useful tool for timing LR after NeoCTx. We present solid data on the increase in APRI ? ALBI during NeoCTx, suggesting a direct correlation between the marker and induced liver damage. Interestingly, this increase was not affected by the number of NeoCTx cycles administered (R = -0.115; P = 0.474), suggesting that the susceptibility of patients to the development of CALI might be more relevant than the duration of NeoCTx itself.
Furthermore, a decrease in APRI ? ALBI during the time of chemotherapy cessation before surgery was observed. Indeed, this finding is consistent with recent reports on the amelioration of CALI after chemotherapy cessation. 46 Correspondingly, APRI ? ALBI might be a relevant tool for timing and postponement of LR. Indeed, sequential assessment of APRI ? ALBI might allow dynamic monitoring of CALI and hence potentially delay surgery until the liver has adequately recovered. Although high-risk patients according to APRI ? ALBI were shown to have severely reduced postoperative outcome, a transition to the low-risk group after a certain period might prevent the patient from these complications. Hence, evaluation of APRI ? ALBI could be used to guide the physician's decision concerning when to operate on a patient. However, the exact time frame might differ between patients, which makes fine-meshed monitoring of this marker essential for high-risk patients.
In conclusion, evaluation of APRI ? ALBI represents an easy instrument for preoperative risk stratification of patients undergoing LR after NeoCTx. Because this marker is based on routine laboratory parameters, it is easily assessable and can readily be implicated in clinical routine. Notably, the summative combination of APRI and ALBI was found to improve the predictive potential compared with both scores assessed individually. Furthermore, evaluation of APRI ? ALBI seems to allow personalized scheduling of LR after completion of NeoCTx. Ultimately, this might help to reduce the incidence of chemotherapyassociated complications after LR.
ACKNOWLEDGEMENTS Open access funding provided by Medical University of Vienna.
DISCLOSURES The authors declare that they do not have anything to disclose regarding conflict of interest with respect to this manuscript. No funding or financial support was received for this work.
OPEN ACCESS This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.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. | 2019-01-22T22:28:38.360Z | 2019-01-07T00:00:00.000 | {
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269747149 | pes2o/s2orc | v3-fos-license | A solution for fillet quality: Slaughter age's effect on protein mechanism and oxidation
Physico-chemical properties of fish flesh are reliable predictors of fillet quality and nutritional value. In our study, the age-related variations of the chemical composition, pH, water activity (aw), water holding capacity (WHC), color and texture analysis, protein thermal stability, myofibrillar fragmentation index (MFI), glycogen content, protein oxidation and protein profiles were investigated in Oncorhynchus mykiss (rainbow trout) fillet. The results revealed that protein denaturation temperatures (Tmax1 and Tmax2) decreased by 2 % and 11.6 % depending on fish age. Tmax1 and Tmax2 values in the same groups were raised 71 % at 11 months' fish and this increase was 58 % at 23 months’ fish. An age-related reduction by 66.6 % and 31.25 % was noticed for protein oxidation markers sulfhydryl groups and disulfide bonds. MFI value increased by 86.6 % connected with age. The characteristics of fish meat quality are complex and are influenced by various factors that affect the degree of freshness of the product and its acceptance in the market. Taking into account the different demands of the consumer, this study has shown that age at slaughter has an impact on final product quality and that the recommended age at slaughter, taking into account market weight, positively affects meat quality.
Introduction
Micronutrient composition as well as protein quality and polyunsaturated fatty acids (PUFA) are important in consumer health and * Corresponding author.
Contents lists available at ScienceDirect preference.Chemical and nutritional properties of seafood provide high reliable way to assess fillet quality.In addition to its high protein and low carbohydrate content, seafood products contain essential amino acids, omega-3 fatty acids, vitamins, mineral substances and have a low cholesterol/calorie value, which increases their importance in a balanced diet day by day.The chemical composition of fish is very close to that of other terrestrial animals.The main components of fish are moisture (66-88 %), protein (15-24 %), fat (0.1-24 %) and ash (0.8-2%).In terms of carbohydrates, fish contains no more than 0.3 % glycogen and 1-2% minerals, 0.5 % calcium, 0.25 % phosphorus and 0.1 % vitamins A, B, C and D in addition to their vitamins in fat [1].Fish protein has a stable composition of essential amino acids with higher lysine and lower methionine and threonine content [2].However, the meat yield and chemical composition of various fish vary.Knowledge of these differences plays an important role in the nutritional and economic preference of these species.In addition to flavor, odor, color and texture as essential attributes of fish quality, other factors including age, size, growth rate, species, seasonal changes, feeding and killing techniques are equally involved in achieving the quality requirements [3][4][5][6][7][8].Aquaculture products, which have an important share among foods with the code "blue food", are the focus of many studies/researchers.These blue foods, which are among the most traded products in the world, are protected by measures taken to ensure the safety and sustainability of the global food system [9][10][11][12].In the ever-expanding seafood-food market, detailed studies have focused on production efficiency and fillet quality, which are important.Physical, chemical and microbiological analyses to determine the quality of processed products are developing in this parallel.The expectation from these analyzes is to provide fast and accurate results in the relevant product in a short time.They are important and pioneering analyses that usually include microbial load, aroma, some chemical (pH, TVB-N and TBARS) and physical (aw, WHC, color and texture) analyses [13].High water activity (aw), high pH and the presence of autolytic enzymes are important factors in the high sensitivity of seafood products.Total volatile basic nitrogen (TVB-N) is one of the most preferred chemical variables for determining the freshness of seafood products.SDS-PAGE method is the most widely applied protein electrophoresis method used in the separation of meat proteins and in some studies to determine the meat species in meat products and mixtures.This method is mostly used to determine the molecular weight of proteins, to control protein purity, to fractionate proteins, to examine the substructure of pure protein [14].
One of the major causes that diminishes fillet quality and shortens shelf life is oxidation.Lipid oxidation in meat and meat products is most commonly measured by Thiobarbituric acid reactive substances (TBARS) analysis.Although lipid peroxidation is the inspiration for a multitude of studies, latest findings claimed that protein oxidation is of equal importance, and is even triggered by the reactive lipid oxidation products [13,[15][16][17][18].
Although the importance of diet, species and season on fillet quality is known, the lack of studies to monitor the effect of age at slaughter on quality constitutes a deficiency in fish quality.Based on the above background explanation, in the present work we addressed for the first time the effect of slaughter age on fillet quality and mechanism of protein oxidation, as well as physico-chemical analysis as well as multiple assessment applications in terms of thermal stability, oxidation and profile of myofibrillar proteins (MPs).
Material and methods
Oncorhynchus mykiss obtained from Atatürk University Fisheries Faculty Inland Fish Research and Application Unit (toxic and disease certified manufacturer), were used as fish material.
A total of 40 male Oncorhynchus mykiss (rainbow trout) with an average age of 11-23 month were used in this study.These ages were preferred because they are the market size (11 months) and breeding stock candidate (23 months) time intervals for trout.At the determined temperatures: 12 ± 0.3 • C, O 2 level was adjusted by increasing the amount of water for survival comfort.Thanks to this setup, O 2 level was kept above 7.5-8 mg L − 1 in all groups.At the end of the time when reached to the determined treatment age under controlled conditions, random samplings were made from the fish belonging to each age group [Group A: Average age of 11 month (A) and, Group B: Average age of 23 month (B)].In order to determine the fillet quality fish were stunned via the fast neck-breaking technique was applied for the safety of the analyses and then decapitated and filleted without skin by hand under sterile conditions.For each age group, 40 fillets from 20 fish were prepared in 2 replicates, and the physico-chemical analysis and electrophoretic techniques were performed on randomly sampled fillets.
Physico-chemical analysis
In fillets divided into age groups, pH and chemical composition, the amount of crude ash, crude oil, crude protein, and dry matter were assessed according to Atamanalp [19].In order to assess the water holding capacity (WHC), 5 g of each sample were centrifuged for 30 min at 4500 g/min and 10 • C. The supernatant was removed and after the pellet part was weighed, the WHC calculations were performed using the following formula [20].
Water Holding Capacity (%) = [1 − (pellet weight / first weight of the sample)] × 100 Water activity (aw) measurements were made with an Aqualab water activity meter (Decagon Devices, model series 3) at 25 • C (±0.2 • C) [21].Before the measurement, the device was calibrated using ready-made package (Decagon Devices, Inc. A. Kara et al.Texture profile analysis (TPA) of the fillet was practiced using texture analyzer (CT3, Brookfield Engineering Laboratories, USA).Cylindrical samples of 20 mm diameter and 20 mm height were extracted from fillets with two press cylinders using a 50.8 mm probe (TA 25/1000, Brookfield Engineering Laboratories, USA) and were analyzed at room temperature.Transaction terms: the pre-test speed was set to 2 mm/s, the test speed and post-test speed were set to 1 mm/s, the time between the first and the second compression was 3 s, and the compression ratio was 50 %.The textural parameters (hardness, adhesiveness, resilience, cohesiveness, springiness, chewiness and gumminess) were assessed [23].
Determination of thermal stability, oxidation and profile of myofibrillar proteins 2.2.1. Thermal stability determination of miyofibrillar protein
Thermal changes of miyofibrillar protein were determined using Differential Scanning Calorimeter (DSC-60, Shimadzu Corp., Japan)].Indium was used to calibrate the device for heat flux and temperature (Tm = 156.6 • C; ΔHm = 28.5 J/g).Approximately 10 mg of the sample was weighed into the aluminum sample cup and hermetically sealed.Heating was applied from 20 • C to 90 • C with a heating rate of 5 • C/min using an empty container placed in the sample device and sealed in the same way as a reference.The thermal changes of the analyzed samples were acquired on thermogram [13].
Protein oxidation a. Muscle glycogen levels and lactic acid concentration
The glycogen level was estimated via the potassium hydroxide (KOH)/anthrone method, using a commercial kit (Abcam, Glycogen Assay Kit), within the first 6 h after following sampling, from the moment the rigor was formed, and was expressed as mg glycogen/g muscle [16].The absorbance was read with UV-Vis spectrophotometer (Shımadzu) at 620 nm.Lactic acid concentration was assigned following the muscle stiffness began to develop (within the first 6 h after sampling) using the commercial kit (ChemBio, CB55560).
b. Myofibrillar protein (MPs) extraction and assessment of myofibrillar fragmentation index (MFI)
Samples homogenized with cold MFI buffer [40 mL of 0.02 M potassium phosphate buffer (pH 7.0)] containing 100 mM KCl, 1 mM EGTA, 1 mM MgCl 2 and 1 mM NaN 3 , were centrifuged for 15 min at 1000×g and 4 • C.After the supernatant was removed and the pellet was resuspended in the same cold MFI buffer, the absorbance was measured using an UV-Vis spectrophotometer (Shımadzu) at 540 nm.The MFI was expressed by multiplying the absorbance by the dilution factor (200) [24,25].
c. Determination of sulfhydryl groups and disulfide bonds in myofibrillar protein
In order to assess the sulfhydryl groups and disulfide bonds content of MPs, the 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) reaction was considered [26].The MPs solution was mixed with 4.5 ml of 0.2 M Tris-HCl, 3 mM, ethylenediamine tetra acetic acid (EDTA), 1 % sodium dodecyl sulfate (SDS) buffer containing 8 ml of urea.Subsequently, 0.5 ml of buffer B (10 mM Tris-HCl, 10 mM DTNB, pH 8.0) was added over 4 ml of this mixture and incubated at 40 • C for 25 min.At the end of the incubation, the absorbance of the supernatant was measured at 412 nm, and the molecular absorbance coefficient of 13600 M − 1 cm − 1 was used to calculate the sulfhydryl content and the disulfide bonds content [16].
d. Carbonyl concentration
In the MPs samples the carbonyl content was determined by considering the 2,4-dithiophenylhydrazine (DNPH) method.For this purpose, MPs suspension was added to 10 mM DNPH solution and incubated for 1 h at room temperature.The obtained mixture was washed with 20 % trichloroacetic acid (TCA) and centrifuged at 10,000 rpm for 5 min.After the supernatant was discarded, the remaining pellet was resuspended in 3 ml of 6 M guanidine and incubated at 37 • C.After cooling, the absorbance was measured at 370 nm via spectrophotometer and the results were expressed as nM carbonyl/mg protein [27].
e. Determination of protein concentration
The amount of MPs was determined by the Biuret method [28].For this purpose, 2 ml MP and oxide were taken as a sample, mixed with 3 ml of Biuret reagent and incubated at 37 • C for 15 min.Following the incubation, the mixture was centrifuged at 3500 rpm for 5 min and the absorbance of supernatant was read at 540 nm via UV-Vis spectrophotometer.
Determination of protein profile
Protein profile analysis (PPA) of the obtained homogenates was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method [29].500 μl of commercial ready mix marker (Precision Plus Protein All Blue Prestained Protein Standard, Cat.No: 1610373) was used.Electrophoresis was performed in constant current mode (20 mA/gel) for approximately 90 min, gels were transferred in Oriole fluorescent gel stain for 2 h and gel imaging software system, Bio Rad Gel Doc XR was used to A. Kara et al. visualize the gel.
Data analysis
In all trials, measurements were repeated three times and values were expressed as mean ± standard deviation (SD).Homogeneity tests were applied for all output using generalized linear models, differences were subjected to Student's t-test comparison tests (SPSS Ver.22.0) and interpreted at p < 0.05.
Results and discussion
In seafood sector, although there are studies on fillet quality and shelf life, the effect of slaughter age on fillet quality and protein oxidation is still unclear.In this sense, our current research, in which detailed data is presented, provides important data for this gap as well as being a modeling study.However, there are no similar studies that can be compared with the research findings.
Interpretation of physico-chemical analysis
For fish products, pH serves as spoilage indicator.In our study, the pH value between the groups differentiated by age was found to be statistically important (p < 0.05).The pH value was determined as 6.79 ± 0.16 in the fillets belonging to group A and 6.46 ± 0.01 in group B (Table 1).The final pH value is a major marker for deciding about fillet quality.The higher pH in group A fillets could be due to distinctions in muscle fiber kinds or low muscle glycogen content.Similar results were previously reported by Si [30], which found that young animals may have higher final pH values than older animals because of a lack of glycogen.
The chemical ingredients of fillet are considered crucial reference marks for the evaluation of meat functionality.Fat, minerals and proteins are key components that show fillet quality, and moisture plays an essential role in maintaining the processing potential, quality and shelf life of seafood products.Age has a distinct effect on the fat and protein content of meat.In our study, When the effect of the slaughter age on the chemical composition of the fillets was examined, the ash, fat and moisture values were found to be statistically significant, but the changes in the protein content were not significant at the p < 0.05 level (Fig. 1).Chemical composition was significantly influenced by the age difference of the fish from the two experimental groups.Meats with high water-holding capacity (WHC) are preferred in the meat industry for economic reasons and because of the technology to be applied.The pH value has a significant effect on WHC, especially.Meats with low WHC and water-binding capacities can lead to undesirable changes such as high leakage water and cooking loss, as well as a decrease in cooking yield during processing, cooking, and storage [31,32].
It is thought that physical characteristics of fillets, including tenderness, juiciness and processing quality, may be sensitive to age, especially in the way of moisture conten [33].One of the most important parameters for fish industry is WHC.An increased water content in the muscle has a negative impact on consumer demand by reducing mechanical strength and formation of extremely soft or tender fillets [34].It is known that the net load of MPs, the membrane permeability of the muscle cell and its components, namely myofibrils, cytoskeletal connections, and the amount of extracellular space in the muscle are effective in fillet WHC values [35].In our study, WHC values determined in rainbow trout fillets as 13.2 ± 0.89 and 14.8 ± 0.69 for group A and group B, respectively, were not found statistically significant at the p < 0.05 level (Table 1).
Aw is one of the main factors responsible for food stability and modulation of microbial response.Regarding the shelf life of fish Aw is a dominant parameter and has an important role on the growth of various microorganisms and spores [36].In the present study, although relative changes in Aw activity were observed in fillets depending on slaughter age (group A = 0.9968 ± 0.003 and group B = 0.9965 ± 0.002), these differences were found to be statistically insignificant (Table 1).
Color is an essential tool for freshness and meat quality evaluation.This parameter is assigned by assessing redness (a*), yellowness (b*), lightness (L*) and is dependent on oxidation state -myoglobin concentration, which is affected by diet, breed, storage conditions and age [33].When the effect of the slaughter age on the color quality of the fillets was examined, it was determined that it had an Lowercase letters (a,b,c) show differences between groups determined in each parameters.a p < 0.05, NS: not significant (p > 0.05) A: 11-month and B: 23-month.
A. Kara et al. important effect on the L* and a* values (p < 0.05) (Table 1).
As seen in Table 1, group B fillets were defined by darker (lower L*) and redder (higher a*) scores than group A. A darker color is likely due to the effect of muscle fiber type with age and increasing myoglobin content.Regarding muscle type, there are reports that the light scattering properties of some muscles are increased by postmortem protein degradation, which promotes an increase in the L* value [30].Colored components are often formed by protein oxidation and degradation, and a high degree of protein oxidation and degradation causes meat discoloration during aging [37,38].In our study, a* value was found to be higher in group B. This is mainly associated with the presence of a natural reduction system in meat after slaughter and the presence of metmyoglobin.During aging, it is reduced to myoglobin by metmyoglobin reductase.However, metmyoglobin reductase activity gradually decreases with aging and the flesh color can change from red to brown [37,38].
Tissue parameters such as springiness, hardness, stickiness, chewiness and flexibility are generally used indicators for assessing the quality of fish products [39].When the effect of slaughter age on texture of fillets was examined, it was seen that age had an important effect on all parameters (p < 0.05) (Table 2).An weaker connective tissue strength may be due to lower pH of tender fillets.It has been reported that higher pH of muscle has favorable effects for fillet tissue index, including firmness, chewiness, and springiness [40].Other studies revealed that muscle fiber condition can have a significant positive correlation with fish stiffness [37,38].Due to textural and structural changes in the postmortem period, the distribution and mobility of water in myofibrils may undergo changes.These affects the WHC and sensory properties of fillets [41].In this process, changes in texture, flavor and color parameters are observed in fillets as a result of the effect of endogenous enzymes [42].Our research findings support this situation in terms of pH and hardness.In our study, most relevant ascertainment is that the high hardness, chewing and stickiness values observed may be due to the higher WHC.Similar results were reported in carp fillets by Liu [43].
Thermal stability, oxidation and profile interpretation of myofibrillar proteins
The endothermic transition peaks (Tmax) and denatured enthalpy (▵H) of the protein during heating are often used as reference marks of thermal stability.The two distinct endothermic peaks (Tmax1, Tmax2) assigned to the denatured temperature of MPs and the denatured enthalpy of myosin head for fillets of each age group and their corresponding enthalpies (▵H1, ▵H2) are given in Table 3.The presented results show that Tmax2 for group A and B fillets increased by 72 % and 58.5 %, respectively, compared with MPs by slaughter age.This increase may be due to irreversible cross-bridge formation between myosin and actin [37,38].In accordance with the previously presented results regarding the thermal stability of miyofibrillar protein, the energy required for the proteins Lowercase letters (a,b,c) show differences between groups determined temporally.a p < 0.05, NS: not significant (p > 0.05), A: 11-month and B: 23-month.
A. Kara et al. denaturation gradually increased in an age-related manner, and then decreased at a similar rate.These increases may indicate inhibition of proteolytic enzymes in denaturation, reduction in enthalpy and lower peaks at higher temperatures, denaturation of fillet proteins [13].
Heat-induced raised muscle protein-protein interactions and decreased water-holding capacity take place in 2 phases [44].Among 30 and 50 • C coagulation of MPs occurs and the greatest reduce in WHC is obvious.From 55 to 90 • C, contraction of muscle fibers in the connective tissue network and increased interaction of the coagulated actomyosin system cause less water escape [45].In our study, a significant and age-dependent decrease in Tmax2 values (p < 0.05), indicates a greater sensitivity of MPs in an age-related manner.Our findings are similar with those reported by Li [46] and Wang [37,38].
After the death of fish, the absence of oxygen transfer to the cells results in the body's reactions occurring under anaerobic conditions; at this stage, glycogen breaks down into lactic acid [16,47].The degree of glycogen in muscle is the main marker of postmortem pH due to lactic acid from anaerobic glycolysis [48].In our study, glycogen levels were determined as 0.32 ± 0.04 and 0.37 ± 0.00 in A and B group fillets, respectively, and this difference was found to be insignificant at the p < 0.05 level (Table 4).An overall statistical analysis of the obtained results revealed that the glycolytic substrate increased and, as a result, the muscle pH decreased in group B fillets, which were defined by higher muscle glycogen stores.In addition, the lower glycogen level might be explained by a lower protein oxidation rate [49].Following capture, even though fish have lost their vital functions post-capture, they are still exposed to post-mortem biochemical reactions with existing energy sources in the muscle tissue, such as ATP, glycogen, and other chemical compounds.Post-mortem changes in fish muscle can develop quickly or slowly depending on the fish species, size, and ambient temperature.In a living fish, the energy required for muscle contraction is provided by ATP produced during glycolysis.In this case, tissue ATP consumption and regeneration, contraction, and relaxation events continue constantly, whereas after post-mortem, with the cessation of blood circulation and oxygen supply in the tissue, the amount of ATP decreases rapidly, and contraction and relaxation events also continue in a limited manner during this decrease [16].Our research has determined that the same killing technique used for each age group of the same fish species did not significantly affect the glycogen level and lactic acid amount, causing relative changes that the killing techniques applied did not have a significant impact.It is known that the killing technique has a significant effect on fish in endocrine responses and post-mortem biochemical processes (ATP breakdown and anaerobic glycolysis).It is thought that thrashing and struggling behaviors are effective in faster glycogen consumption.Decreases in muscle glycogen concentrations among age groups are due to the accumulation of lactic acid [47].Increases in glycogen levels are thought to be caused by a low metabolic rate [50].MFI is an index of protein degradation and meat tenderness [51].It has been reported that the higher the MFI value, the worse the integrity of the internal structure of myofibrils [52].In our study, the MFI showed that the totality of myofibrils and proteins clearly deteriorated with age, the spaces between myofibrils getting larger, and thus leading to severe fragmentation.The increment of MFI value may be due to the degradation of myonectin and accompanying actin in the I-band of the MPs sarcomere.It has also been shown that fragmentation of myofibrils may be as a result of integrity disruption near the Z line [53].
Myosin and actin are affluent in thiol groups, which will be turned into disulfide bonds after being attacked by intermolecular and intramolecular oxidation.A great number of sulfhydryl groups in the protein are located in the inner region of the molecule.Nevertheless, under the MDA oxidation system, the spatial structure of the protein changes, the structure expands and a large number of sulfhydryl groups are exposed, which can be lost following oxidation [37,38].In our study, the age-related decrease of sulfhydryl content in MPs may be caused by the degradation of myosin, which leads to alterations in the spatial structure of the protein and causes the exposure and oxidation of embedded sulfhydryl groups [53].
Mainly, the sulfur atoms' high reactivity to oxidation, the oxidative conversion of cysteine to disulfide bonds and cysteine oxyaic are effective in this reduction [54,55].Carbonyl compounds are the main products of protein oxidation during meat processing [56].
As shown in Table 4, the carbonyl content of MPs increased remarkably in an age related-manner, but statistically insignificant (p < 0.05).The augmentation in carbonyl content is probably caused by carboxylation of the side chains of certain amino acids in myofibrils [55].Increasing the carbon content and decreasing the sulfhydryl content can cause protein crosslinks that affect the constitution and spatial adjustment of MPs and reduce WHC [54].The WHC results reported in our study are compatible with the previous reports.Thus, as a result of the MPs oxidation, validated by the increase of carbonyl content and the decrease of sulfhydryl content, the protein configuration and polarity change, affecting the water recombination of MPs.
Determination of protein profile
Oxidation can cause structural changes, including the structure of covalent bonds and the degradation of high molecular weight proteins to light molecular weight proteins.For this reason, SDS-PAGE is appreciated as a powerful technique for the assessment of the proteins oxidation extent [57].β-mercaptoethanol, one of the components of this electrophoretic method, is a reducing agent that can break the disulfide bonds in SDS-PAGE [37,38].
In our study, examining the influence of slaughter age on rainbow trout quality fillets, the conventional model for MPs consisting of two main bands is clearly seen (Fig. 2).One of these bands is thought to be actin with a weight of about 42 kDa.Actin bands appear to be more intense than other bands, which is highly correlated with protein oxidation.However, the low-intensity bands in the obtained SDS-PAGE image are estimated to be protein chains produced by degradation of major histocompatibility complex (MHC).While dense band images indicate heavy molecular chain, protein cross-linking and protein accumulation, the appearance of light molecule chain can be interpreted as oxidation of the protein, which leads to degradation of the heavy molecular weight proteins [58].
Considering the research findings and literature to date, it has been noticed that the effect of harvest age on the quality of aquatic products has not been fully evaluated with a holistic approach, nor has any strategy been developed regarding this situation.Therefore, it is thought that the current study data will consider this parameter an important quality element in the processing of aquatic products and the food industry in the production of unprocessed, processed, semi-processed, and advanced processed products.
Conclusion
The study the influence of the slaughter age on the quality of the fillet.The authors identified undesirable changes in fillet properties such as chemical composition, color, and texture.Authors also reported alterations of pH, water holding capacity, oxidation, and thermal stabilization were related to slaughter age.Our results show that age at slaughter can affect important physical and sensory aspects of rainbow trout fillet quality.Overall, the findings indicated that increasing age has an impact on fillet quality serving the consumer market.In the collected findings, it was found that the myofibrils and proteins clearly deteriorated with age and slaughter age caused texture changes.In addition, the brightness of the fillets negatively affected with slaughter age.
Physical, chemical and microbiological changes in seafood after death such as rigor mortis, and enzymatic changes have a very important effect on fillet quality.In the postmortem period, the distribution and mobility of water in myofibrils may change due to textural and structural changes.This affects the water holding capacity and sensory attributes of fillets.Changes in texture, flavor and color parameters are observed in fillets as a result of the effect of endogenous enzymes.Nevertheless, the effect of slaughter age on protein oxidation and thermal stability, especially in fish, and their influence on fillet quality, should be further clarified.
Our study covers important gaps regarding the influence of the slaughter age on the quality of the fillet.Undesirable changes of fillet properties such as chemical composition, color and texture were noticed, which can lead to a diminution in consumer demands.In addition, other important alterations of pH, water holding capacity, oxidation and thermal stabilization were related to slaughter age.The findings obtained with the present research constitute a modeling study and created a useful data library for the elucidation of the mechanism by which the slaughter age can influence the quality of the fillet.Besides, these results offer a strong foundation for further advanced research of slaughter age in terms of lipid oxidation and microbial spoilage in the food industries.influence the work reported in this paper.A. Kara et al.
Fig. 2 .
Fig. 2. SDS-PAGE image of miyofibrillar protein of slaughter age on rainbow trout fillets A: 11-month B: 23-month and Marker.
Table 1
pH and color values of slaughter of different age on rainbow trout fillet.
Table 2
Texture profile analysis of slaughter of different age on rainbow trout fillet.
Table 3
Change in the thermal stability of MPs of slaughter of different age on rainbow trout fillet.Different lowercase letters indicate significant difference in each column for each parameters (P < 0.05).Abbreviation: Δ H, denaturation enthalpy; SD: standard devition of the means (n = 12); T max , peak temperature, A: 11-month and B: 23-month.
Table 4
Biochemical values of slaughter of different age on rainbow trout fillet. | 2024-05-13T15:02:59.253Z | 2024-05-01T00:00:00.000 | {
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216266553 | pes2o/s2orc | v3-fos-license | DETERMINATION OF DAILY WATER CONSUMPTION PATTERN (A CASE STUDY OF KHYBER PUKHTOONKHWA, PAKISTAN)
Water is not only necessary for life but it plays vital role in the social accompanied with economic growth of that specific country especially developing countries. Increasing population and rapid urbanization accompanied with climate change may reduce the supply of fresh water globally in twenty-first century. This study aims to understand current household water use and water use pattern in different five houses of different five places of KPK Pakistan for five months, to improve the efficiency of house hold water use, to encourage sustainable use and conservation of water resources. For the provision of new fresh water facilities. It’s necessary for water supply system planners to comprehend current water consumption behaviors of inhabitant, and how they use water of the new facility in future. The water consumption pattern is differs for the nations and societies and dependent on factors which may vary consumption on daily, weekly, monthly and yearly basis. These factors are availability of water source, economic, cultural, seasonal, climatic, and approachability to these water sources. Daily water consumption pattern for five different houses in different areas of KPK for five months were found by carefully examining the time taken by pump to fill the Household overhead tanks. But in order to increase reliability of the acquired data the pumps were allowed to fill the tank till water flow for one minute at overflow pipe of the water tank was not recorded. During the period of research (March 2018 to July 2018) it was concluded that the average consumption in Charsadda (urban), Charsadda (rural), Mardan (urban), Mardan (rural) and Kohat (urban) was 102.84, 61.81, 105.99, 66.44 and 100 litres per captia per day (LPCD) respectively. JOURNAL OF MECHANICS OF CONTINUA AND MATHEMATICAL SCIENCES www.journalimcms.org ISSN (Online) : 2454 -7190 Vol.-15, No.-2, February (2020) pp 48-56 ISSN (Print) 0973-8975 J. Mech. Cont.& Math. Sci., Vol.-15, No.-2, February (2020) pp 48-56 Copyright reserved © J. Mech. Cont.& Math. Sci. Zohaib Hassanz et al 49 Interestingly it was also observed that the trends of water consumption were almost the same in urban and rural areas of different districts of KPK.
I. Introduction
Fresh water sources are not abundant in nature. Although over 2/3 part of our planet consists of water but the quantity of fresh water sources availability on the earth is not in abundance for use. Out of which only three percent is potable, two percent is frozen in glaciers and polar ice caps, which leaves only 1 percent as useable water and the rest 97 percent is saltish water (sea water) [I]. Now a day freshwater shortage is not only a global challenge for sustainable development but it also affects human intervention, inadequate freshwater supply, and inappropriate management which causes a rapid deterioration of existing fresh water sources [IX]. There is a vastcompetition for the use of fresh water consumption due to agricultural crops, industries and urbanization. By the end of year 2050,more water in future required to produce foodin demand of the Earth's population as demand increased to 9 billion [X]. Thus, reuse of municipality wastewater is now becoming a critical issue because of increasing water demand for human and agricultural sector consumption [IX]. Pakistan, which is previously a country of water-surplus quantity, currently considered as a country approaching towards water deficiency [VII]. The gross demand of water in Pakistan for the purpose of nonirrigation estimated in 2007approximately 8.5 billion m 3 , expected to increase about 11.2 billion cubic meter by the year of 2025, fallowing an increasing rate 1.5 percent due to rapid urbanization and industrialization [XI]. The intensity of rainfall is currently in-adequate to encounter the rapid increasing requirement of water consumption. In addition, it is currently expected that these constant incremental increase in the consumption of water will reaches to severe condition promoting scarcity in the quantity of fresh water sources in future [II].
Per person water availability in Pakistan per year reduced from 5,000 cubic meters in 1951 to 1038 cubic meter per year by 2010 which was somewhat above is the internationally recognized water scarcity level of 1000 cubic meter [IV].
Moreover, as per the water stress index, if the fresh water availability in a country drops below 1000 m 3 per person per year, it is considered as a water-scarce country and if it decreases from 500 m 3 then country is in absolute water scarcity [III].
It is estimated that 67% of the global population will face moderate to high water stress and half of the population will suffers limitations in t supply of fresh water by 2025 [VII].
Supply of fresh water sources to the entire population of world from now to 2030 as absolutely the finite supply is recognized as a clearly mentioned and significant challenge due to the fact that the quantity of fresh and drinking water sources are only one percent of the world's total water which is managed in a poorand haphazard manner. Population growth is one of the key factor affecting water scarcity, besides this improving the level of living standards, urbanization and variability in supplies at consumer end due to climate change will also add unbearable pressure on fresh water sources which leads insufficiency in numerous parts of the world [VI].
Everyone in the world, whatever their approach of development, social and economic situations, have the right to have access to potable water in adequate quality and quantity as per their basic needs. Standards for domestic water use differ with climatic conditions, life style, culture, technology and economy and people's behaviour to treats fresh water during their diurnal activities [V].
In households' water is used for drinking, preparation of food, bathing, flushing toilets, washing of clothes, and watering of gardens and lawns. As per the guidelines of WHO; Drinking Water Quality, Defines use of water for domestic purpose as the "the usage of water in usual for all the domestic purposes counting the drinking water consumption, for bathing and the preparation of food" [III].
The three important variables which greatly affect the domestic consumption of water rate which is affected bythe size of Household, Social and economic status accompanied with Seasonal and climatic variations [IX].
The consumption of daily water consumption in Dhaka city was determined via a questionnaire and a for domestic water a unique typebucket was used for households having different activities this research study presents 200 to 300 litres per person per day consumption of potable water in the middle class families [I]. As per the recommendation of WHO50 to 100 litres of water (LCPD) is required meeting the needs of daily domestic activities i-e for personal hygiene, cleaning of floors along with washing of cloths etc. [IX].
Besides the size of household, social and economic status, seasonal variationalong with climatic changes there are some other issues associated with consumption rate such as shared overhead tanks, shared taps education and water faucets. And through a cluster of data acquired from metered data and storage container inventories based on the above-mentioned factors it was concluded that most of the houses used water below 135 LPCD except a very few houses [VI].
Household's daily and activity wise water use is greatly affected by the availability of water source, its quality, duration of availability and frequency of water supply. A research was conducted where the size of the vessels used for the measurement were used for domestic used where stored water in vessels were measured along with observing water from running tap, the time in a specific tap was used and consumed quantity of water measured unit of flow per minute flowing out from the specific tap was measured. These results revealed the average daily water consumption was 117.0 LPCD. While in low-income family the observed consumption of water was 97 LPCD [X].
Brief description of study areas
This research study was conducted out at the same time for five different households in three different districts as shown in Table 1 having different particulars i-e rural, urban, source of fresh water, economic status, education level of family members and water source accessibility for a period of five month starting from March and ending at July.
Estimation of daily water consumption
As all the households involved in the research had their own water sources along with their own overhead storage tank. The volume of their storage tank was found along with the determination of discharge of their pumps using container of known volume and stopwatch. The household water tank was filled and after that the time for which the supply pump delivers water to the storage tank was noted precisely till next 24 hours in order to find out the volume of consumed water within 24 hrs. The same at which it was previously filled. Furthermore, to acquire greater accuracy the pump was allowed to fill the tank till flowing water was not observed at overflow pipe of the storage tank for at least one minute.
Further analysis of the acquire data was carried out using Microsoft excel. Where daily per captia consumption, their patterns along with monthly peak factors were determined for various days of a week. In order study the variation of daily water consumption at different days of a week in a month.
III. Results and Discussion
Daily water consumption was obtained by the multiplication of water supplying pump discharge rate with the time taken by storage tank to fill till flowing water at the overflow pipe was not noticed for at least one minute.
Estimation of daily water consumption
Daily water consumption for the representative households were observed and determined for the study period without any discontinuity. Water consumption within the month of march shown in Figure 1 representing a water consumption in rural area's ranges between 46.16 to 62.65 LPCD while water consumption in Urban area's ranges between 59.72 to 97.72 LPCD.
Fig. 1: Daily Water Consumption in study Area's for March 2018
Water consumption within the month of April shown in Figure 2 representing a water consumption in rural area's ranges between 54.39 to 64.65 LPCD while water consumption in Urban area's ranges between 77.81 to 99.48 LPCD. Increase in consumption was observed due to the increase in temperature as increase in temperature results an increase in the consumption of water in Households. An increase in water consumption were found during study period both in urban and rural areas represented by Figure6. Besides increase in temperature water consumption in household depends upon the demographic characteristics, education, income, cost of fresh water su infrastructure of water distribution system and metering.
The actual daily water consumption observed during the researc less than the one which water supply facilities planners were used for the design of water supply systems. The trends of water consumption were almost the same in the urban and rural areas of different districts. There is also possibility that the water consumption rate drops for the households having no individual water supply sources and storage reservoirs. Besides seasonal changes, huge impacts on daily water consumption were observed due to cultural and demogr
Water supply planners need to adopt the actual water consumption while designing fresh water facilities to reduce the depletion available fresh water sources and to promote sustainability. Further study is required design of the continuous water supply and to use water in a conservative manner. Water consumption patterns for a specific area is not appropriate to use every in the world due to cultural, seasonal, demogra
55
in water consumption were found during study period both in urban and rural areas represented by Figure6. Besides increase in temperature water consumption in household depends upon the demographic characteristics, education, income, cost of fresh water supply, water quality, types of water supply system, infrastructure of water distribution system and metering.
Daily Water Consumption in study Area's for Study Period
The actual daily water consumption observed during the research period is far less than the one which water supply facilities planners were used for the design of The trends of water consumption were almost the same in the urban and rural areas of different districts.
There is also possibility that the water consumption rate drops for the households having no individual water supply sources and storage reservoirs.
Besides seasonal changes, huge impacts on daily water consumption were observed due to cultural and demographic variations.
Recommendations
Water supply planners need to adopt the actual water consumption while designing fresh water facilities to reduce the depletion available fresh water sources and to promote sustainability.
Further study is required for determining diurnal consumption pattern for design of the continuous water supply and to use water in a conservative manner.
Water consumption patterns for a specific area is not appropriate to use every in the world due to cultural, seasonal, demographic and socio-economic variations. in water consumption were found during study period both in urban and rural areas represented by Figure6. Besides increase in temperature water consumption in household depends upon the demographic characteristics, education, pply, water quality, types of water supply system, Daily Water Consumption in study Area's for Study Period h period is far less than the one which water supply facilities planners were used for the design of The trends of water consumption were almost the same in the urban and rural There is also possibility that the water consumption rate drops for the households having no individual water supply sources and storage reservoirs.
Besides seasonal changes, huge impacts on daily water consumption were Water supply planners need to adopt the actual water consumption while designing fresh water facilities to reduce the depletion available fresh water sources for determining diurnal consumption pattern for design of the continuous water supply and to use water in a conservative manner.
Water consumption patterns for a specific area is not appropriate to use every economic variations. | 2020-03-12T10:22:18.357Z | 2020-02-27T00:00:00.000 | {
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88519355 | pes2o/s2orc | v3-fos-license | The skew-t factor analysis model
Factor analysis is a classical data reduction technique that seeks a potentially lower number of unobserved variables that can account for the correlations among the observed variables. This paper presents an extension of the factor analysis model by assuming jointly a restricted version of multivariate skew t distribution for the latent factors and unobservable errors, called the skew-t factor analysis model. The proposed model shows robustness to violations of normality assumptions of the underlying latent factors and provides flexibility in capturing extra skewness as well as heavier tails of the observed data. A computationally feasible ECM algorithm is developed for computing maximum likelihood estimates of the parameters. The usefulness of the proposed methodology is illustrated by a real-life example and results also demonstrates its better performance over various existing methods.
variables; see, for example, Lawley and Maxwell (1971). The correlations between the variables under consideration are explained by their linear dependence on a usually much smaller number of unobservable (latent) factors. In particular, FA can be considered as an extension of principal component analysis (PCA), both of which are widely used statistical tools for reducing dimensionality by constructing linear combinations of the variables. Unlike PCA, which forms only a set of linearly uncorrelated representations explaining the most variance of the variables, FA seeks the most correlation among the variables by including additive independent errors on the observed variables. FA can also be viewed as a clustering method where the variables are described by the same factors which are grouped together.
FA has been applied successfully to numerous problems that arise naturally in many areas, see Basilevsky (2008) for a literature survey. In the FA framework, errors and factors are routinely assumed to have a Gaussian distribution because of their mathematical and computational tractability. However, the traditional FA approach has often been criticized for the lack of stability and robustness against non-normal characteristics such as skewness and heavy tails. Statistical methods which ignore the departure of normality may cause biased or misleading inference.
To remedy this weakness, authors such as McLachlan et al. (2007), Wang and Lin (2013) and Zhang et al. (2013) considered the use of the multivariate t (MVT) distribution for robust estimation of FA models, known as the tFA model. To our knowledge, there is little extended work on simultaneously accounting for asymmetry and heavy-tailedness in such models.
When the data have longer than normal tails or contain atypical observations (the so-called outliers), the multivariate t-distribution has been shown to be a natural extension of the normal for making robust statistical inference (Lange et al., 1989;Kotz and Nadarajah, 2004) as it has an extra tuning parameter, the degrees of freedom (df), to regulate the thickness of tails. In many biological applications (e.g., Pyne et al., 2009;Rossin et al., 2011;Ho et al. 2012) and other applied prob-lems, however, the data often involve observations whose distributions are highly asymmetric as well as having fat tails. One way is to use mixtures of factor analyzers (MFA) as proposed by Ghahramani and Hinton (1997). This approach was developed further in McLachlan and Peel (2000; and McLachlan et al. (2003;. To further reduce the number of free parameters to be estimated, Baek et al. (2010) and Baek and McLachlan (2011) introduced MFA with common component factor loadings (MCFA) before rotation of the factors to be white noise.
Over the past two decades, there has been a growing interest in proposing more flexible parametric families that can accommodate skewness and other non-normal features. In particular, the family of multivariate skew t (MST) distributions (Azzalini and Capitaino, 2003;Jones and Faddy, 2003;Sahu et al., 2003;Azzalini and Genton, 2008) had receive recent attention. This family contain additional skewness parameters for modeling asymmetry and includes the MVT family as a special case. This paper presents a robust extension of FA model by replacing the normality assumption for the latent factor and errors with the restricted multivariate skew t (rMST) distribution, hereafter referred to as the skew t factor analysis (STFA) model. The rMST distribution is reduced to the restricted multivariate skew normal (rMSN) distribution when the df approaches infinity. Both of which were originally used in Pyne et al. (2009) based on a restricted variant of the skew-elliptical distributions of Sahu et al. (2003). A comprehensive overview of their characterizations together with their conditioning-type and convolution-type representations can be found in Lee and McLachlan (2013a;. The proposed STFA model is a good alternative to deal with dimensionality reduction of multivariate data that have fat tails with strong degrees of asymmetry. The STFA includes the classical FA and tFA models as special cases and thus would be more widely applicable.
The remainder of this paper is organized as follows. In Section 2, we establish the notation and briefly outline some preliminary properties of the rMSN and rMST distributions. Section 3 discusses the specification of STFA model and presents the development of ECM algorithm for obtaining the ML estimates of model parameters.
In Section 4, we describe two simple ways of computing the standard errors of STFA model parameters based on the information-based method and the parametric bootstrap procedure. In Section 5, we illustrate the usefulness of the proposed method with a real-life data set. Some concluding remarks are given in Section 6 and technical derivations are sketched in Supplementary Appendices.
Preliminaries
We begin with a brief review of the restricted version of the MSN and the MST distributions and a study of some essential properties. To establish notation, we let ϕ p (·; µ, Σ) be the probability density function of N p (µ, Σ) (a p-variate multivariate normal distribution with mean µ and covariance matrix Σ); Φ(·) be the cumulative distribution function (cdf) of the standard normal distribution; t p (·; µ, Σ, ν) be the pdf of t p (·; µ, Σ, ν) (a p-variate MVT with location µ and scale covariance matrix Σ and degrees of freedom ν); T (·; ν) be the cdf of the Student's t distribution with df ν; T N (µ, σ 2 ; (a, b)) be the truncated normal distribution for N (µ, σ 2 ) lying within a truncated interval (a, b); M 1/2 denote the square root of a symmetric matrix M ; 1 p denote a p × 1 vector of ones; I p be the p × p identity matrix; Diag{·} be a diagonal matrix created by extracting the main diagonal elements of a square matrix or the diagonalization of a vector and vec(·) for a operator that vectorizes a matrix by stacking its columns vertically.
The restricted multivariate skew normal distribution
A p-dimensional random vector Y is said to follow a rMSN distribution with location vector µ ∈ R p , scale covariance matrix Σ and skewness vector λ ∈ R p if its pdf is where Ω = Σ + λλ T . In usual notation, we shall write Y ∼ rSN p (µ, Σ, λ) for a random vector with density (1). If λ = 0, the density of Y will be reduced to N p (µ, Σ) density.
Writing V = |X 0 |, a hierarchical formulation of (2) can be represented as As a consequence, the expectation and covariance matrix of Y are The result follows immediately from the first two moments of truncated normal distributions (Lin et al., 2007, Lemma 2) and the law of iterated expectations.
The restricted multivariate skew t-distribution
Formally, a p-dimensional random vector Y is said to follow a rMST with location vector µ ∈ R p , scale covariance matrix Σ, skewness vector λ ∈ R p and df ν ∈ (0, ∞), denoted as rSt p (µ, Σ, λ, ν), if it can be represented by where gamma(α, β) stands for a gamma distribution with mean α/β. If λ = 0, the distribution of Y reduces to t p (µ, Σ, ν) and to rSN p (µ, Σ, λ) as ν → ∞. In addition, this class of distributions also includes the multivariate normal distribution, recovered by setting λ = 0 and ν → ∞. Combining the strengths of the MVT and rMSN distributions, the rMST distribution offers a robustness mechanism against both asymmetry and outliers observed in the data.
Proposition 1. Let X | w ∼ N p (µ, w −1 Σ) and W ∼ gamma(α, β), then the joint pdf of X and w has the following relationship: where f G (·; α, β) denotes the pdf of a gamma distribution with mean α/β and the Proposition 2. For any scalar a ∈ R, ) .
Proof: The result is a special case of Lin (2010, Appendix A) when p = 1.
Supplementary Figure 1 presents various scatter diagrams and contours together
with their histograms of the density associated with a bivariate rMST distribution, , λ = (λ 1 , λ 1 ) T and ν = 4. The values of λ 1 , λ 2 and ρ reveal different shapes of bivariate rMST distributions. It is apparent that these plots are not elliptical and can be highly correlated and skewed toward different directions depending on their choices of parameters.
Using (4) and the law of iterated expectations, the mean and covariance matrix The mean and covariance matrix do not exist when ν ≤ 1 and ν ≤ 2, respectively.
3 Skew-t factor analysis model
Model formulation
Suppose that Y = {Y 1 , · · · , Y n } constitutes a random sample of n p-dimensional observations. To improve the robustness for modelling correlation in presence of asymmetric levels of sources, we consider a generalization of the tFA model in which the latent factor is described by the rMST distribution defined in (8). The model considered here is being a scaling coefficient. An appealing feature of model (11) is that which coincide with the conditions under the tFA model. According to (7), the STFA model has a two-level hierarchical representation: Derivation of the marginal distribution of Y can be accomplished by direct calculation which leads to where It therefore follows from (9) and (10) that With the STFA model (11), the parameters estimates of µ, B, D and ν can be used to recover the sample mean and sample covariance for both the tFA and STFA models in such a way that the models are likely comparable. Like the traditional FA models, the STFA model enjoys the scale invariance property (Anderson, 2003) and can be reduced to tFA model by imposing zero skewness for U j .
For a hidden dimensionality q > 1, the STFA model also suffers from an identifiability problem associated with the rotation invariance of loading matrix B, since model (11) The other commonly used technique is to constrain the loading matrix B so that the upper-right triangle is zero and the diagonal entries are strictly positive (e.g., Fokoué and Titterington, 2003;Lopes and West, 2004). Both methods impose q(q − 1)/2 constraints on B. Therefore, the number of free parameters to be estimated is m = p(q + 2) + q − q(q − 1)/2 + 1.
Maximum likelihood estimation via the ECM algorithm
To help the derivation of the algorithm, we adopt the following scaling transforma- Clearly, the model remains invariant under the above transformation. It follows from (3) and (13) that the STFA model can be formulated in a flexible hierarchical representation as follows: Consequently, applying Bayes' rule, it suffices to show where In what follows, define c j (r) = {(ν + p + r)/(M j + ν)} 1/2 , where r = −2, 0, 2.
We establish the following proposition, which is crucial for the calculation of some conditional expectations involved in the proposed ECM algorithm.
Proposition 4. From (16), we have the following conditional expectations: and Proof: See Supplementary Appendix C.
The expectation-maximization (EM) algorithm (Dempster et al., 1977) is For ML estimation of the STFA model, we resort to the ECM algorithm (Meng and Rubin, 1993) in which the M-step is replaced by a sequence of computationally simper conditional maximization (CM) steps while sharing all appealing advantages of the standard EM algorithm.
For notational convenience, let y = (y T 1 , . . . , y T n ) T be the observed data. Moreover, we let U = (U T 1 , . . . , U T n ) T , V = (V 1 , . . . , V n ) T and W = (W 1 , . . . , W n ) T , which are treated as missing values in the complete data framework. In light of (15), the complete data log-likelihood function for θ = (µ, B, D, λ, ν) given To calculate the expected complete data log-likelihood, called the Q-function, it involves the calculation of the following conditional expectations: which are directly obtainable from using (17)-(23) given in Proposition 4. As a result, the Q-function can be written as which contains free parameters µ andB. In summary, the implementation of the ECM algorithm proceeds as follows: (25), for j = 1, . . . , n.
CM-step 4: Updateλ (k) by maximizing (26) over λ, which giveŝ CM-step 5: Calculateν (k+1) by maximizing (26) over ν, which is equivalent to solve the root of the following equation: where DG denotes the digamma function and In the above CM-step 5, the R function 'uniroot' is emplyed to obtain the solution of ν. To facilitate faster convergence, the range of ν is restricted to have a maximum of 200, which does not affect the inference when the underlying distribution of factor scores are near-normality. Upon convergence, the ML estimate of θ is denoted by θ = (μ,B,D,λ), whereB =BΛ 1/2 andΛ = I q + (1 −ν −2 νâ 2 ν )λλ T . Consequently, the estimation of factor scores through conditional prediction is obtained bŷ can be evaluated via (18) with θ replaced byθ andâ ν is a ν in (12) with ν replaced byν.
We further make some remarks on the implementation of the proposed ECM algorithm.
Remark 1. To monitor the convergence based on the monotonicity property of the algorithm, a simple way is to repeat iterations after a certain number of iterations, say K, or until the difference between two successive log-likelihood evaluations is small enough, say ℓ (k+1) − ℓ (k) < ϵ, where for brevity of notation ℓ (k) means the log-likelihood value evaluated atθ (k) and ϵ is a user-specified tolerance. In our analysis, we use K = 5, 000 and ϵ = 10 −6 .
Remark 2. As analogous to other iterative optimization procedures, one needs to search for appropriate initial values to avoid divergence or time-consuming computation. A direct way of deriving the initial estimate for mean vector, factor loading and error covariance matrix can be obtained by performing a simple FA fit using the factanal command in the R package. The resulting estimates are taken as initial values, namelyμ (0) ,B (0) andD (0) , respectively. Next, compute the factor scores via the conditional prediction method. The initial skewness vectorλ (0) and dfν (0) are obtained by fitting the rMST distribution to the sample of factor scores via the R package EmSkew .
Remark 3. For model selection and determination of q, the fitting results are compared based on the Akaike's information criterion (aic; Akaike, 1973) and the Bayesian information criterion (bic; Schwarz, 1978), which are defined as aic = 2m − 2ℓ max and bic = m log n − 2ℓ max .
where ℓ max is the maximized log-likelihood and m is the number of free parameters in the considered model.
Provision of standard errors
Under regularity conditions (Zacks, 1971), the asymptotic covariance matrix ofθ can be approximated by the inverse of the observed information matrix; see also Efron and Hinkley (1978). Specifically, the observed information matrix is defined as the Hessian of the negative of the log-likelihood function To obtain I(θ; y) numerically, Jamshidian (1997) suggested using the central difference method. Let G = [g 1 ; · · · | g m ] be a m × m matrix with the cth column being where s(θ; y) = ∂ℓ(θ; y)/∂θ is the score vector of ℓ(θ; y), e c is a unit vector with all of its elements equal to zero except for its cth element which is equal to 1, h c is a small number, and m is the number of parameters in θ. Explicit expressions for the elements of s(θ; y) are summarized in Supplementary Appendix D.
Since G may not be symmetric, we suggest using to approximate I(θ; y) The asymptotic standard errors ofθ can be calculated by taking the square roots of the diagonal elements of [Ĩ(θ; y)] −1 .
Notably, the inverse of (28) is not always guaranteed to yield proper (positive) standard errors. The parametric bootstrap method (Efron and Tibshirani, 1993), although computationally expensive, is often used instead to obtain estimates of the standard errors. Letf (y;θ) be the estimated density function of (14) obtained from fitting the STFA model to the original data. Obtaining bootstrap standard error estimates consists of the following four steps.
A numerical illustration
As an illustration, we apply the proposed technique to the Australian Institute of Sport (AIS) data, which were originally reported by Cook and Weisberg (1994) and subsequently analyzed by Azzalini and Dalla Valle (1996), Capitaino (1999, 2003) and Azzalini (2005), among others. The dataset consists of p = 11 physical and hematological measurements on athletes in different sports which are almost equally bisected between 102 male and 100 female. Table 1 about here For simplicity of illustration, we focus solely on n = 102 observations of male.
A summary of 11 attributes along with their sample skewness and kurtosis is given in Table 1. It is readily seen that most of attributes are noticeable moderately to strongly skewed and leptokurtotic in nature. Next, we are interested in comparing the ML results of STFA with those obtained under three reduced models, namely the FA, tFA and SNFA. The data have been standardized to have zero mean and unit standard deviation to avoid variables having a greater impact due to different scales. We fit these models with q ranging from 1 to 6 using the ECM algorithm developed in Section 3. Notice that the choice of maximum q = 6 satisfies the restriction (p−q) 2 ≤ (p+q) as suggested by Eq. (8.5) of McLachlan and Peel (2000). Observing the unrotated solution of factor loading displayed in the 3-6th columns of Table 3, the first factor can be labelled general nutritional status, with a very high loading on lbm, followed by Wt, Ht and bmi. The second factor, which loads heavily on rcc, Hc and Hg, might be called a hematological factor. The third factor can be viewed as overweight assessment indices since the bmi, ssf and Bfat load highly on this factor. The fourth factor is not easily interpreted at this point.
The comparison process is also conducted for the original (non-standardized) data. Clearly, as shown in Supplementary Figure 2, the STFA still provides the best overall fit, followed by tFA and SNFA. The fit of FA is the worst, indicating a lack of adequacy of normality assumptions for this dataset. It is also noted that both criteria prefer four-factor solutions under all scenarios. the best fitted STFA model. A visual inspection reveals that the fitted contours adapt the shape of the scattering pattern satisfactorily. To summarize, the implementation of STFA procedure tends to be more reasonable for analyzing this data set.
Conclusion
We introduce an extension of FA models obtained by replacing the normality assumption for the latent factors and errors with a joint rMST distribution, called the STFA model, as a new robust tool for dimensionality reduction. The model accommodates both asymmetry and heavy tails jointly and allows practitioners for analyzing data in a wide variety of considerations. We have described a four-level hierarchical representation for the STFA model and presented an analytically simple ECM algorithm for ML estimation in a flexible complete-data framework. We demonstrate our approach with a real data set and show that the STFA model may provide better performance than several existing competitors.
In the situation with the occurrence of missing data, our algorithm can be easily modified to account for missingness based on the scheme proposed in . Due to recent advances in computer power and availability, it is worthwhile to develop Markov chain Monte Carlo (MCMC) methods Lin and for carrying out Bayesian inference of the STFA model. It is also of interest to consider a finite mixture representation of STFA models. Our initial work on the latter problem has been limited to mixtures of factors with a skew-normal distribution .
Also, it should be noted in other unpublished work involving mixtures of factor models (Murray et al., 2013a;) that the skew t-distribution adopted is different to the skew t-distribution considered in our paper. Rather it is the limiting form of the generalized hyperbolic distribution, which has some quite different properties.
For example, it has one exponential tail and one polynomial tail instead of two polynomial tails as with the usual skew t-distribution. Also, as the skewness parameters in its formulation tend to zero, it does not become a skew normal distribution; that is, it does not nest the skew normal distribution as a special case. The unrestricted skew t-distribution is considered in Murray et al. (2013c). But as in Murray et al. (2013a), the factor analytic representation applies only to the error terms in the presence of the skewing variables and not the factors. | 2013-12-03T14:49:46.000Z | 2013-10-20T00:00:00.000 | {
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14363608 | pes2o/s2orc | v3-fos-license | New Small Molecules Targeting Apoptosis and Cell Viability in Osteosarcoma
Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS.
Introduction
Osteosarcoma (OS) is an orphan disease with an incidence of approximately 0.4 per 100,000 population per year [1]. The rarity of the disease and thus the limited availability of biopsy material complicate the possibility of large-scale analyses of these tumors. In addition, the genetic complexity of OS up-to now hampered the identification of "druggable" OS-specific targets [2]. Although several genetic alterations have been described to occur in OS at varying frequency, OS are generally characterized by highly complex karyotypes [3,4], at least in a subset of tumors resulting from chromothripsis [5]. Thus, so far researchers have failed to identify an OS-specific mutation or a pathway.
These circumstances may partly explain that therapy in OS has not significantly improved in the last three decades. The standard therapy involves multidrug chemotherapy (methotrexate, doxorubicin, cisplatin and ifosfamide) in combination with radical surgery [6]. This treatment yields positive outcomes in many patients with an overall 5-year survival rate of approximately 70%. However, the prognosis drastically worsens in patients with apparent metastatic spread or recurrent disease with overall survival rates generally below 20% [7].
In the last years a great effort has been done to develop new therapeutic strategies for OS patients. Several studies and clinical trials have been testing multimodal therapies and dose variations of classical drugs. However, a great majority of these studies fail to enter phase III clinical trials [8]. Beside the challenge of establishing an adequate trial design in such a rare disease due to the lack of resources and the limited number of patients fulfilling all requirements, several studies had to be stopped due to low efficacy and safety [8,9].
Most molecular biology studies have focused on drugs that target single alterations associated with OS with the attempt to develop personalized therapies. Among the most promising drugs are small molecule kinase inhibitors, for example the inhibitor of insulin-like growth factor 1 receptor (IGF-1R) belonging to the family of receptor tyrosine kinases (RTK) [10], as well as p53-interacting drugs [11]. Insulin-like growth factors 1 (IGF1) and 2 (IGF2) stimulate certain pathways via the IGF receptor regulating cell growth and survival, pathways that are frequently deregulated in OS [10,12].
Likewise, p53 mutations were associated with genomic instability in OS [13]. However in most of the tumors so far no loss-of-function mutations have been observed in the p53 protein.
Consequently, the reactivation of the tumor suppressor function of p53 by the nutlin small molecule inhibitors of MDM2-p53 interaction seem very promising [11]. Recently, structural variations in intron 1 of the TP53 gene were reported that could explain the absence of general p53 protein mutations but rather the altered activity of the p53 in OS [14,15]. Thus alterations in p53 binding affinity may cause the activation of different pathways that could contribute to malignant transformation [16]. Problems arising from the targeted therapies include amongst others the development of resistance, which is true for some RTK inhibitors [17], and the lack of predictive biomarkers to validate a positive outcome in patients [10]. Likewise, some drugs simply fail due to improper preclinical target validation and not lastly by the limited number of patients in which the respective target has been revealed [9].
Consequently, phenotypic drugs that target OS cell proliferation or metastasis are subjects of great interest in the development of new drugs [2,8,18]. Although in the past target-based approaches have been the gold standard in small-molecule high-throughput screens focusing on compound-target interactions [11,19], within the last decade more and more phenotypic screenings have been applied to discover small molecule inhibitors with new mechanisms of action [18,[20][21][22].
Besides the discovery of new molecules from e.g. natural sources handled at the in vitro level, other promising agents such as maltonis are currently tested in phase I/II [23]. Still, the only new therapeutic agent in the treatment of OS that has been clinically approved in the recent years is muramyl tripeptide (MTP), an immunomodulatory drug [24]. Consequently, there is still a need for compounds from new sources and of diverse structural characteristics that would offer manipulation capability and could be tested in drug discovery evaluation processes.
In this study, we describe a phenotypic screening strategy that enabled us to screen a large set of diverse compounds (25,000) with regard to their effectiveness in OS cell lines. The stepby-step strategy allowed us to reduce the number of eligible compounds to a manageable number of compounds. We used this strategy to investigate compound actions with regard to their selectivity and apoptosis inducing potential in OS cells and compared these effects to wellknown drugs that are used in clinical OS treatment.
Screening strategy and compound identification
In order to identify new compounds that specifically target OS cells we screened three diversity libraries obtained from different suppliers (see Materials and Methods) that altogether comprised 25,000 diverse compounds. Fig 1 describes the workflow of the screening strategy used for compound identification. In a primary screen, the effects of compounds on cell viability were assessed in U2OS cells, a well-characterized OS cell line [25], using Celltiter Blue (CtB) assay (Fig 1, step 2). The primary screen at a final concentration of 10 μM resulted in 320 positive hits that reduced the cell viability of U2OS cells from 65 to 20% (Fig 2A). To identify those compounds that selectively act on OS cells, compounds were counter-screened in different OS and non-OS control cells ( Table 1) using again cell viability as read-out (Fig 1, step 3). Here we used various well-known OS cell lines, including ZK-58 and MNNG-HOS as well as immortalized human osteoblasts hFOB1.19 [25,26]. From these results we performed a hierarchical clustering according to cell type and compounds with regard to their effects on cell viability. The graphic shows the number of compounds (triangle on the left), their characteristics (mid row) and the respective approach (right) that was applied for compound selection. Arrows from top to bottom indicate the "timeline". *described in [19]. Clustering resulted in two main groups separating compounds with weak or no effects from those with strong effects on cell viability (Fig 2B). One group highlighted by a red circle could be further sub-divided into the compounds that generally reduced viability in all cell lines and those with the highest effects specifically in OS cell lines (Fig 2B, red circle). This latter subgroup consisting of 58 compounds specific to OS cell lines was further short-listed based on Lipinski´s rules on their medicinally relevant physical chemical properties [27]. The criteria included the solubility, cell permeability, assay reproducibility but also the ease of synthesis and the possibility for diverse chemical manipulations of the core structures. In total 29 compounds were selected (S1 Table) which showed low inter-replicate variability in primary and counterscreen with standard deviations (SD) < 30% ( Fig 2C) (Fig 1, step 5).
Subsequent hit validation and analysis of dose-response relationship of the remaining 29 compounds in three OS cell lines (U2OS, HOS, Saos-2) and two non-OS control cell types (HepG2-hepatocellular carcinoma cell line, and hOB-primary human osteoblasts) finally identified two most prominent compounds, namely A13 and H12 with different structural characteristics (Fig 2D and 2E). The compound A13 had pyrazole and thiophene heterocyclic cores bridged by a benzamide moiety. The compound H12 contained a pharmacologically potent and biologically relevant chromene scaffold. These two amphiphilic compounds showed reasonable assay reproducibility with SD of 2% ( Fig 2C).
The compound A13 strongly reduced viability of OS cells (U2OS and HOS) at concentrations ranging between 0.31 and 20 μM (Fig 3A). In contrast, viability of HepG2 and primary hOB was not significantly affected. A strong selective effect (p < 0.001) in U2OS and HOS Compound selection process and compound chemical structures. A) Scatter plot shows distribution of 25,000 compounds in regard to cell viability (%) in U2OS cell line with compounds not significantly affecting viability (grey dots), those that reduced viability from 75 to 20% (blue dots), and defined active compounds that were selected for further analysis with viability ranging from 20 to 65% (green dots). Cell viability was determined using Celltiter Blue (CtB) assay. B) Hierarchical clustering of 320 defined active compounds. Compounds were screened on various OS and control cell lines. Respective cell lines are indicated on the left, color key shows values according to cell viability ranging from 20-100%. The red circle marks the cluster that was short-listed for subsequent medicinal chemistry analysis. C) The graph shows inter-replicate variability in the primary and the counter screen of the 29 short-listed compounds. A low inter-replicate variability was used as a selection criteria in medicinal chemistry analysis. The two confirmed hits are highlighted in red and blue, respectively. D) and E) show structures of the two confirmed OS-selective hits, namely compounds A13 (D) and H12 (E). compared to primary hOB cells was observed at concentrations from 0.31 μM to 20 μM. Treatment with the compound H12 resulted in a significant dose-dependent decrease in cell viability of U2OS and HOS cells ( Fig 3B). The compound H12 showed a significant selectivity (p < 0.001) in U2OS and HOS observed at concentrations between 1.25 μM and 20 μM when compared to primary hOB cells, but also in HepG2 ( Fig 3B). Of note, in both treatments the p53-negative OS cell line Saos-2 showed almost no sensitivity. IC50 values for cell viability have been calculated for the compounds A13 and H12 (S1 Fig), and for staurosporine and are shown in. Table 2 The IC50 values for compound A13 and H12 clearly reflect the differences in sensitivity to compound treatments of the different cell types. The effectiveness of the new compounds was compared to two well-known drugs: Staurosporine, a pan-inhibitor of kinases, is a well-known research compound often used as a classical , or with classical drugs staurosporine (C) and doxorubicin (D), respectively. Cell viability was assessed using CtB assay. Shown is fold change of cell viability normalized to DMSO-treated control for each cell type after incubation with increasing concentrations (μM) of the respective substance for 24h. Data are expressed as mean +/-SD from duplicates of three independent experiments. Differences of means were calculated using multiple t test for all cell lines versus primary hOB and all highly significant differences with #p < 0.001 are indicated by hash key (#). Differences of means to control were calculated for all bars and all were significantly different from control with *p < 0.05 indicated by the lines on top of the bars except those bars that are marked with ns = not significant (p > 0.05). apoptosis-inducing agent [28], and doxorubicin that is used in clinical OS treatment and induces strong cytotoxicity [29]. Importantly, the U2OS cell line showed reduced sensitivity to this drug [30]. In our assay, staurosporine reduced the cell viability of all cell types at concentration of 20 μM or higher ( Fig 3C). In contrast, doxorubicin only slightly affected cell viability at similar concentration ranges ( Fig 3D). However, results showed that both the A13 and H12 compounds were effective in OS cell lines at concentrations lower that those observed for staurosporine and doxorubicin ( 1.25 μM vs. 20 μM, respectively). Taken together, the A13 and H12 compounds identified by our screening strategy had the most potent effects in U2OS and HOS cells
Mechanistic aspects of compound action
To gain more insight into the mechanistic aspects of action of the two active compounds, a multiparametric assay was applied combining high-throughput imaging and quantification techniques. In this assay, several characteristic morphological changes are measured: cell loss, nuclear shrinkage, cell membrane disruption and mitochondrial dysfunction.
The compound A13 induced in U2OS and HOS a loss in the number of cells, disruption of cell membrane, mitochondrial changes (increase of mitochondrial mass) and reduction in nuclear size at concentrations of 5 μM and 10 μM (Fig 4). Almost no dead cells and no changes in nucleus area were observed in Saos-2 and in the control cells hOB (Fig 4).
Treatment with the compound H12 resulted in cell loss in U2OS and HOS, decreased mitochondrial mass and nucleus area and a slight increase in membrane permeability (Fig 5).
In general, these parameters indicate that the compounds A13 and H12 are capable to induce cell death in OS cell lines. Reduction of nuclear size most probably results from nuclear condensation indicating the induction of apoptosis by the two compounds. However, treatment with compound H12 showed reduced effects in OS cell lines compared to the compound A13.
The apoptosis signaling in OS cells after compound treatment
To analyze in more detail pathways involved in cell death mechanism, we determined caspase 3/7 activity and cell lysis in U2OS cells. Cells were treated for 8 and 24h with compounds and caspase 3/7 activity was determined and compared to the activity of cells treated with staurosporine and doxorubicin (Fig 6). Caspase 3/7 activation was strongly increased after treatment with compound A13 at a concentration of 10 and 20 μM, while H12 only slightly induced caspase activity at a concentration of 20 μM for 24h ( Fig 6A). Both compounds also led to a moderate induction of caspase 3/7 activity in primary hOB (2-fold) and Saos-2 (3 to 4-fold) after , and membrane permeability (%) (E) were analyzed after treatment with compound H12 at 10 μM for 24h. Unpaired t test showed *p < 0.05, **p < 0.01, ***p < 0.001, and not significant (ns) for U2OS vs. hOB and HOS vs. hOB, respectively, and in E) #p < 0.001 relative to DMSO-treated control of the respective cell type (H12 -). Data are expressed as means +/-SD from duplicates of two independent experiments and are shown as percent or as fold change relative to DMSO-treated control. 24h (S2 Fig). In comparison, staurosporine strongly induced caspase 3/7 already after 8h, and doxorubicin most effectively induced caspase 3/7 activity after 8 and 24h treatment (Fig 6A). Despite strong caspase activation by compound A13, the observed cell lysis was rather low (up to 3-fold at the concentration of 20 μM after 24h) and was similar to that observed for compound H12 (Fig 6B). In cells treated with staurosporine, however, classical apoptosis induction was observed by initial caspase activation followed by time delayed strong cell lysis (Fig 6A and 6B). Doxorubicin showed dose-dependent increase of cell lysis at both time points (Fig 6B). Taken together, both compounds were capable to induce caspase 3/7 activity. To test whether the compound effects are dependent on caspase activity we investigated the effects of several caspase inhibitors (pan-caspase-, caspase 8-, and caspase 9 inhibitors) in cotreatment with compound A13 and H12. We observed a significant decrease of cell number in cells treated with 20 μM of A13 and H12, respectively (Fig 6C). These effects were significantly reduced in the presence of the caspase 9 inhibitor Z-LEHD-FMK. The pan-caspase inhibitor Z-VAD-FMK also reversed the effects of compound H12 in a significant manner while the caspase 8 inhibitor Z-IETD-FMK had no effect (Fig 6C). The results indicate an action of the compounds on caspase 9 and the involvement of intrinsic apoptosis pathways in the drug responses.
Discussion
In this study, we demonstrate two new compounds identified by our screening strategy as potential candidates for OS treatment. The two compounds, namely A13 and H12, had strong and dose-dependent effects on cell viability in the OS cell lines U2OS and HOS, but did neither significantly affect the control cell lines from hepatocellular carcinoma (HepG2) nor primary human osteoblasts. The compounds induced changes in cell morphology that included nuclear condensation and changes in the mitochondrial mass. In addition we observed a strong (A13) and a moderate (H12) induction of caspase 3/7 activation in U2OS cells. These results indicated the potential of apoptosis induction in OS cells by the two new compounds.
Induction of apoptosis is an important issue in therapy development not lastly because this cell death mechanism prevents an inflammatory response that is the case in necrosis [34]. Consequently, the potential of a compound to induce necrosis should be limited to the minimum since inflammation induced by cells dying from necrosis causes severe side effects in patients. Morphological indicators of apoptosis are amongst others nuclear condensation and intact cell membrane whereas hallmarks of necrotic cells are cell lysis and mitochondrial swelling [35]. In vitro the late stages of apoptosis also include cell lysis [36]. In our study, a strong cell lysis subsequent to caspase activation was observed for the well-known apoptosis inducing agent staurosporine. Our data showed that the compounds did not induce strong necrosis in OS cells at the concentrations investigated since the cell lysis observed was rather low. Moreover, the morphological changes that we observed in OS cells are associated with apoptosis at least at the doses investigated.
The results obtained from the caspase 3/7 activity assay suggested the time point of apoptosis induction to be around 24h after compound treatment at concentrations of 10 to 20 μM. Using western immunoblotting, however, we could not detect cleavage of the apoptosis marker Poly (ADP-ribose) polymerase (PARP) at 10 μM after 24h, neither after 48 nor 72h (data not shown). In contrast, higher compound doses (30 μM) induced a strong PARP cleavage in HOS and Saos-2 cells and also in osteoblasts (hFOB1.19) (S3 Fig). Almost no protein was detected in U2OS cells, which may result from strong cell lysis at this high compound doses in the sensitive U2OS cell line [37]. We conclude that the cell line-selective effects that were strongly suggested using the cell viability assay (Fig 3, S1 Fig) and IC50 values ( Table 2) are apparent at low compound concentrations (< 10 μM), while at higher doses the effects also affect other cell lines. In addition we performed Annexin V staining in combination with the dye Yo-Pro that stains lysed cells. We only observed Annexin V positive cells after A13 and H12 treatment between 5 to 10% Annexin V positive/ Yo-Pro negative cells in U2OS, HOS and Saos-2, whereas Annexin V positive cells were not induced after treatment in osteoblasts and HepG2 (S4 Fig). We conclude that the additional experiments support our findings that the compounds are capable of apoptosis induction, however the effects on apoptosis induction are rather mild which may argue for specificity of the effect without inducing general toxicity. More important, at lower doses the two compounds were almost not toxic to cells derived from hepatocellular carcinoma (HepG2), a cell line often used in drug discovery to exclude hepatocytotoxicity [38], and human osteoblasts.
The two compounds strongly reduced cell viability, whereas the induction of caspase 3/7 and cell lysis by the compounds were rather low. In contrast, staurosporine and doxorubicin strongly induced caspase 3/7 and cell lysis but effects on cell viability were only observed at higher concentrations (Fig 3). We assume that the strong induction of caspase 3/7 and cell lysis by doxorubicin (Fig 6) could be a bias of the fluorometric/luminogenic assays which leads to an overestimation of the signal, as has already been reported [39]. Our data, however, indicate that the cell viability assay, which measures the metabolic activity of a cell, implicates a strong reduction of cell viability in OS cells already at low concentrations. In contrast, an apparent induction of cell death was not detectable at low compound concentrations in the other assays. The discrepancy between the strong effects on cell viability (Fig 3A and 3B) and the modest effects on apoptosis induction (Fig 6A and 6B) and loss in cell number (Fig 5B) is explainable by the time course of the process. The cell viability reflects the metabolic activity of a cell that is not necessarily directly linked to apoptosis as an outcome. The effects on viability are present already at early time points and at lower compound concentrations, while apoptosis induction or other cell death mechanisms that finally lead to a detectable loss in cell number occur subsequent to that initial event [40]. Moreover, in Fig 6C we used increased concentrations of compounds A13 and H12 (20 μM) that led to a significant loss in cell number. We conclude that, beside a possible overestimation of doxorubicin effects, the two new molecules were capable to affect OS cells already at lower concentrations when compared to the well-established drugs.
The multiparametric assay also indicated that the A13 compound led to the increase of mitochondrial mass. In contrast, the H12 compound decreased the mitochondrial mass. Interestingly, the effects of the two compounds on cell number were significantly abrogated in the presence of the caspase 9 inhibitor Z-LEHD-FMK (Fig 6C), and also partly by the pan-caspase inhibitor Z-VAD-FMK, which indicates the involvement of intrinsic apoptosis pathways in the drug responses of the two compounds. For detailed analysis of apoptosis mechanisms the compound action on mitochondria will be further investigated in future studies. This is of particular interest because mitochondria are important players in mediating cell death and apoptosis and promising target structures in cancer therapy [41]. For example, analysis of mitochondria isolated from treated cells could give more insights into mitochondrial conditions [38]. Recently, Ma and colleagues described a pancratistatin analogue JC-TH acetate-4 (JCTH-4) and showed that this agent induced apoptosis in OS cell lines via mitochondrial targeting. Addition of the natural herb curcumin, that has anti-proliferative properties, further enhanced the apoptotic effects [42]. Moreover, they showed that JCTH-4 also induced autophagy in OS cells, a mechanism that has distinct outcomes in therapeutic usage and also depends on the p53 status of a cell [43]. Further downstream, autophagy may influence the PI3K/AKT/mTOR pathway that is also implicated in OS [44,45] thus making JCTH-4 a promising agent, although the effect of autophagy in cancers yet is not well characterized [34]. Thus it would be interesting to test whether OS cells treated with the two new molecules described in our study would also induce changes associated with autophagy and whether this would involve the PI3K signaling pathway.
With regard to the important role of p53 in many cancers and especially in OS the p53-negative OS cell line Saos-2 exhibited lower (compound A13) or almost no sensitivity (compound H12) to the newly identified compounds. This makes p53 and its associated pathways an interesting starting point for further investigations on the compound actions. It has to be noted that the OS cell lines used in this study are of different p53 status [25] and in some cell lines p53 expression has been associated to MDM2 (murine double minute-2) gene amplifications [25,46]. Moreover, differentially activated forms of p53 protein in Saos-2, U2OS and MNNG-HOS cells have been implicated in drug sensitization of OS cells [47] which makes the role of p53 very complex and difficult to elucidate. A greater sensitivity of p53-wild type cells towards PI3 kinase inhibitor has been already reported in glioma cells [48]. PI3 kinases that act downstream of p53 in the PI3K/AKT/mTOR pathway are implicated in OS thus being interesting targets of small molecule inhibitors [2,9,49]. In future studies we will investigate the important link between p53 and the sensitivity of OS cell lines to the two new compounds identified in this study. Here, we aim to test these two compounds after the knockdown of p53 in OS cells or after the re-introduction of an active p53 into Saos-2 to further elucidate the role of p53 [48].
Considering the severe side effects induced by the multidrug chemotherapy applied in OS, new molecules exhibiting such selective effects as described in our study are of special interest in drug development. In recent years, many molecules discovered for therapeutic application have evolved from phenotypic screens by identification of compounds exhibiting a certain action of interest, such as an anti-proliferative or an apoptosis inducing effect [50]. Thus, the compounds identified in our study could crucially contribute to the development of new drugs in OS.
Summary
The discovery of new drugs and the improvement of treatment strategies still is an important issue in cancer disease because such new drugs could support or even substitute a conventional therapy when its effectiveness is compromised. In this study, we used a phenotypic highthroughput approach to screen the effectiveness of a 25,000 small-molecule diversity library against osteosarcoma cells. We characterized a large set of diverse compounds in U2OS cell lines and selected compounds that actively reduced the cell viability. By applying various OS and non-OS cells, we then step-by step could exclude inactive or overall toxic compounds and tested the remaining compounds for their chemical properties and robust dose-response. Finally, we identified two new small molecules that selectively reduced viability and induced apoptosis signaling in OS cell lines in a dose-dependent manner. The two compounds were robust in medicinal chemistry analysis and are susceptible to chemical manipulations due to their structural properties. Thus, we provide promising substances affecting OS cell viability that offer a possibility for further drug development in OS.
Library buildup and compound preparation
The composition of the 25,000 diverse-compound library has been described earlier [19]. Compounds were either dissolved in dimethylsulfoxide (DMSO) arranged in 384-well micro-plates for high-throughput screening (HTS) or delivered as a dry powder in glass vials for individual assays. All compounds were diluted in DMSO (product number AE56.2; Roth, Karlsruhe, Germany) and stored tightly sealed at -20°C. The two compounds found in the screening were obtained from ChemDiv (San Diego, CA), and can be re-ordered using the compound IDs for A13 (compound ID: 6228-2300) and H12 (compound ID: 5948-4347), respectively. Staurosporine (s5921) and Doxorubicin (d1515) were both obtained from Sigma-Aldrich (Taufkirchen, Germany) and diluted in DMSO and H 2 O dest., respectively.
Screening instruments and performance
Cell seeding and assays were performed in black 384-well microplates (Greiner Bio-One, Monroe, NC; 781091) for fluorometric assays, and white 384-well Optiplate (PerkinElmer-PE, Waltham, MA; 6007290) for luminescence-based assays, respectively. Cells were seeded 24h before treatment in 384-well microplates with a cell number of 2000 cells/well for highly proliferating cells and 3000 cells/well for low proliferating cells ( Table 1) [51]. This resulted in an intermediate confluency after 24h (time of compound addition) and in a confluent layer after approximately 48h. Plate handling, automated cell seeding and compound addition was performed using following HTS devices: Plate handling was performed using a Sciclone G3 with a Twister II robotic arm (PerkinElmer, USA) and a Flexdrop (PerkinElmer, USA) liquid handling system. CellTiter Blue (G8081; Promega, Mannheim Germany) measurements were performed using an EnVision Multilabel Reader (PerkinElmer, USA).
Fluorometric cell viability assay
Cell viability was determined using Celltiter Blue (G8081; Promega, Mannheim Germany). This assay measures the reducing potential according to metabolic activity in cells. Cells were seeded 24h before treatment in black 384-well plates. Following 24h incubation at 5% CO 2 , 37°C, compounds (10 mM) were added to the cells in a final concentration of 10 μM (0.1%. Cells were incubated for further 22h. Pre-warmed Celltiter Blue reagent was added to the medium (1:5 dilution), plates were gently shaked and incubated for 2h at 5% CO 2 , 37°C. Prior to measurement, plates were again gently shaked and cell viability was measured at the Envision Multilabel Reader (PE) at 560 Exc /590 Em nm (4 areas per well measurement). Data were normalized to vehicle control-treated cells (1% DMSO).
Fluorometric cytotoxicity assay
The CellTox Green Express Cytotoxicity Assay (G8731; Promega) contains a fluorescent dye that binds to DNA of cells with impaired membrane integrity (cell lysis). Binding of the dye to cell DNA results in fluorescence signal which is detectable at 495 nm. For determination of cell lysis, cells were seeded manually in black 384-well microplates and incubated 24h at 5% CO 2 , 37°C. Medium was replaced by fresh medium containing the fluorescent dye diluted by 1:500 dilution. Compounds and control substances were diluted in the respective medium/dye mix and added to the cells. For positive control of cell lysis, 30 μM digitonin was added to the cells 15 min prior to measurement. Background signal (medium and CellTox Green dye) was subtracted and data were normalized to vehicle control (1% DMSO).
Luminescent caspase 3/7 activity assay
The caspase 3/7 assay (G8091, Promega) uses a DEVD-linked luminogenic substrate that is cleaved upon exposure to active caspase 3 and 7. Cleavage of the substrate results in a luminescent signal generated by activated luciferase. For apoptosis induction, cells were seeded in white 384-well microplates and incubated for 24h at 5% CO 2 , 37°C. Subsequently, compounds and control substances were added to the cells in a dilution series and incubated for further 8 and 24 h, respectively. One hour before end of drug exposure time, Caspase-3/7-Glo reagent was added to the cells in 1:2 dilution as described by the manufacturer and incubated for one hour at room temperature in dark. Luminescence was measured at 700 nm. Background signal (medium and Caspase-3/7-Glo reagent only) was subtracted and data were normalized to DMSO-treated cells.
Multiparametric cytotoxictity assay
The multiparametric assay determines cell number, cell membrane integrity and nuclear fragmentation in an image-based quantification using the Operetta/Harmony high-throughput imaging platform (PE) [38]. Cells were seeded into black 384-well micro-plates 24h prior to treatment. For co-treatment of cells with compounds and caspase inhibitors the cells were pretreated with pan-caspase (Z-VAD-FMK), caspase 8 (Z-IETD-FMK) or caspase 9 inhibitor (Z-LEHD-FMK; all obtained from BioVision Incorp., CA) in a 1:1000 dilution 30 min prior to compound addition. Compounds were added in a serial dilution to the cells and plates were incubated for 24h at 5% CO 2 , 37°C. For DNA counterstaining, NucBlue, a dye similar to Hoechst 33342, was used, mitochondria were stained using MitoTracker deep red and dead cells were stained using Po-Pro iodide (product numbers: R37605, M22426, and P3585, respectively; all by Life Technologies). Dyes were diluted in medium and added to living cells. After 45 min incubation at 5% CO 2 , 37°C, images were recorded using the automated Operetta microscope using the 40x NA objective for high-resolution images. For quantification, two images of each condition in duplicates and for each cell type were recorded using a 20x objective. This resulted in a cell number of approximately 500 cells of each condition. Quantification on cell size was performed using the Harmony software (PE).
Statistical analysis
All the data were collected in the given precision in tab-separated text formats and Microsoft Excel. Hierarchical clustering of cell viability data was performed using the R software environment for statistical computing and graphics version 3.0.1 (www.r-project.org, package 'cluster', function: agnes, agglomerative hierarchical clustering, metric: Pearson correlation, method: complete linkage). In order to determine differences of means the t-test (unpaired, two sample test, p values less than 0.05 were considered to be significant) was applied using GraphPad Prism (GraphPad Software 6.0f, La Jolla California, USA, www.graphpad.com). In the case of multiple tests we corrected the raw probability values by the FDR method [53]. In the drug discovery process IC50 values (inhibitory concentrations) were calculated to evaluate the suitability and performance of a drug [54]. The calculation of the IC50 values were performed with GraphPad Prism and followed a nonlinear regression model applied to the sigmoidal dose-response curves of the cell viability data. The values were log-transformed before fitting the model. Details to the procedure can be found in the Graphpad Prism User Guide (www. graphpad.com/guides/prism/6/user-guide).
Supporting Information S1 Fig. Dose-response curves of cell viability in the five cell lines used for hit validation (HepG2, primary hOB, U2OS, HOS, Saos-2) shown for the compound A13 and H12. Curves were generated from nonlinear regression and IC50 values have been calculated. Cell viability has been determined using the Celltiter Blue Assay after compound incubation for 24h. Data represent means and SD of two (HepG2, hOB) to three (U2OS, HOS, Saos-2) independent experiments always performed in duplicates. (TIFF) S2 Fig. Caspase 3/7 activity and cell lysis assays for primary hOB and Saos-2 cells 24h after compound treatment. Primary hOB and Saos-2 cells were treated with 10 or 20 μM of compound A13 or H12 or with vehicle-control (DMSO). Slight to moderate induction of caspase 3/ 7 activation was observed for the two cell types (2-fold for hOB, 3 to 4-fold for Saos-2). Cell lysis was significantly increased in hOB (2-to 3-fold). Bars show means and SD of duplicates of one representative experiment of two. Fc = fold change relative to DMSO-treated cells, Ã p < 0.05, ÃÃ p < 0.01, ns = not significant p > 0.05. Table. Cell viability of various cell lines from primary and counter screen treated with the 29 short-listed compounds. Viability (%) is shown for the indicated cell lines after treatment with the compounds named after their hit-picking well-ID. The table also shows the chemical formula and the molecular weight for each of the 29 compounds. (XLSX) S1 Text. Additional materials and methods. (DOCX) support. Financial support to HKP from Helmholtz Zentrum München is gratefully acknowledged. | 2018-04-03T01:10:13.522Z | 2015-06-03T00:00:00.000 | {
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119008971 | pes2o/s2orc | v3-fos-license | Efficient scheme for quantum entanglement, quantum information transfer, and quantum gate with three-level SQUID qubits in cavity QED
A novel scheme is proposed for realizing quantum entanglement, quantum information transfer and a set of universal quantum gates with superconducting-quantum-interference-device (SQUID) qubits in cavity QED. In the scheme, the two logical states of a qubit are the two lowest levels of the SQUID. An intermediate level of the SQUID is utilized to facilitate coherent control and manipulation of quantum states of the qubits. The method presented here does not create finite intermediate-level population or cavity-photon population during the operations. Thus, decoherence due to spontaneous decay from the intermediate levels is minimized and the requirement on the quality factor of the cavity is greatly loosened.
A novel scheme is proposed for realizing quantum entanglement, quantum information transfer and a set of universal quantum gates with superconducting-quantum-interferencedevice (SQUID) qubits in cavity QED. In the scheme, the two logical states of a qubit are the two lowest levels of the SQUID. An intermediate level of the SQUID is utilized to facilitate coherent control and manipulation of quantum states of the qubits. The method presented here does not create finite intermediate-level population or cavity-photon population during the operations. Thus, decoherence due to spontaneous decay from the intermediate levels is minimized and the requirement on the quality factor of the cavity is greatly loosened. Cavity QED has been extensively studied to implement quantum information processing (QIP) with a variety of physical systems such as atoms, ions, quantum dots and Josephson junctions [1][2][3][4][5][6]. A well-known reason for this is that compared with those non-cavity proposals where significant overhead is needed for coupling distant qubits, the cavity-based schemes is preferable since the cavity mode acts as a "bus" that can mediate long-range fast interaction between any qubits, which enables one to perform two-qubit gates involving any desired pair of qubits.
Recently, a scheme has been proposed for obtaining a complete set of universal quantum gates, quantum information transfer, and entanglement with superconductor quantum interference devices (SQUIDs) in cavity QED [7]. Technically speaking, the SQUID-cavity QED scheme may be among the most promising candidates for demonstrating QIP because placing SQUIDs at desired positions is straightforward in a cavity and superconducting qubits have been demonstrated to have relatively long decoherence time [8][9][10]. In Ref. [7], the gates were performed by inducing transitions to the intermediate level |a [see Fig. 1(a)] via microwave pulse and cavity field. However, though the cavity mode is not populated during the operation, the population of the SQUIDs in the intermediate levels is non-zero. Thus, the operation must be done within a much shorter time than the energy relaxation time of the intermediate level to maintain coherence. Another key point is that the operation in [7] requires rapid adjustments of level spacings of SQUIDs, which might be undesirable in experiment.
In this letter, we propose a significantly improved approach to achieve entanglement, information transfer and universal gates with three-level Λ-type SQUID qubits in cavity QED. The new method has three major advantages: (a) during the gate operations, the intermediate level is unpopulated and thus decoherence induced by spontaneous emission from the intermediate level, is greatly suppressed; (b) no transfer of quantum information between the SQUIDs and the cavity is required, i.e., the cavity field is only virtually excited and thus the requirement on the quality factor of the cavity is relaxed; (c) there is no need to adjust the level spacings during the operation.
Let us first introduce the Hamiltonian of a SQUID qubit coupled to a single-mode cavity field and a classical microwave pulse with B µw (r, t) = B µw (r) cos ω µw t.
Here, B µw (r) is the amplitude of the magnetic component and ω µw is the carrier frequency. The qubits considered in this letter are rf SQUIDs each consisting of a Josephson tunnel junction in a superconducting loop (typical size of an rf SQUID is on the order of 10 µm−100 µm). The Hamiltonian of an rf SQUID (with junction capacitance C and loop inductance L) can be written in the usual form where Φ, the magnetic flux threading the ring, and Q, the total charge on the capacitor, are the conjugate variables of the system (with the commutation relation [Φ, Q] = ih), Φ x is the static (or quasistatic) external flux applied to the ring, and E J ≡ I c Φ 0 /2π is the maximum Josephson coupling energy (I c is the critical current of the junction and Φ 0 = h/2e is the flux quantum). The quantized Hamiltonian of the cavity mode is given by H c =hω c (c + c + 1/2) , where c + and c are the photon creation and annihilation operators; and ω c is the frequency of the cavity mode.
Consider a Λ-type configuration formed by the two lowest levels and an excited level of the SQUID, denoted by |0 , |1 and |a with energy eigenvalues E 0 , E 1 , and E a , respectively [ Fig. 1(a)]. For the sake of concreteness, we choose the following device and control parameters: C = 90 fF, L = 100 pH, I c = 3.75 µA, Φ x = 0.4995 Φ 0 for the SQUID qubit in the rest of this letter. Suppose that the coupling of |0 , |1 and |a with the other levels via the cavity mode and the microwave is negligible (e.g., by adjusting cavity size, microwave frequency, or level spacings of the SQUID). Under this assumption, it is easy to find that when the cavity mode is coupled to the |0 ↔ |a transition but far-off resonant with the |0 ↔ |1 and |1 ↔ |a transitions, and when the microwave pulse is coupled to the |1 ↔ |a transition while far-off resonant with the |0 ↔ |1 and |0 ↔ |a transitions, the Hamiltonian of the system can be written as: where g is the coupling constant between the cavity mode and the |0 ↔ |a transition; Ω is the Rabi-flopping frequency corresponding to the |1 ↔ |a transition; and σ ij = |i j| (i, j = 0, 1, a). The expressions of g and Ω are given by [7] where S is any surface that is bounded by the SQUID ring, r is the position vector on S, and B c (r) is the magnetic component of the normal mode of the cavity. Consider a situation in which the cavity mode is largely detuned from the |0 ↔ |a transition , i.e., ∆ c = ω a0 − ω c ≫ g, and the microwave pulse is largely detuned from the |1 ↔ |a transition, i.e., . Under this condition, the intermediate level |a can be adiabatically eliminated [11,12]. Thus, the effective Hamiltonian in the interaction picture becomes [11,12] where σ 01 = |0 1| , σ + 01 = |1 0| , δ = ∆ c − ∆ µw , and g ef f = Ωg 2 ( 1 ∆c + 1 ∆µw ). The first two terms are ac-Stark shifts of the levels |0 and |1 induced by the cavity mode and the microwave pulse, respectively. The last two terms are the familiar Jaynes-Cummings interaction, describing the Raman coupling of the two lowest levels of the SQUID.
Effective Hamiltonian for two SQUID qubits in cavity. To simplify presentation, let us consider two identical SQUIDs I and II ( the method is also applicable to nonidentical SQUIDs). The two SQUIDs are coupled to the same single-mode microwave cavity and each driven by a classical microwave pulse B i µw (r, t) = B i µw (r) cos ω µw t (i = I, II) [ Fig. 1(c)]. The separation of the two SQUIDs is assumed to be much larger than the linear dimension of each SQUID ring in such a way that direct interaction between the two SQUIDs is negligible. Also, suppose that the coupling of each SQUID to the cavity mode is the same (this can be readily obtained by setting the two SQUIDs on two locations r 1 and r 2 where the cavityfield magnetic components B c (r 1 , t) and B c (r 2 , t) are the same). In this case, it is obvious that based on Eq.
(3), the Hamiltonian for the system in the interaction picture can be written as Under the condition that δ ≫ g 2 ∆c , Ω 2 ∆µw , g ef f , there is no exchange of energy between the SQUIDs and the cavity mode. The effective Hamiltonian is then given by [13][14][15][16] where the third and fourth terms describe the photonnumber dependent Stark shifts induced by the offresonant Raman coupling, and the last two terms describe the "dipole" coupling between the two SQUIDs mediated by the cavity mode and the classical fields. The parameter γ = g 2 ef f /δ characterizes the strength of Stark shift and inter-qubit coupling. If the cavity is initially in the vacuum state, then the effective Hamiltonian reduces to Note that the Hamiltonian (6) does not contain the operators of the cavity mode. Thus, only the state of the SQUID system undergoes an evolution under the Hamiltonian (6), i.e., no quantum information transfer occurs between the SQUIDs and the cavity mode. Therefore, the cavity mode is virtually excited. The state |0 I |0 II is unaffected under the Hamiltonian (6). From (6), one can easily get the following state evolution where γ ′ = γ − Ω 2 ∆µw . In the following, we show that Eq. (7) can be used to create entanglement, to implement quantum information transfer, and to perform quantum gates.
Generation of entanglement. The two logical states of each SQUID qubit are represented by the two lowest energy states |0 and |1 . From (7), one can see that if the two SQUID qubits are initially in the states |0 I and |1 II , they will evolve to the following maximally entangled state after an interaction time π/(4γ) where the common phase factor e −iχπ/4 (χ = γ ′ /γ) has been omitted. Quantum information transfer. Suppose that the SQUID qubit I is the original carrier of quantum information, which is in an arbitrary state α |0 + β |1 . The quantum state transfer from the qubit I to the qubit II initially in the state |0 is described by (9) which can be realized in the following two steps.
Step (i): Apply two microwave pulses to the two SQUIDs I and II, respectively, so that the states of the two SQUIDs undergo an evolution under the Hamiltonian (6) for an interaction time π/(2γ).
The states after each step of the above operations are listed below: It is clear that the two-step operation transfers quantum information from the SQUID qubit I to the SQUID qubit II.
Single SQUID qubit operations can be achieved via various schemes [7,17,18]. In Ref. [17], it has been shown that by applying two microwave pulses to induce twophoton Raman resonant transition between the qubit levels, any single-SQUID-qubit logic operation can be realized, without real excitation of the intermediate level. It is noted that during the present single-qubit operation inside a cavity, the cavity mode can be decoupled from the qubits without adjusting the qubits' level spacings. The reason for this is that one can choose the frequencies of the applied microwave pulses so that two-photon Raman resonant transition between the qubit levels |0 and |1 is satisfied, while the cavity mode is highly detuned from either pulse [see Fig. 1 (b)].
Quantum logical gates. A non-trivial and universal two-qubit controlled NOT (CNOT) can be realized by combining the Hamiltonian (6) with single-qubit operations. We find that the CNOT gate |i I |j II → |i I |i ⊕ j II (i, j ∈ {0, 1}) acting on the two SQUID qubits I and II can be achieved through the following unitary transformations where the common phase factor e −iχπ/4 is omitted, U I,II is a two-SQUID-qubit joint unitary operation defined by U I,II (γt) = exp[− ī h H ef f t] with γt = π/4, σ y is the Pauli operator, S results in a single-qubit phase-shift |0 → e −iχπ/8 |0 while |1 → e iχπ/8 |1 , in the single-qubit Hilbert subspace formed by |0 = (0, 1) T and |1 = (1, 0) T . It is well known that at least three CNOT gates are needed [7] to construct a two-qubit SWAP gate. Note that information transfer (9) is equivalent to a transformation |i I |0 II → |0 I |i II (i ∈ {0, 1}). Thus, to simplify the gate operation, a two-SQUID-qubit SWAP |i I |j II → |j I |i II (i, j ∈ {0, 1}) can be realized through the following procedure: It is necessary to give a discussion on the effect of the Stark shift. From (6), one can see that after omitting the Stark-shift terms, the Hamiltonian (6) reduces to H ′ ef f =hγ[σ + 01I σ 01II + σ 01I σ + 01II ]. For an arbitrary twoqubit state |ψ = α 0 |00 + α 1 |01 + α 2 |10 + α 3 |11 , the probability of the gate error caused by discarding the Stark shift is given by where F = ψ| U −1 U ′ |ψ 2 is the fidelity of the operation described by U ′ = exp(−iH ′ ef f t/h), in contrast to the operation described by a real unitary operator U = exp(−iH ef f t/h). The equation (13) shows that for the special case of γ ′ t = 2nπ or α 0 = α 3 = 0, i.e., |ψ = α 1 |01 + α 2 |10 , the gate error is zero. However, it is noted that in general, one has P e = 0, i.e., the gate operation will be affected by the Stark shift. In particular, for α 0 = α 3 = 1 √ 2 , one has P e = sin 2 (γ ′ t), which is unity for γ ′ t = (n + 1/2)π.
Finally, we show that parameters necessary for the experimental realization of the proposed schemes are achievable. For the SQUIDs with the parameters given above and with junction's damping resistance R > 1 GΩ [19], the level |a 's energy relaxation time T 1 ≃ R 60MΩ · µs would be ∼ 15 µs. The transition frequency is ω a0 /(2π) ≃ 30 GHz. Hence, we choose ω c /(2π) = 29.7 GHz as the cavity-mode frequency. For a 10 × 1 × 1 mm 3 cavity and a SQUID with a 50 × 50 µm 2 loop, a simple calculation shows that the coupling constant is g ≃ 1.8 × 10 8 s −1 , i.e., about 0.1∆ c . By choosing the frequency and amplitude of the microwave pulse appropriately such that ∆ µw = 10Ω and g = 1.2Ω for each SQUID, we have δ ≃ 10g ef f ≃ 3.0 × 10 8 s −1 . Then the typical time needed for the SQUID-cavity interaction is on the order of T s−c = πδ/(2g 2 ef f ) ≃ 0.5 µs, which is much shorter than the level |a 's effective decay time T 1 /P a ≥ 1.5 × 10 3 µs for T 1 = 15 µs, where P a ≤ 0.01 is the occupational probability of the level |a for the present case of ∆ c = 10g and ∆ µw = 10Ω. The photon lifetime is given by T c = Q c /ω c where Q c is the quality factor of the cavity. In the present case, the cavity has a probability P c ≃ 0.01 of being excited during the operation. Thus, the effective decay time of the cavity is T c /P c ≃ 10 µs ≫ T s−c for Q c ≃ 2 × 10 4 , which is realizable as demonstrated by recent experiments [20].
Note that the method described above does not require two SQUIDs with identical parameters. In the case of non-identical SQUIDs I and II, one has δ I = ω I a0 − ω I a1 − ω c +ω I µw and δ II = ω II a0 −ω II a1 −ω c +ω II µw , which can always be set to equal by adjusting the frequencies, ω I µw and ω II µw , of the two microwave pulses applied to the SQUIDs. The present scheme has the following advantages: (i) During the operation, the intermediate level is unpopulated and thus gate errors caused by energy relaxation is greatly suppressed. (ii) The cavity field is virtually excited and thus the required quality factor of the cavity is greatly loosened. (iii). No tunneling between the qubit levels |0 and |1 is needed and thus the rate of spontaneous decay from the level |1 can be made negligibly small, by the use of higher potential barrier between the two qubit levels. (iv) No adjustment of level spacings is needed during logic operations, since the qubit-qubit interaction required for the two-qubit operations is via the cooperative actions of the cavity mode and the microwave pulses. (v) The method can be extended to perform QIP on many SQUID qubits in a cavity, because the cavity mode can mediate long-range coherent interaction between SQUID qubits. Also, the proposal can be applied to any other type of solid state qubits which have a Λ-type three-level configuration.
In summary, we have explicitly shown how quantum entanglement, quantum information transfer, and universal quantum gates can be realized with SQUID qubits in cavity. We stress that in our analysis, all Stark shift terms, which may significantly affect the gate fidelity, are included. In addition, we have shown that the method is feasible with experimentally demonstrated qubit and cavity parameters. Thus, it provides a realistic approach for robust quantum information processing with superconducting qubits, and we hope that this work will stimulate further theoretical and experimental activities in this emerging research field. CPY is very grateful to Prof. Shi-Biao Zheng for many fruitful discussions and very useful comments. This research was partially supported by National Science Foundation (EIA-0082499), and AFOSR (F49620-01-1-0439), funded under the Department of Defense University Research Initiative on Nanotechnology (DURINT) Program and by the ARDA. 1. (a) The potential and level diagram of an rf SQUID with a Λ-type three levels |0 , |1 and |a . The cavity field is detuned from the classical microwave pulse by δ = ∆ c − ∆ µw . (b) Illustration of single-qubit operation. The two microwave pulses with frequencies ω 0 and ω 1 are applied to induce two-photon Raman resonant transition between the qubit levels |0 and |1 with ω 10 = ω 1 − ω 0 , for the purpose of single-qubit logic operation. (c) Schematic illustration of two SQUIDs (I, II) coupled to a single-mode cavity field and manipulated by microwave pulses. The two SQUIDs are placed along the cavity axis (the Z axis) and in the X-Z plane. B c , B I µw and B II µw are in Y direction. | 2019-04-14T03:19:17.529Z | 2003-05-22T00:00:00.000 | {
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42556361 | pes2o/s2orc | v3-fos-license | The prognostic role of nutrition risk score (NRS) in patients with metastatic or recurrent esophageal squamous cell carcinoma (ESCC)
The purpose of this study was to elucidate the prognostic value of nutritional risk score (NRS) in patients with metastatic or recurrent ESCC. A total of 187 patients who undergoing S1 based or paclitaxel based salvage chemotherapy were enrolled in this retrospective study. Nutritional status was evaluated by NRS. The relationship between NRS and clinicopathological variables and post-treatment outcomes were assessed by univariate and multivariate analysis. NRS was significantly associated with weight loss (P<0.001), BMI (P<0.001), chemotherapy regimens (P=0.038) and treatment response (P=0.013). The Kaplan-Meier survival curves indicated that patients with NRS ≥ 3 had worse overall survival (OS) compared to patients with NRS < 3 (P<0.001). Multivariable regression revealed that weight loss, NRS and treatment response were three prognostic factors (P<0.05). These results suggest that NRS is a promising indicator of poor prognosis in patients with metastatic or recurrent ESCC who received S1 based or paclitaxel based salvage chemotherapy.
INTRODUCTION
Esophageal cancer remains the eighth most commonly diagnosed cancer worldwide. Although the five-year overall survival rate for patients with localized disease approaches 85% [1], the survival rate for metastatic or recurrent disease is only 5% [2]. Most patients are died of nutritional problem that leading to metabolic and physiological changes.
Esophageal squamous cell carcinoma (ESCC) is considered to be the predominant type in eastern Asia countries, with increased incidence recently [3]. Patients are often presented with obstructive symptoms, such as dysphagia and unintended weight loss. Furthermore, the psychological influence of cancer diagnosis and treatment can cause low mood or depression, which also reduce patients' appetite [4]. Most patients could be affected by malnutrition during diagnosis, cancer treatment and follow-up. A consensus exists that weight loss is one of the most important criteria for malnutrition. Weight loss, poor life-style associated factor, is a common symptom in 60% of patients before diagnosis [5]. Significant weight loss resulted in systemic inflammation [6], higher rate of treatment complications and lower quality of life. Many studies reported that weight loss is associated with cancer recurrence in gastric cancer [7] and breast cancer [8]. However, weight loss alone does not identify the full effect of malnutrition on physical function [9] and is not a perfect prognostic factor [10]. Nutrition risk score (NRS) is a novel method for distinguishing high risk patients who will suffer from malnutrition and related with survival in gastric cancer [11]. Therefore, we were interested in that if this new method can be used in metastatic or recurrent ESCC patients who received palliative chemotherapy.
The aim of this study was investigate the association between NRS and other nutrition variables and the prognostic value of NRS in patients with metastatic or recurrent ESCC.
Patients' characteristics
A total of 187 patients with metastatic or recurrent ESCC were analyzed. Baseline characteristics were presented in Table 1. Most patients were male (61.5%) with the median age of 61 years (range: 38-78 years). Most patients (66.8%, 125/187) had metastatic disease and 13 patients (7.0%) both had metastatic and recurrent disease. More than half of the patients (62.6%, 117/187) had experienced subtotal transthoracic esophagectomy and regional lymphadenectomy with curative intent, and other 70 patients (37.4%) had experienced radical radiotherapy. Most patients (57.8%) received S1 based chemotherapy and the remaining patients (41.2%) received paclitaxel based chemotherapy. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee. This study was approved by the institutional review board of the hospital. Informed consent was obtained from all patients included in the study.
Relationship between NRS and clinical features
According to NRS, 52.4% (n=98) had a score ≥3. The median NRS for all patients was 3. NRS was significantly associated with weight loss (P<0.001), BMI (P<0.001), chemotherapy regimens (P=0.038) and treatment response (P=0.013), whereas there was no significant association between gender (P=0.495), age (P=0.722), first line treatment (P=0.924) and tumor location (P=0.722). Correlation of NRS with BMI, weight loss, serum albumin level, hemoglobin and tumor markers are presented in Table 2. Spearman's correlation revealed that NRS had a positive correlation with weight loss (P<0.001) and serum albumin level (P<0.001).
Association of NRS with survival
After a median follow-up duration of 23 months, the estimate 1-year and 2-year overall survival rates in all patients were 42.3% and 9.4%, respectively. One hundred and sixty-four patients died due to tumor progression or malnutrition. Weight loss, treatment response and NRS were significantly related with OS ( Table 3). The Kaplan-Meier survival curves indicated that patients with NRS ≥ 3 had worse OS compared to patients with NRS < 3 (P<0.001, Figure 1). In further analyses, NRS ≥ 3 was significantly associated with shorter OS for patients who received S1 based chemotherapy (P=0.005, Figure 2A) and patients who received paclitaxel based chemotherapy (P<0.001, Figure 2B). One-year survival probability was 28.7% for NRS ≥ 3 patients versus 54.2% for NRS < 3 patients with S1 based chemotherapy, and 23.9% for NRS ≥ 3 patients versus 60.0% for NRS < 3 patients with paclitaxel based chemotherapy. Similarly, NRS ≥ 3 was significantly associated with shorter OS for female patients (P<0.001, Figure 3A) and male patients (P=0.016, Figure 3B).
Then we performed multivariable regression using a Cox proportional hazards models revealed that weight loss, NRS and treatment response were three prognostic factors in patients with metastatic or recurrent ESCC (P<0.05). Patients with NRS ≥ 3 had an elevated risk of death compared to those with NRS < 3. The hazard ratio was 2.14 (95% confidence interval [CI] 1.25-3.68) for death (Table 4).
NRS in the validation cohort
When the cutoff value of 3 was used in the validation cohort, 68 (51.5%) of the ESCC patients were observed to have NRS ≥ 3. The age distribution, tumor location, treatment response, and NRS were well balanced between the two patients' cohorts (P>0.05). The patients in the validation cohort with NRS ≥ 3 exhibited decreased OS (P=0.016) compared with the patients who had NRS < 3 ( Figure 4). The multivariate COX regression analysis showed that NRS ≥ 3 and treatment response were independent predictors (Table 4).
DISCUSSION
In this present study, we investigated NRS in metastatic or recurrent ESCC and its correlation with prognosis. Our data demonstrated that NRS was significantly associated with weight loss and serum albumin level. NRS was an independent prognostic factor related with overall survival in metastatic or recurrent ESCC. As predicted, the OS was better for patients with NRS < 3 than for patients with NRS ≥ 3.
Currently, the TNM staging system is a standard method to predict patients' prognosis. However, clinicians need an accurate tool to manage the treatment and predict survival when patients developed distant metastasis or local-regional recurrence. Tumor markers, such as CEA, SCC and CYFRA2-11, are not precisely associated with outcome in metastatic or recurrent ESCC patients [12]. In our study, tumor markers (CEA, CA125, CA199 and CA724) were not associated with overall survival according to univariate analysis (P>0.05). Several studies reported that the nutritional and immunologic conditions of patients could influence the post-operative complications and outcome with cancer [13][14][15]. NRS, calculated based on patients' weight loss and BMI, is a significant predictor for malnutrition according to Cox et al' report [16]. The nutritional status was also shown recently to be an indicator for complications in patients with esophageal cancer [17,18]. Early nutritional support could significantly reduce severe complications which related with high morbidity, such as pulmonary complications and anastomotic leakage [19]. Although www.impactjournals.com/oncotarget Another important result of this study was that in patients with metastatic or recurrent ESCC, salvage chemotherapy with paclitaxel-based regimens could slightly improve overall survival (P=0.071). In Yang et al' report, chemotherapy with paclitaxel-based regimens demonstrated higher efficacy with less toxicity in patients with ESCC compared with the fluorouracil based regimens [20]. The median PFS in patients received paclitaxelbased regimens were significantly longer than in patients received S1-based regimens (13.0 m Vs 6.5 m, P=0.034). In our study, the estimate 2-year OS rates were 16.7% and 5.9% in patients received paclitaxel-based regimens and S1-based regimens. It seems in salvage chemotherapy using paclitaxel-based regimens was a promising treatment in patients with metastatic or recurrent ESCC. However, further large scale randomized clinical trials are needed to confirm this result.
There are several limitations of this study. The major limitation is that the information of post-treatment local recurrence or metastasis was insufficient. One of the least convincing things in this study are lack the data of disease free survival, although overall survival is the standard indicator in the cancer prognosis study. Another limitation is the number of study samples was relatively small. Also, some nutritional parameters, such as TSF (triceps skin fold), MAC (mid-arm muscle circumference), HGS (handgrip strength) and MAMA (mid-arm muscle area), were insufficient. Therefore, we cannot compare NRS with other nutritional scores, like sPG-SGA [13].
The results of this study suggest that NRS are strongly related with BMI, weight loss and treatment response in patients with metastatic or recurrent ESCC. Additionally, NRS can be used as a possible marker to predict overall survival. Patients with NRS ≥ 3 had an
Patients
This study in patients with metastatic or recurrent ESCC was conducted at Zhejiang cancer hospital, Hangzhou, China. A total of 187 patients were enrolled in this study. Only patients with histologically confirmed diagnosis of ESCC were included. Patients with the following characteristics were excluded from our study: patients showed a severe functional impairment of vital organs who cannot tolerate chemotherapy; those whose expectancy life less than 3 months. The nutritional status evaluation was performed by two independent investigator at the first outpatient visit after verified metastatic or recurrent ESCC diagnosis. Clinical details such as gender, age, tumor histopathology, tumor site and TNM stage were collected from hospital records. This study was approved by the institutional review board of the hospital. All patients provided informed consent before treatment.
We then used the validation cohort to test the result in predicting prognosis in patients with metastatic or recurrent ESCC. The validation cohort data were collected in the same hospital of patients who received salvage chemotherapy between October 2016 and March 2017. Finally, we identified 132 cases in the validation cohort that fit the inclusion criteria.
Nutritional assessment
Nutritional assessment was evaluated by nutrition risk score (NRS) [11,13,21]. NRS consisted of the combination of weight loss, body mass index (BMI), age and severity of disease. The final score ranges from 0-7. According to the previous report, NRS ≥3 was considered high nutritional risk [13]. On admission to our department, the patients' height and weight were documented and detail information about weight loss was obtained using a structured questionnaire. BMI was calculated using the well-known formula. Weight loss was defined as exceeding five percent of habitual body weight in the preceding three months or ten percent in the preceding six months. Blood samples were taken from each patient routinely before treatment to measure serum albumin, hemoglobin and tumor markers (CEA, CA125 and CA199).
Treatment
Chemotherapy consisted of four to six cycles of S1 based chemotherapy or paclitaxel based chemotherapy according to patients' performance status score and their preference. If patient's PS score=1 or 2, S1 based chemotherapy was recommended, otherwise paclitaxel based chemotherapy was recommended. Chemotherapy was stopped when there was unacceptable toxicities and disease progression. Best support care (BSC) was given to all patients, including pain management, nutritional treatment, esophageal dilation or stent placement and blood product transfusions.
Assessment and follow-up
Tumor assessment was performed 6 weeks after treatment or earlier in cases of clinical suspicion of progression. The objective response to treatment was defined using the Response Evaluation Criteria in Solid Tumors (RECIST 1.1) [22]. For this analysis, patients with complete response (CR) or partial response (PR) were classified as responders, and those with stable disease (SD) or progressive disease (PD) were defined as nonresponders.
All patients received standardized follow-up at a 2-month interval for the first 2 years after operation, a 6-month interval in the third year and yearly thereafter. Evaluation comprised physical examination, complete blood count, chest computed tomography, esophagogram and abdominal ultrasound.
Statistical analysis
Overall survival (OS) was defined as the time interval from the initial event (diagnosis) to the death or censoring. The chi-square test was performed to evaluate the association between the clinicopathological variables and NRS. The correlations between NRS with BMI, weight loss, pretreatment albumin, pretreatment hemoglobin, pretreatment red blood cell count and tumor markers (CEA, CA125, CA199 and CA724) were estimated by linear correlation analysis. Survival curves were estimated by the univariate Kaplan-Meier method. The log-rank test was applied to check the significant differences in the curves among groups. Furthermore, we used the Cox proportional hazards model for multivariate analysis. All statistical calculations were performed with SPSS 21.0 for Windows (Chicago, IL). Two-sides P values of < 0.05 were considered statistical significance. | 2018-04-03T06:25:50.247Z | 2017-08-24T00:00:00.000 | {
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110757219 | pes2o/s2orc | v3-fos-license | The Required Competencies of ParaProfessionals in Library Services of Sarawak State Libraries
Para-professionals in the library services are library associates, library technicians, or library assistants who assist the professional librarians in library-related task. Mining the literature unveiled that studies investigating the required competencies on para-professional are still very limited. To address this gap, this paper reports a study that was conducted with the purpose to identify the required competencies of para-professionals in the library services. Adopting the survey research methodology, data were collected using questionnaires from 502 para-professional librarians working with the Sarawak State Library in Sarawak, Malaysia. The findings suggest that all the seven sets of competencies, namely general competencies; Agency and Organizational Knowledge; Reference and Research; Collection Management; Content Organization and Structure and Library Technology Management are either moderately or highly required by the para-professionals. Based on these findings, the appropriate training needs could be identified.
Introduction
The importance of mastering a specific set of competencies for any given jobs has been well documented in the literature.It is because of this reason that researchers have developed various competencies models and framework for various kinds of professions (e.g.Vakola, Soderguist & Prastacos, 2007;Schroeter, 2008;Yaszay et al, 2009).According to Griffiths & King (1985), competency is the generic knowledge, skills and attitude of a person related to affective behavior as showed through performance.
Within the library and information management field, various studies have investigated the required competencies of the professional librarians (e.g.Abriza, 1998;Zaiton et al., 2004).Providentially, the findings of these studies have helped to further deepen our understanding of the topic.However, upon further scrutiny, most of these studies have focused on professional librarians.Very few studies have attempted to explore the required competencies among para-professionals of the library services.Questions on whether their required competencies are comparable to professional librarians seem to be unanswered.To this effect, this paper reports the findings of a study which was conducted with a view to identifying the required competencies among paraprofessionals in the library services.
Para-Professional in Library Services
Para-professionals in the library services are library associates, library technicians, or library assistants who assist the professional librarians in library-related task.The duties performed by them include database management, cataloguing, ready reference and serials and monograph processing.In terms of qualifications, the para-professionals of the library services usually hold college diplomas but not library-related degrees.Within the context of the Malaysian government public services (inclusive of the Malaysian State Government), para-professionals in the public libraries are those staffs holding the post of "Assistant Library Officer"; "Senior Assistant Library Officer"; "Library Attendant" and "General and "General Assistant".
Competencies
Competencies have been defined as the interplay of knowledge, understanding, skills and attitudes required to do a job effectively from the point of view of both the performer and the observer (Murphy, 1991).
The Federal Library and Information Centre Committee Library of Congress or FLICC (2008) defines competencies as the knowledge, skills and abilities that define and contribute to performance in a particular profession.Knowledge refers to having information about; knowing or understanding something; skill is the ability to apply knowledge effectively and attitude refers to the individual's mental or emotional approach to something.
One can distinguish between professional competencies which are generic skills, attitudes and values.Helmick and Jaguszewski (2006) suggest that in personal career development terms, competencies can also be thought of as flexible knowledge and skills that allow the librarian to function in a variety of environments and to produce a continuum of value-added, customized information services that cannot be easily duplicated by others.At a time when professionals in all fields are being encouraged to invest in themselves and to prepare for employment as independent contractors, it is critical that librarians define their competencies and that they continue to improve the range of professional and personal competencies that will form the basis for their future careers.
Standards of Librarianship
The importance of competencies standard has been well advocated in the literature.For instance, Eells & Jaguszewski (2008) and Kenny (2008) identified the importance of these competencies standards as (i) creating a common bond of understanding and a common language for defining professional standards, (ii) competencies are the foundation for competency-based management and continuous process improvement ensuring that librarians have the knowledge, skills and abilities to accomplish mission requirements, (iii) they can be used as tools to design and develop training and educational programs, position descriptions and performance evaluation instruments and for alignment with strategic objectives.
Within the library profession, currently, there are two professional competencies standards namely the ALA's Core Competencies of Librarianship (American Library Association, 2009) and the Federal Librarian Competencies (Library of Congress, 2008).Between the two, the later is considered more comprehensive compared to the later as it is more comparable to the various competencies model, such as Management Competency Model; Holistic Competency Model and Professional Competency Model (Lester, 1995;Cheetham & Chivers, 1996;Vitala, 2005).Table 1 presents the domain and sub-domain of the Federal Librarian Competencies.
Library Technology Management
Technology Library and Content Management Systems Information Assurance and Security Specialized Subject Knowledge
Studies on Required Competencies
Mining the literature unveiled that various studies have been carried out to investigate the training needs of the professionals and para-professionals in the library services.These studies are: Cronin (1983);Goulding et.al. (1999); Musher (2001); Braun (2002);Soo (2005) and Ashcroft (2004).These studies have employed various methodologies, some of which were surveys, interviews and focus groups discussions.Due to the descriptive nature of the knowledge, skills and abilities of the professionals and para-professionals employed in the libraries, survey questionnaires together with face-to-face interviews were the most frequently employed as seen in studies by Buttlar & Du Mont (1996);Soo (2005); Griffiths (2005); Krissof & Konrad (2005); Lou (2007); Rice-Lively & Racine (2008).One of the aims of the studies was identification of competencies needed by the librarians and information professionals in the new millennium.
In the context of Malaysia, there are three previous studies carried out on competencies of librarians.Abriza's study (1998) examined the role of teacher librarians in secondary schools as perceived by three groups of people directly involved in school librarianshipteacher librarians, library educators and education officers.Her study aimed at determining the essential competencies and the education required for teacher librarians to perform their role.In 2004, the National Library of Malaysia conducted a study to identify the gaps between the present knowledge and skills, and those required for excellent performance by the professionals and para-professionals employed by the National Library.One of the aims of the study was to design training and development plan of those involved.On the other hand, Zaiton et al. (2004) examined the manpower needs of IBIMA Business Review 4 professionals and non-professionals in the libraries of Malaysia.
Research Methodology
The conduct of this study involves the use of survey research methodology.A questionnaire was developed and used to collect the research data.The instrument developed by Federal Library and Information Center Committee of Library of Congress (Library of Congress, 2008) were adopted and adapted for developing the questionnaires.In addition to that, several other measurements developed by previous studies, such as Buttlar & Du Mont (1996); Abriza (1998); Zaiton et al. (2004);Soo (2005); Griffiths (2005); Krissof & Konrad (2005); Lou (2007) and Rice-Lively & Racine (2008) were also referred to.The developed questionnaire used in the study measured seven aspects of competencies as shown in Table 2. Likert scale was used for all questions measuring competencies.The anchoring used was NR for "Not Relevant"; 1 for "Weak", 2 for "Fair" and 3 for "Good".Prior to actual data collections, the questionnaires were pre-tested and pilot tested with 30 library personnel at PUSTAKA Negeri Sarawak.Several questionnaires were also sent to library personnel at the Perbadanan Perpustakaan Awam Negeri Selangor.In the pilot study exercise, the participants were requested to evaluate and appraise the questionnaires in terms of content accuracy, clarity, length, and overall presentation.They were also encouraged to comment and criticize constructively.However, prior to the evaluation, all participants were briefed on the research questions, research objectives, scope and context of the study.Based on the feedback obtained from the pilot study, the researchers modified the questionnaire accordingly.The questionnaire was sent to every library personnel across all public libraries in Sarawak.However, out of 552 questionnaires that were sent, 507 were returned but only 502 were found useful.The remaining were either unreturned or unusable i.e. incomplete.
Demographic
Table 3 shows the demographic profiles of respondents who participated in the study.Out of 502 respondents, 100 or 19.9% of the respondent were male, while the females were 402 or 80.1%.In terms of their age, the age group between 36 and 40 were the highest respondents which stood at 22.5%; followed by age group between 41 and 45 (20.3%) and age group between 31 and 35 (17.1%).The least number of respondents came from the age group above 55 (2.0%).With regard to their designation, 50.8% reported to hold the "General Assistant" post, while 46.4% indicated as holding the "Library Assistant" post.The remaining 2.8% responded as holding the "Assistant Library Officer" post.In this study, three categories of job status were reported by respondents, namely permanent, contract and part-time.Evidently, the majority of the respondents indicated that they were permanent staff (54.6%) while 28.9% and 16.5% indicated that they were part-time and contract respectively.Finally, in terms of their length of service, the highest number of respondents had been working for between 6 and 10 years (22.5%).The least number of respondents indicated to have been working for more than 30 years (4.6%).2% respondents indicated that the required general competencies was high while 9.9% indicated that their level of general competencies was low.About 5.8% of the respondents responded that the listed general competencies were irrelevant to their current job.In terms of most required competency, the highest responses was 'teamwork and collaboration' (59.4%), followed by 'interpersonal skills' and 'ethical framework' which scored 52.4% and 50.4% respectively.In terms of the lowest required competency, the highest responses were 'mathematical reasoning' (20.5%), followed by 'external awareness' (14.7%) and 'foundational knowledge' (13.9%).With regard to competencies that are not relevant to their current job post, the three top scoring were 'external awareness' (14.7%), 'mathematical reasoning (13.9%) and 'leadership' (12.7%).Table 6 showcases the required level of library leadership and advocacy competencies profiles of the respondents measured in terms of percentage of responses.Top three highest responses for the most required competencies were 'understanding of principles and practices of team building and team work' (46.4%), 'ability to deliver library program(s) and service(s)' (44.8%) and 'the ability to understand the library's vision' (42.4%).In terms of moderate mediocre required competency level, the top three highest responses were 'ability to understand the principles of library facilities planning and space management' which recorded 51.8%, followed by 'ability to communicate and implement the library's vision in daily practices' which stood at 46.0% and 'ability to apply the results of customer surveys or studies in daily practices' which recorded 45.8%.In terms of the lowest required competencies, the top three responses were 'ability to understand the library's Continuity of Operations Plan (COOP) and risk management program' (31.9%), 'knowledge of the role of alliances and collaborative relationships in program development and outreach' (30.7%) and 'knowledge and practices of outreach to existing and potential clienteles' (27.9%).The top three highest responses for the not needed competencies were 'knowledge of and ability to use staff training programs' (29.7%), 'ability to understand and use library licenses and other agreements' (25.7%) and 'knowledge of principles and practices of human resources management and labor relations in a diverse workforce' (25.5%).Overall, the majority of respondents indicated that the required competency for library leadership and advocacy aspects was mediocre (36.8%).In contrast 15.0%, 18.8% and 29.4% responded that the competencies were not needed, low and high respectively.Based on the findings, it was discovered that majority of the respondents indicated that the required competency for the research and reference aspect was moderate (32.0%), while 23.4% indicated that the required skill was low and 23.6% was high.The remainder (21.1%) indicated that the competencies were not required with the current job.The top three highest responses of most required competencies were 'ability to use the library's existing reference and research products, services and programs' (30.3%), 'understanding of selection and dissemination tools and technologies for continuous information flow' (27.1%) and 'knowledge of current technologies and information resources' (25.9%).The three highest responses for the moderate level were 'ability to use the library's existing reference and research products, services and programs' (39.4%), 'understanding of and ability to apply search strategies to retrieve information' (37.8%) and 'knowledge of current technologies and information resources' (36.3%).The top three highest recorded least-needed competencies were 'ability to understand and apply the principles of verification and evaluation of Information Resources' (28.7%), 'Knowledge of database structure and organization' (28.3%) and 'understanding of data mining techniques' (26.9%).The top three responses for the not needed competencies were 'understanding of data mining techniques' (31.3%), 'knowledge of reference and research principles and methodologies' (31.1%) and 'knowledge of the principles and practices of bibliographic instruction' (27.3%).Table 8 shows the respondents' required level of collection management competencies measured in terms of percentage of responses.The top three responses for the most required competencies were 'understanding of the concepts and best practices of circulation and access to library resources', 'knowledge of the library's acquisitions policies and procedures', 'knowledge of the concepts, principles and guidelines of library resource sharing' which recorded 32,7%, 29.9% and 28.1% respectively.The top three responses for moderate-required level were 'knowledge of the library's acquisitions policies and procedures, 'knowledge of the concepts, principles and guidelines of library resource sharing',' knowledge of the theory, principles, and standards and practices in the life cycle of IBIMA Business Review 12 library collections' which stood at 35.9%, 35.5% and 35.5% respectively.The top three responses for least required competencies were 'knowledge of theories, trends and practices of conservation, preservation, or archiving of resources' (33.5%), 'knowledge of disaster planning and the library and organization's plan(s)' (30.7%) and 'knowledge of disaster planning and the library and organization's plan(s)' (30.3%).The top three responses for not needed competencies were 'ability to understand and use resource sharing tools' which scored 45.0%, followed by 'knowledge of the licenses or agreements governing access to the library's electronic collections' and 'knowledge of the publishing and information industry in relation to collection assessment and development' which stood at 26.7% and 26.5% respectively.(28.7%), followed by 'knowledge of systems analysis principles and techniques' and 'knowledge of standard performance measures for library technology applications' which measured at 25.9% and 25.5% respectively.About 34.1% respondents also noted that 'knowledge of the process of system certification and acceptance' was not needed in their current job while 32.9% indicated that 'knowledge of authentication protocols and their application' was also not required.The lowest required competencies reported by respondents are the library technology management competencies.These competencies which measure, among others, skills and knowledge on systems analysis; information infrastructure; data standards; library content management systems; library technology tools and practices etc, may not be very relevant and appropriate to some of the respondents as their workplace is located in quite a rural area and furthermore, the facility and the nature of their work does not require them to practice such competencies.Perhaps, due to this reason, the library technology management competencies recorded the lowest scoring among the seven listed competencies.
IBIMA Business Review 6
Other than the general competencies and the library technology management competencies, the scoring of all other competencies is almost identical, which can be considered as moderately needed or required.Given that the environment of the respondents' workplace which is very diverse with some working in the urban areas, others working in rural areas, the nature of the required competencies could be different.Likewise, the type of library that these respondents are serving also could be an influential factor.As for those who work in the rural library where the facility is not as extensive compared to the state library, the need for technologyrelated competencies may not be very demanding.On the other hand, in the state libraries or any other libraries located in the urban part where the libraries are far better equipped with ICT facilities, the need of technology-related competencies could be very demanding.The demographic profiles of the community using the library services could also be an important factor.In a rural library settings, where most users are not well exposed to ICT related services, the need to have human-related competencies would be far higher compared to urban settings.
Conclusion
The conduct of this study has been to investigate the required competencies among para-professionals in the library services in the state of Sarawak, Malaysia.The findings of this study suggest that the competencies needed by the paraprofessionals are almost identical to the competencies of the professional librarians which incorporate general competency; organizational knowledge; library leadership and advocacy; reference and research; collection management; content organization and structure and library technology management.The findings of this study will help the authorities concerned to identify the training needs required by these para-professionals.In addition, academic institutions offering library management programs will also find the findings to be useful when devising their curriculum.Just as in other studies, this study is not without limitations.Firstly, the study used perceptual measures to gauge respondents required competencies.In addition, the questionnaire is also very lengthy which to some extent affect the concentration of the respondents when IBIMA Business Review 16 answering.These limitations can be exploited by future researchers, in case they intent to conduct similar study.
Table 4 : Required Level of General Competencies
Persuades others to accept recommendations, cooperate, or change their behavior; works with others towards an agreement; negotiates to find mutually acceptable solutions. | 2018-11-23T20:19:21.707Z | 2012-09-01T00:00:00.000 | {
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139250600 | pes2o/s2orc | v3-fos-license | Top and Bottom Spin Valves With Ni-Fe-Mn Antiferromagnetic Layer
Structure, magnetic and magnetoresistive properties of spin valves with Ni-Fe-Mn antiferromagnet as a pinning layer have been studied. A technique of fabrication of spin valves with an enhanced thermal stability and improved hysteretic characteristics has been elaborated.
Introduction
Bilayers of permalloy/antiferromagnetic triple Ni-Fe-Mn alloy have been studied, as well as magnetic and magnetoresistive properties of spin valves (SVs) with Ni-Fe-Mn antiferromagnet (AF) as a pinning layer. A technique of fabrication of bottom spin valves based on Ni-Fe-Mn ordered AF with an enhanced thermal stability and improved hysteretic characteristics has been elaborated.
To apply the ordered Ni-Fe-Mn AF phase in spin valves, a definite deposition order of permalloy and manganese layers is necessary. Notably, the permalloy layer should be deposited on manganese or the manganese containing alloy [1]. With this deposition order, the authors of [2] fixed the high value H ex = 110 Oe after the annealing of the FeMn(15nm)/NiFe(15nm) bilayers. It should be noted that the formation of this ordered system is only possible with the thermal treatment of the films that contain the permalloy and manganese layers or the layers of the manganese containing alloy. The annealing of Ni 48 Fe 12 Mn 40 and Ni 32 Fe 8 Mn 60 films which after magnetron sputtering from the targets of corresponding compositions, were in the state of a homogeneous ternary solid solution did not result in the formation of an ordered antiferromagnetic phase. After annealing at 400°C, the decomposition of a ternary solid solution into the nickel based solid solution and almost pure manganese was revealed. A similar result was found in [3].
Experimental details
The samples were made by DC magnetron sputtering and by electron-beam evaporation on glass (Corning) substrates and single-crystalline sapphire (1012). To form a unidirectional anisotropy the magnetic field of 110 Oe was applied in the process of nanostructures growth, and the thermal-magnetic treatment was performed at pressure of 10 -4 Pa in permanent magnetic field of 2 kOe applied in the sample plane at 260 ºC for 4 h. The exchange bias field temperature dependence was measured in the temperature range of 20-260 ºC. Etching of samples for preparation of bottom SVs was carried out in a device for reactive ion-plasmic etching PlasmaPro NGP 80 RIE Oxford.
Results and discussion
According to the results of the studies carried out, the bilayers with antiferromagnetic triple alloy Ni 14 Fe 6 Mn 80 are characterized by medium blocking temperature T b = 150 ºС ( Fig. 1) and moderate energy of exchange interaction J ex = 0.05 erg/cm 2 [4].
Higher T b = 270 ºС ( Fig. 1) and J ex = 0.27 erg/cm 2 were obtained for the annealed manganese/permalloy bilayers. In this case, an ordered AF phase of Ni-Fe-Mn is formed [5]. The ordered AF phase formation is testified by an appearance of super-structural Debye rings (100), (110), (210), (211) (indicated by arrows in Fig. 2a) in electron diffraction patterns of sample Al 2 O 3 /Mn(50 nm)/Ni 77 Fe 23 (30 nm)/Ta(5 nm) after its annealing in the magnetic field at T ann = 260 о С for 4 h. In this case in the electron-microscope images one can see the columnar structure, and an intermediate layer between manganese and permalloy layers is absent (Fig. 2b). To study the magnetoresistance dependence of a spin valve on the copper layer thickness the samples with t AF = 25 nm were prepared.
With increasing copper layer thickness the magnetoresistance ΔR/R s at first increases and then decreases [4]. The dependence obtained is nonmonotone and demonstrates qualitative agreement with the data published in [6].
The maximal value of ΔR/R s = 7.30 % corresponds to t Cu = 2.8 nm (Fig. 3). The magnetoresistance sensitivity determined as an average value for the ascending and descending hysteresis loop of the free layer Ni 80 Fe 20 /Co 90 Fe 10 is Δ(ΔR/R s )/ΔH = 0.75 %/Oe. The data obtained demonstrate possibility of using disordered alloy Ni 14 Fe 6 Mn 80 as a pinning layer in top spin valves. To fabricate a bottom SV with the ordered AF phase Ni-Fe-Mn a technological cycle has been worked out, including the following operations: 1) Formation of the ordered AF phase Ni-Fe-Mn in a sample Al 2 O 3 /Ni 77 Fe 23 (5 nm)/Mn(50 nm)/Ni 77 Fe 23 (30 nm)/Ta(5 nm) by the thermal-magnetic treatment at 260 о С for 4 h.
2) Ion etching for 20 min of the annealed sample for the surface layer removing.
The ion etching duration was chosen to assure the removal of the contaminated surface layer and retaining the ferromagnetic area together with the ordered AF phase required for the formation of the unidirectional anisotropy in the FM layer sputtered on the sample after etching.
3) Magnetron sputtering of the layered structure consisting of ferromagnetic Co 90 Fe 10 layers separated by Cu on the as-prepared sample Al 2 O 3 /Ni-Fe-Mn.
The magnetoresistance of the as-fabricated spin valve Al 2 O 3 /Ni-Fe-Mn/Co 90 Fe 10 (5.5 nm)/Cu(3.6 nm)/Co 90 Fe 10 (5.5 nm)/Ta(5 nm) is ΔR/R s = 3.8 % (Fig. 4). This value is significantly higher than the effect obtained in [1], since the replacement of permalloy in the free and pinning layer by Co or the Co 90 Fe 10 alloy leads to an increase in spindependent scattering and an increase in the giant magnetic resistance effect (GMR) in the spin valves [7].
Conclusion
A possibility of application of the Ni-Fe-Mn AF for SV devices is demonstrated on a sample of composition Ta/Ni 80 Fe 20 /Co 90 Fe 10 /Cu/Co 90 Fe 10 /Ni 14 Fe 6 Mn 80 /Ta. A technology of fabrication of a spin valve based on the ordered AF phase Ni-Fe-Mn has been worked out. A spin valve with giant magnetic resistance exceeding the GMR of previously known structures has been created, | 2019-04-30T13:07:46.030Z | 2018-01-01T00:00:00.000 | {
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270654505 | pes2o/s2orc | v3-fos-license | Bear Bile Powder Inhibits the Release of NLRP3 by Activating the cAMP/PKA/CREB Signaling Pathway to Treat Dextran Sulfate Sodium-induced Colitis in Mice
Background and objectives: Ulcerative colitis (UC) is a chronic autoimmune disease that mainly affects the rectum and colon. The symptoms primarily include abdominal pain, diarrhea, and bloody stools. The incidence of UC continues to increase each year. Bear bile powder (BBP) is a well-known traditional medicine that remains in use due to its outstanding efficacy. This study aimed to elucidate the therapeutic effects and molecular mechanisms of BBP on dextran sulfate sodium (DSS)-induced UC. Methods: DSS-induced UC model mice were created and then randomly assigned to the following groups: control, DSS-treated, 5-amino salicylic acid-treated, BBP low dose, and BBP high dose. Treatment was administered by gavage. Disease activity index, body weight loss, colon histopathology, colon length, and the expression of inflammatory cytokines were measured. Samples of the intestinal content were collected, and differences in the gut microbiota were analyzed by 16S rDNA sequencing. Results: The experimental results demonstrated that BBP significantly alleviated the symptoms and histopathological scores in UC mice, reduced the production of interleukin-6, interleukin-1β, tumor necrosis factor-α, malondialdehyde, nitric oxide, and myeloperoxidase, and upregulated the expression of cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP-response element binding protein. Moreover, 16S rRNA sequencing revealed that the gut microbiota of mice in the DSS-treated group was disordered compared to the control group. The abundance of gut microbiota in the treatment groups improved to varying degrees. Conclusions: Together, these results indicate that BBP significantly improves the inflammatory symptoms of mice with acute colitis, which may be related to its upregulation of the cAMP/protein kinase A/cAMP-response element binding protein signaling pathway, inhibition of NOD-like receptor thermal protein domain associated protein 3 inflammasome secretion, and
Introduction
Ulcerative colitis (UC) is a chronic disease characterized by pain, diarrhea, and blood stool.At present, the etiology of UC is unclear; however, genetic, immune, environmental, and psychological factors, as well as intestinal mucosal barrier function, inflammation, and gut microbiota, may contribute to its development. 1 Currently, the clinical treatment of UC mostly involves the use of aminosalicylic acid, glucocorticoids, biological agents, etc. 4 However, the long-term issue of these agents can result in systemic adverse reactions.Although biological agents are effective in the short term, their costs and the risk of immunosuppression are significant concerns. 5cholars are increasingly interested in using traditional Chinese medicine for the treatment of inflammatory bowel disease (IBD).Bear bile powder (BBP) is a preparation obtained by freeze-drying the bile of black bears. 6Chemically, it is mainly composed of bile acids (BAs), with tauroursodeoxycholic acid, ursodeoxycholic acid (UDCA), and deoxycholic acid (DCA) being its main active ingredients.Studies have found that tauroursodeoxycholic acid and UDCA can reduce intestinal inflammatory response and oxidative stress, thereby alleviating intestinal symptoms. 7In traditional Chinese medicine, BBP is used to protect the liver and improve eyesight. 8Cyclic adenosine monophosphate (cAMP) is a classical second messenger that mediates many important signaling pathways. 11esearch has indicated that increased levels of cAMP reduced inflammation in rats with colitis, while lower colonic cAMP levels in UC patients result in abnormal production of inflammatory intestinal cytokines. 12Many studies have highlighted the importance of cAMP-response element binding protein (CREB) in UC, showing that activation of protein kinase A (PKA) leads to phosphorylation and upregulation of CREB. 13This, in turn, decreases inflammatory factors and upregulates the expression of anti-inflammatory signals.PKA further activates the downstream CREB, which undergoes nuclear translocation into the nucleus, thus regulating the cellular response.It inhibits the release of the inflammasome NOD-like receptor thermal protein domain associated protein 3 (NLRP3).In one study, activating the vasoactive intestinal peptide/cAMP/PKA pathway improved the diversity of the gut microbiota and protected the intestinal barrier, effectively alleviating experimental colitis. 14A gut ecological imbalance, defined as a state of microbial imbalance, is considered an important pathogenic factor in many diseases. 15An intestinal ecological imbalance is closely related to IBD.It has been demonstrated that the gut microbial community is a crucial link in the host's physiological and pathological processes.The gastrointestinal tract is home to the largest number of bacteria in the body.BAs are the end products of cholesterol catabolism.The gut microbiota undergoes multiple BA biotransformation reactions, and the composition and abundance of the gut microbiota are sequentially influenced by BAs. 16urrently, few studies have examined the treatment of UC with BBP, and the underlying mechanism of action remains elusive.Therefore, this study aimed to explore the protective effects of BBP on DSS-induced UC in mice and elucidate its molecular mechanisms.
Animals
Eight-week-old SPF grade C57BL/6 mice, weighing 18-22 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd.(2107162111089).The mice were housed in the Experimental Animal Center of South-Central Minzu University under SPF-grade barrier facilities and standard environmental conditions (temperature 22 ± 2°C, humidity 40-60%, 12 h light/dark cycle).The mice were allowed free access to water and food throughout the experiment.The animals were acclimatized and fed for seven days before the experiment commenced.All experimental protocols in this study were approved by the Institutional Animal Care and Use Committee of South-Central Minzu University (2020-SCUEC-023).All animal experiments conformed to the Management Rules of the Chinese Ministry of Health and were performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.
Induction of colitis
As shown in Figure 1, all groups, except for the NC group, were provided with 3% DSS drinking for seven days. 17The treatment groups were intragastrically administered 5-ASA or BBP for 10 days.Mice were sacrificed by CO 2 inhalation or cervical dislocation at desired time-points, and all efforts were made to minimize suffering.
Assessment of the disease activity index
During the experimental period, the Disease Activity Index (DAI) was employed as a quantitative indicator to assess the severity of colitis damage. 18Daily monitoring and recording of body weight loss, fecal characteristics, and hematochezia were performed.The DAI was calculated based on established parameters (Table 1). 19
Colonic length and splenic index measurements
After the mice were sacrificed, the colon and spleen tissues were collected, and the length of the colon was measured, photographed, and recorded.The spleen dry-to-wet ratio was recorded.The spleen index was calculated as follows 20 : spleen index (mg/g) = spleen mass/mouse body mass.
Histological analysis of the colon
Firstly, the mice were dissected, and the colonic tissues of each group were collected.Secondly, colonic tissues were soaked in 4% paraformaldehyde for 72 h, embedded in paraffin, and sectioned, stained with hematoxylin and eosin staining solution, dried, sealed, microscopically examined, and scored.The scoring included the degree of intestinal epithelial cell damage and inflammatory infil-Tian H. et al: The effect of bear bile powder on ulcerative colitis Future Integr Med tration to evaluate the degree of colonic tissue damage (Table 2). 21
Cytokines analysis by enzyme-linked immunosorbent assay (ELISA)
Colon tissue was obtained and homogenized thoroughly on ice using an electric homogenizer at a weight-to-volume ratio of 1 g (tissue): 9 ml (PBS, pH 7.4).ELISA kits were used to detect TNF-α, IL-1β, and IL-6 according to instructions.
Oxidative stress index assay
Using ELISA kits and following the provided instructions, T-SOD, MDA, and MPO levels in the colonic tissue, as well as NO levels in the serum, were measured.
16S rRNA sequencing
Following the extraction of total DNA from colon stool samples, primers were designed to target conserved regions.A sequencing junction was added to the end of the primers to facilitate PCR am-plification.Subsequently, the amplified products were purified, quantified, and homogenized to construct a sequencing library.The constructed library was first subjected to quality control.The qualified library was sequenced by Illumina NovaSeq 6000.The raw image data files obtained from high-throughput sequencing were transformed into Sequenced Reads by Base Calling analysis, and the results were stored in FASTQ (fq for short) file format, which contains sequence information of sequenced sequences (Reads) and their corresponding sequencing quality information.
Based on the Illumina NovaSeq sequencing platform, a Paired-End library was constructed and paired for sequencing.The species composition of the sample was revealed by filtering, clustering or de-noising, species annotation, and abundance analysis of Reads.
Western blot analysis
For the western blot assay, a whole-cell lysis kit (KeyGene Bio-Tech, China) was used to extract proteins from the colonic tissue samples.The protein concentrations were determined using a BCA kit (KeyGene BioTech, China).Equal amounts of protein were separated on 10% SDS-PAGE gels and subsequently transferred to PVDF membranes (Merck Millipore, Germany).Following this, the membranes were blocked with 5% skim milk for 1 h, then incubated with primary antibodies at 4 °C overnight, followed by secondary antibodies for 1 h.Detection was performed with enhanced chemiluminescence (BioRad, USA).Blots were quantified using Image Lab software.
Statistical analysis
All data are expressed as the mean ± standard deviation.Statistical analysis was performed using SPSS 21.0 software.Group differences were assessed using one-way ANOVA.P-values less than 0.05 were considered statistically significant (*p < 0.05, **p < 0.01).
BBP ameliorates DSS-induced colitis
Mice were orally administered 3% DSS water to induce UC, serving as an acute inflammatory model.The effect of BBP on DSS-induced colitis was evaluated by weight loss (Fig. 2a), the DAI score (Fig. 2b), colon length (Fig. 2c-d), and histopathological analysis of the colon tissue (Fig. 2e).The average weight of the NC group showed a stable increasing trend, while the DSS-treated group started to lose weight on day 3.The weight of the BBP-treated group increased compared to the DSS-treated group.Compared to the NC group, the DAI score of the model group was significantly increased from day 3, and the colon became extensively congested and edematous, exhibiting shortening.The DAI of the BBP-treated group was significantly lower than the model group starting from day 5, and the BBP treatment significantly inhibited the shortening of the colon compared to the DSS-treated group.Compared to the NC group, the DSS-treated group showed pathological changes in the colon, with damaged or even apoptotic intestinal epithelial cells, reduced cupped cells and intestinal glands, crypt abscesses, and massive infiltration of inflammatory cells in the lamina propria and submucosa.The BBP treatment alleviated these pathological changes in colon tissue.Significant differences were observed in all treated groups, and the histological scores were lower than those of the DSS-treated group.The BBP-L and BBP-H groups exhibited similar effects.
Splenic index
UC is considered an autoimmune disease.Research has demonstrated that the weight of the spleen increases with the aggravation of inflammation. 22In this study, there was a significant increase in the spleen index in the DSS-treated group compared to the NC group (p < 0.01), indicating that the spleen, the immune organ, of mice in the colitis DSS-treated group showed a decline.Compared to the DSS-treated group, there was a significant decrease in the spleen index in the 5-ASA-treated, BBP-L, and BBP-H groups (p <0.01), indicating significant protective effects of the treatments on the immune organs of mice with colitis (Fig. 3).Among them, the BBP-H group exhibited the best effect compared to the BBP-L and 5-ASA-treated groups.
BBP suppresses the release of inflammatory cytokines in UC mice
Inflammatory injury is the main pathological feature of UC.IL-1β, TNF-α, and IL-6 are pro-inflammatory factors that may be increased in UC. 23 As shown in Figure 4, the main pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were detected by ELISA.
Compared to the NC group, the levels of TNF-α, IL-1β, IL-6, and other pro-inflammatory cytokines were significantly higher in the DSS-treated group.The levels of these pro-inflammatory cytokines were significantly lower in the BBP administration groups compared to the DSS-treated group, indicating that BBP has an anti-inflammatory effect in the treatment of UC.The BBP-L and BBP-H groups exhibited similar effects.
BBP decreased oxidative stress in DSS-induced colitis mice
Studies have shown that oxidative stress is closely related to the pathogenesis of colitis.As shown in Figure 5, the NO, MPO activity, and MDA content were significantly increased (p < 0.01), and the T-SOD activity was significantly decreased (p < 0.01) in the DSS-treated group compared to the NC group.Compared to the DSS-treated group, the BBP group showed a significant decrease in the MDA content and the NO and MPO activities, and a significant increase in T-SOD activity (p < 0.01).This indicates that BBP can effectively alleviate the oxidative stress level of DSS-induced colitis mice.The BBP-L and BBP-H groups exhibited similar effects.
BBP upregulation the cAMP/PKA/CREB signaling pathway to inhibit the expression of the NLRP3 inflammasome
As shown in Figure 6, the protein expression levels of cAMP, PKA, CREB, and p-CREB in the UC model mice induced by DSS were significantly reduced, and the NLRP3 inflammasome was activated.However, after the 5-ASA and BBP treatments, the protein expression levels were significantly increased.This indicated that BBP can inhibit the activation of the NLRP3 inflammasome by upregulating the cAMP/PKA/CREB protein pathway to exert its anti-colitis effect.
The effect of BBP on the abundance and diversity of the intestinal microbiota
In recent years, it has become evident that the intestinal flora plays a role in intestinal immunity. 16Further, recent research has demonstrated that an imbalance in the gut microbiota is a crucial mechanism implicated in the pathogenesis of colitis. 24Therefore, we wondered whether BBP can regulate the gut microbiota to induce its anti-colitis effect.Accordingly, 16SrRNA analysis of the microbiota was performed on the intestinal contents of the experimental mice to investigate the possible mechanism underlying the effect of BBP in the treatment of UC.As shown in Figure 7, compared to the NC group, the Shannon and Simpson indices of the UC group showed decreasing trends, but there were no statistically significant differences (p > 0.05).Compared to the DSS-treated group, the Shannon and Simpson indices of the BBP treatment group exhibited upward trends, but there were no significant differences (p > 0.05).
Next, beta diversity analysis was performed to evaluate the similarity of the mouse gut microbiota community.Principal component analysis, principal coordinates analysis, and nonmetric multi-dimensional scaling are important indicators of beta diversity.Here, the weighted UniFrac algorithm based on the out number was utilized to analyze the beta diversity of the samples.Principal component analysis and principal coordinates analysis showed that compared to mice in the DSS-treated group, the intestinal microflora composition of mice in the BBP treatment group showed significant changes (Fig. 8).The longer the distance between the different groups, the greater the difference in the intestinal microflora.In the non-metric multi-dimensional scaling analysis, stress < 0.05 indicates that the data are highly representative.Thus, it can be seen that the microbial communities of the NC and DSS-treated groups were significantly separat-ed, while the microbial communities of the NC and BBP groups were closer.
LEfSe analysis was performed to identify the bacterial communities. 25The differences in the abundance of microbial communi- ties were analyzed from the phylum to species level.As shown in Figure 9, linear discriminant analysis combined with effect quantity measurement was performed to analyze the differential microbiota in colitis mice.The results showed that Parabacteroides and Mucispirllum were the main dominant bacterial groups in the NC group compared with the DSS-treated group.Mucispirillum antagonizes the virulence of Salmonella to protect mice against colitis, 26 while parabacteroides exert a positive regulatory influence on glucose and lipid metabolism. 27Therefore, DSS changed the composition of intestinal flora in mice.The comparison between the BBP group and the DSS-treated group showed that the main differential dominant bacteria in the BBP group were Mucispirillum and Eubacterium.Corynebacterium and Ruminococcaeae were the main differential bacteria in the DSS-treated group.Eubacterium plays a pivotal role in the body's nutritional metabolism, maintenance of intestinal homeostasis, and metabolism of BAs and cholesterol. 28Corynebacterium was found to be enriched in the duodenum of food-responsive diarrhea dogs pretreatment. 29Active IBD is usually accompanied by an increase in Ruminococcus gnavus, 30 inducing dendritic cells to secrete TNF-α.This result indicates an increase in harmful bacterial components in the DSS-treated group, while the main advantage of the BBP treatment group is the beneficial bacterial community, indicating that bear bile powder has the effect of regulating the composition of intestinal microbiota.
Discussion
UC is a chronic intestinal, mainly characterized by recurrent and persistent chronic non-specific inflammatory changes of the mucosa and submucosa.The main pathological processes include excessive apoptosis of the intestinal epithelium, infiltration of inflammatory cells, disruption of the intestinal microenvironment, bacterial infection, and eventually, recurrent ulceration.Effective treatment strategies for UC aim to inhibit the apoptosis of colonic epithelial cells, promote the repair of damaged mucosa, and reduce the infiltration of inflammatory cells.5-ASA is a classical drug for the treatment of UC.It is taken orally and works by inhibiting lipid oxidase and cyclooxygenase, thereby interfering with arachidonic acid.
This inhibition reduces the synthesis of lipoxygenase and cyclooxygenase, exerting anti-inflammatory effects and alleviating intestinal pathology.DSS can destroy the integrity of intestinal epithelial cells and break the mechanical barrier of the intestine, which can induce acute UC.2][33] In this experiment, an injury model of UC in mice was established through DSS administration.
Its main active ingredient is lithocholic acid (LCA), which is often reduced in IBD patients. 35Research has shown that both LCA and UDCA can alleviate colitis. 36Currently, BBP is used in numerous Chinese patent medicines included in the Chinese Pharmacopoeia and serves as a major ingredient in clinical practice.In this study, we comprehensively investigated the therapeutic effects of BBP on UC and its potential underlying mechanisms.
In the present experiment, mice in the DSS-treated group exhibited symptoms similar to those of UC patients, including diarrhea, blood in the stools, inflammatory infiltration, weight loss, and colonic ulceration.In contrast, the mice in the BBP treatment group exhibited significant improvements in macroscopic damage, such as reduced blood in the stools, diarrhea, weight loss, and colon shortening, suggesting a potential anti-colitis effect of BBP.In addition, the mechanism underlying DSS-induced intestinal inflammation is closely associated with epithelial cell layer damage and abnormal alterations in colonic inflammatory mediators.In the present experiment, BBP effectively reduced the infiltration of inflammatory cells in colonic tissues and decreased the apoptosis of the colonic epithelium, thereby reducing the inflammatory response, preserving colon length, and alleviating colitis symptoms.
An increase in oxygen free radicals (OFR) is an important factor in the local inflammatory response and damage to the colonic mucosal tissue in UC.Low levels of SOD and high levels of NO in the body are key pathogenic factors in DSS-induced colitis.When the inflammatory response occurs in the intestine, increases in tissue OFR and lipid peroxides occur, indicating lipid peroxidation.The end product of lipid peroxidation is MDA, so increases in OFR and MDA indicate an increase in oxidative reactions in the body.SOD, an important antioxidant enzyme in the antioxidant system, can block the action of OFR and prevent lipid peroxidation, effectively protecting cell membranes.Thus, SOD activity can be used as an important indicator of the antioxidant capacity of the body.The MPO level in tissues provides a direct assessment of the content of neutrophils in that tissue, which is positively correlated with the severity of UC. 37 The findings of the current study indicated that the T-SOD level was lower and NO, MDA, and MPO levels were higher in the DSS-treated group compared with the NC group, suggesting an increased oxidative response and inflammatory cell infiltration.BBP significantly increased antioxidation and reduced colitis cell infiltration.Thus, the results of the present study suggest that continuous treatment with BBP significantly reduces the severity of colonic injury and oxidative stress damage induced by DSS.
The abnormal immune response of UC is mainly manifested as an imbalance in cytokine release, including an increase in pro-inflammatory factors and a decrease in anti-inflammatory factors. 38,39IL-6 is a glycoprotein involved in inflammatory response and inflammatory cell chemotaxis.IL-1β, secreted by macrophages, lymphocytes, and monocytes, can induce the expression of TNF-α and other inflammatory factors, promoting an inflammatory response in the body.IL-1β is secreted by macrophages, lymphocytes, and monocytes, which can induce the expression of TNF-α and other inflammatory factors, promoting the inflammatory response. 40The results of this study demonstrated that the levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α were significantly increased in mice in the DSS-treated group compared with the NC group.However, these levels were significantly decreased in the treatment group compared with the DSS-treated group, indicating that BBP has a strong anti-inflammatory effect in UC, comparable to the effect in the 5-ASA-treated group.
The gut microbiota is closely related to UC, 41 and dysbiosis of the gut microbiota affects the intestinal mucosal environment.The results of this experiment showed that harmful bacterial components increased in the DSS-treated group, while the main dominant bacterial group in the BBP treatment group was beneficial bacteria, indicating that bear bile powder regulates the composition of intestinal microbiota.Research to date has shown a close relationship between BA metabolism and the gut microbiota, as well as the progression of IBD. 16BBP, as a BA drug, contains BAs as its main active ingredients.We speculate that BBP's regulation of the gut microbiota may be related to the BA components of BBP; however, the specific connection and mechanism require further in-depth research.In summary, we believe that the efficacy of BBP in the treatment of colitis may be closely related to its promotion of the mutual conversion between BAs and intestinal flora and the regulation of the intestinal flora composition.However, further research is still needed.
Recent studies have shown that the cAMP/PKA/P-CREB signaling pathway has an inhibitory effect on the inflammasome NLRP3.Recent studies have shown that NLRP3 inflammasome, mucosal immune response, and intestinal homeostasis exhibit complex interactions. 42Further, studies have demonstrated that cAMP-specific PDE4 can significantly inhibit cytokines of various inflammatory cells, including macrophages, neutrophils, and 44 The results showed that compared with the NC group, the protein expression levels of cAMP, PKA, and P-CREB were significantly reduced in the DSS-treated group, while the expression of NLRP3 inflammasome protein was significantly increased.Compared with the DSS-treated group, the expression of cAMP, PKA, and P-CREB proteins was significantly increased in the BBP treatment group, while the expression of NLRP3 inflammasome protein was significantly reduced.
The experimental results in this study suggest that the potential mechanism by which BBP exerts its anti-colitis effects may be related to the upregulation of the cAMP/PKA/CREB signaling pathway, which then inhibits the expression of NLRP3 inflammasome-related proteins.In another study, LCA inhibited the activation of the NLRP3 inflammasome through the Takeda-G-protein-receptor-5 (TGR5)-cAMP-PKA axis, thereby improving the development of inflammation.TGR5 is a BA receptor located on the cell membrane that participates in regulating metabolism and inflammation. 10BAs can activate the TGR5 receptor to upregulate adenosine-activating enzyme activity, 45 increase intracellular cAMP levels, and activate the cAMP/PKA/CREB signaling pathway.However, BBP contains a large amount of BA components such as UDCA.Therefore, we speculate that the effects of BBP may also be related to the activation of the cAMP/PKA/CREB signaling pathway by the BA components of BBP.Nonetheless, experimental validation of this hypothesis is needed.At present, research shows that the cAMP/PKA/CREB signaling pathway can be activated by BA receptors, and the gut microbiota is also regulated by BAs.There may be potential connections among them, requiring further in-depth research.
Conclusion
This study examined the anti-colitis effect of BBP.The findings showed that BBP effectively reduced inflammatory cell infiltration, decreased the inflammatory response, inhibited apoptosis of colonic epithelium, regulated the gut microbiota, increased the cAMP/PKA/CREB signaling pathway, and reduced the release of the NLRP3 inflammasome in a mouse model of UC.These findings suggest that BBP may serve as a new treatment approach for UC, warranting further research and promotion.While these findings offer an experimental basis for the development of new anti-UC drugs, more studies are needed to be elucidated.
Fig. 7 .
Fig. 7. Alpha diversity boxplot (note: the horizontal axis represents the group name, and the vertical axis represents the corresponding Alpha diversity index).(a) Simpson's index (The P value between each group is p > 0.05), (b) Shannon's index (The P value between each group is p > 0.05).BBP, bear bile powder; DSS, dextran sulfate sodium; NC, normal control.
Table 2 . Histopathological scoring criteria
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52134076 | pes2o/s2orc | v3-fos-license | Reported Seasonal Dependence of Herbicide Developmental Toxicity in Mice
We are concerned over specific scientific issues reported by Cavieres et al. in the November 2002 issue of EHP (Cavieres et al. 2002). The paper, which has already received considerable attention in the media, presents conclusions that are not supported by the experimental design or by the data; an array of significant inconsistencies and errors are also present in the paper. Accordingly, we believe that Cavieres et al. should retract their paper or the journal should withdraw it until these problems are addressed. In the abstract of the paper, Cavieres et al. (2002) stated that
We are concerned over specific scientific issues reported by Cavieres et al. in the November 2002 issue of EHP . The paper, which has already received considerable attention in the media, presents conclusions that are not supported by the experimental design or by the data; an array of significant inconsistencies and errors are also present in the paper. Accordingly, we believe that Cavieres et al. should retract their paper or the journal should withdraw it until these problems are addressed.
In the abstract of the paper, stated that The data, although apparently influenced by season, showed an inverted or U-shaped doseresponse pattern for reduced litter size, with the low end of the dose range producing the greatest decrease in the number of live pups born. The decrease in litter size was associated with a decrease in the number of implantation sites, but only at very low and low environmentally relevant doses.
The conclusions of were based on a series of seven developmental toxicity studies. Five of the experiments used pregnant mice exposed no earlier than days 5 or 6 of gestation; therefore, treatment started after implantation, so the studies were never capable of assessing effects on implantation. In only two studies were mice exposed during or before implantation, but those studies did not include the "very low doses" that Cavieres et al. claim preferentially decreased implantation sites with an inverted U dose-response curve. Thus, the basis for the authors' conclusions is in error.
Our second concern is raised by discrepancies between the paper by and Cavieres' dissertation (Cavieres 2001). The dissertation and the paper both appear to report the results of the same experiments, as evidenced, for example, by identical tables describing analytical confirmation of the doses for the seven studies. Importantly, there are unexplained discrepancies in the numbers of animals tested and in the outcomes. For example, when the two "preimplantation plus organogenesis" studies were combined in the dissertation, the control group was not significantly different from the treated groups. However, Cavieres et al.'s Table 2 contains data in which nine animals were deleted from the four groups. The authors reported statistical significance for the low-dose group after this manipulation of the data, but they did not explain why. Although the deletion of two control animals caused an increase in the mean litter size for the controls, deletions in other groups caused a decrease in mean litter size for the treated groups. The outcome of these two manipulations accounted for the difference between treated and control animals reported by , but not in Cavieres' dissertation (Cavieres 2001). provided no reliable data to support their conclusion that the difference between litter size and implantations was due to resorptions. They did not count deaths and cannibalizations; therefore, accurate estimates of litter size are impossible.
In their Figure 1 combined data from studies that followed critically different designs and thus should not have been combined. In Cavieres' dissertation (Cavieres 2001), litter size was reported, but it was not significantly different for the low-dose "organogenesis" animals. However, combining those animals with the "preimplantation plus organogenesis" animals in Table 1 (Cavieres 2001) created a significant difference from controls. were not consistent in their presentation of data. In Figure 1 of their paper, Cavieres et al. show that 62 control dams were used to determine litter size, but in Table 2 they show 64 control dams for the same end point. Although the authors should not have combined the various experiments, they indicated no reason for having a different number of animals in two representations of the same end point from the same collection of experiments. also reported potential influence of seasonality on the experiments. A more plausible explanation is typical variability between experiments and that the findings are not related to treatment. The authors did not study seasonality in a systematic manner.
Finally, one of the most prominent conclusions of is a substantial overstatement of the findings of the experiments. They compared the pattern of the "dose response" with the inverted U, but the pattern in this case was inconsistent, even in the most favorable light. When the experiments are separated, as they should be, there is no reproducible dose-response pattern. It appears that the authors used novel theories to describe inconsistent data. They also used very selective citation of the literature to support the inverted U doseresponse hypothesis, without citing the many unsuccessful attempts to replicate findings supportive of the hypothesis.
In conclusion, the paper by contains numerous discrepancies and inconsistencies, as well as disagreement between the results presented in their paper and in the dissertation (Cavieres 2001) upon which this paper was based. We believe that should revise and correct their paper or it should be withdrawn.
Editor's note: In accordance with journal policy, Cavieres et al. were asked whether they wanted to respond to this letter, but they did not provide a response.
Reported Seasonal Dependence of Herbicide Developmental Toxicity in Mice
In their paper published in the November 2002 issue of EHP, exposed pregnant mice to a commercial herbicide mixture and determined pregnancy outcomes. Separate experiments were conducted for each season of the year; the spring exposure occurred between gestation days (GD) 0 and 15, whereas in the other seasons, exposure was postimplantation (GDs 5-15). The authors concluded that, although apparently influenced by season, the results showed an inverted or U-shaped dose-response pattern for reduced litter size and reduced implantation sites. These decreases were reported to occur "only at very low environmentally relevant doses of the herbicide mixture." These findings are of interest to us, but we are concerned about several inconsistencies in the reporting and also about the merging of different groups of data. As a result, it is not possible to reconstitute the original data for independent analysis. For example, the numbers of observations per group are different in Cavieres et al.'s Figure 1 and Table 2, and among Tables 2, 3, and 4. It is also unclear why reported a higher number of implantation sites than the number of litter size recordings (e.g., the summer high-dose group), especially when the authors stated in the text that implantations were only recorded for a subset of litters. The authors did not explain why they analyzed implantations on the basis of covariance with final litter size. The control litter-size data [Figure1B ] has an unusual distribution, with the mode being the most frequent. The authors tentatively rationalized their findings in terms of the chemical treatment causing either preimplantation loss or fetal death. However, preimplantation exposure occurred only in the spring group, and no significant increases in resorptions were observed in any group. Thus, in the fall, winter, and summer groups, herbicide-induced preimplantation loss could not have occurred; therefore, the reported reduced implantations and reduced litter sizes in the absence of an increase in resorptions was an effect that simply could not be the result of herbicide exposure. The situation was further confused by the imperfect correlation between litter size and implantation sites. For example, in the very low-dose summer group, a significant (23%) reduction in litter size was associated with a significant (12%) reduction in implantation sites. However, in the high-dose summer group, the nonsignificant (7%) reduction in litter size was associated with a larger, but nonsignificant (15%) reduction in implantation sites.
Although the test data were tabulated according to season and a seasonal influence on test outcome was noted, the data were merged for all cases where implantation data existed [ Figure 2 ], ignoring the individual seasonal data. These seasonal data are shown in Figure 1, in the format of the Cavieres et al.'s Figure 2 . Significant reductions in litter size are distributed across all the dose groups, with the fall data following a normal monotonic decrease in litter size ( Figure 1). However, Cavieres et al. excluded these monotonic fall data from their Figure 2 because of the absence of implantation data. We suggest that a primary decision should be made regarding whether or not the data are seasonally related. If they are not, the data could be merged and analyzed as such. If they are, the merging of data (as presented by Cavieres et al. in their Figure 2) is invalid, and an explanation for the seasonal influence must be sought. In the extreme, such a seasonal influence would lead to an observed normal monotonic response in the fall and an inverted U response in the summer for the same chemical. The animals used in these experiments were purchased and maintained for 2 weeks before the experiments began to allow them to adjust to the light-dark cycle and temperature of the animal rooms. Similar conditions would have applied in the commercial animal-breeding unit; therefore, the seasonal perceptions of the mice must have derived from the journey between the supplier and the laboratory-an unlikely proposition.
We conclude that substantial uncertainty exists regarding the origin of the litter size effects reported and that the conclusion of a low-dose inverted U-shaped doseresponse curve cannot be made at this stage.
Asthma and Gulf War Exposures
As a physician who treats patients with Gulf War syndrome and multiple chemical sensitivities, and on the basis of my clinical experience, I believe that these diseases are related and reflect valid pathophysiologic and biochemical processes in the body, which have yet to be clearly defined. The article by Lange et al. on respiratory illness among Gulf War veterans (Lange et al. 2002) was industrious in trying to disprove the validity of the veterans' complaints-or at least to infer that they were likely to be psychologically derived. The study has several methodologic shortcomings that need to be made explicit. First, the authors state that [Current] injury and symptoms of major depression, measures that have little or no biologically plausible relationship to oil-fire exposure, were included to serve as control health outcomes.
I have no problems with using "injury" as it was operationally defined in the study. However, "depression" as defined has at least two confounding and confusing correlations: First, if currently depressed subjects were chronically depressed, their perception of "level of preparedness" could be quite low, regardless of how well they were trained, because of their ongoing insecurities and low self-esteem. Second, if, in fact, Gulf War exposures made the subjects feel chronically ill, whether this was physiologic or psychologic in origin, their inability to lead normal lives could exacerbate preexisting depression or produce the reactive depression that is often seen in chronic disabling illness. These considerations make "depression" a poor independent variable. However, its use allows Lange et al. (2002) to graph similar curves Environmental Health Perspectives • VOLUME 111 | NUMBER 9 | July 2003 A 451 for asthma, bronchitis, and depression (their Figure 3), all independent of modeled exposure to oil-fire smoke. The authors ascribed all of this to "recall bias." Furthermore, a reader might easily infer that since the exposure model is "more objective" and shows no significant correlations in this study, and the veterans' complaints of asthma and bronchitis are strongly correlated with depression, all of their complaints might well be psychologic in nature. The second methodologic shortcoming is that nowhere did Lange et al. (2002) mention that oil-fire smoke and particles are only part of the hypothesized causes of Gulf War-induced respiratory complaints. They did not mention that similar studies should be undertaken to correlate symptom reports and demographics with exposure to chemical warfare agents (or their destruction), pesticide use [such as N,N-diethyl-mtoluamide (DEET)], volatile organic hydrocarbon exposure, vaccine administration, or antichemical warfare treatment, among other hypothesized agents. Taking these factors into consideration, the impact of Lange et al.'s study is far more limited than their hypothesis and conclusions would suggest; also, their study has a builtin bias that emphasizes psychologic factors.
Asthma and Gulf War Exposures: Response
As clearly stated in the introduction of our paper (Lange et al. 2002), there are many deployment-related exposures that have been suggested as causes for illnesses observed among veterans of the Gulf War. In our study we investigated the hypothesis that self-reported symptoms of respiratory illnesses after the war may have been related to modeled and self-reported exposures to oil-fire smoke.
Our study (Lange et al. 2002) was not designed to address other exposures of potential significance. In order to recognize the possibility of recall bias, our study included health conditions that had little biological plausibility for a relationship with oil-fire smoke exposure. Gordon takes issue with our use of major depression as one of these conditions. We agree that one can construct a scenario whereby exposure to smoke in 1991 could result in recent symptoms of major depression in 1996 or injury within the past 3 months in 1996. However, these possibilities are remote compared to the pulmonary outcomes of asthma and bronchitis. Beyond recognizing the possibility of recall bias within the self-reported measures used in our study, identifying the nature of this bias or the effect of other exposures was not within the scope of our study.
The author declares he has no conflict of interest.
Department of Occupational and
Environmental Health University of Iowa Iowa City, Iowa E-mail: peter-thorne@uiowa.edu
Communication in Emergencies
In their Commentary "Ethical Perspectives for Public and Environmental Health," paraphrased a 1990 editorial that I coauthored with Lester Lave to create a straw man against which to contrast their proposal that emergency communication should foster understanding and autonomy.
Our text (Morgan and Lave 1990) that referenced reads, There is wide, if not universal, agreement that attempts to manipulate behavior are appropriate when people are faced with large, immediate dangers. Officials are expected to issue explicit orders for action to people living in the path of a hurricane's storm surge or downwind of the spreading chlorine plume from a tank car accident, rather than just provide neutral messages for participants to weigh. Indeed, in such circumstances, if risk communications cannot get people to move out of harm's way, more intrusive measures, such as police, may be used. wrote, In contrast, by embracing the concept of fostering autonomy, the public can formulate and share the imperative. The right to know means that people need to understand the reason behind evacuation, verbal injunctions, or barricades. Manipulation and coercion may save some lives, but they certainly do not foster understanding.
My colleagues and I are strong proponents of providing people with full understanding so that they can make independent informed decisions. Our recent book, Risk Communication (Morgan et al. 2002), is entirely built on this philosophy. In it we define risk communication as communication intended to supply laypeople with the information they need to make informed independent judgments about risks to health, safety and the environment.
Even in crisis situations, officials should try to supply complete and balanced information within the constraints of the situation. But if a chlorine plume is about to engulf my family's house, I want the state police to get them out as quickly as possible; I would be completely satisfied if the only explanation at the time was, "Madam, I'm Officer Jones of the Pennsylvania State Police. There has been a terrible chemical accident, so we must get you and your family out of here right now. We'll explain more later." The author declares he has no conflict of interest.
Department of Engineering and Public Policy Carnegie Mellon University
Pittsburgh, Pennsylvania E-mail: granger.morgan@andrew.cmu.edu
Communication in Emergencies: Response
In our paper ), we did not advocate neutral messages in acute emergency response situations and agreed with that we have a responsibility to prevent people from entering harm's way. However, we presented an argument for fostering autonomy rather than the use of manipulation and coercion in these situations. Hague (1929) distinguished ethical risk communication as an issue of persuasion as opposed to coercion. Faden and Beauchamp (1986) defined these extremes: Persuasion is restricted to influence by appeal to reason, the intentional and successful attempt to induce a person, through appeals to reason, to freely accept-as his or her own-the beliefs, attitudes, values, intentions or actions advocated by the persuader.
According to Faden and Beauchamp (1986), coercion occurs if someone intentionally and successfully influences another by presenting a credible threat of unwanted or avoidable harm so severe that the person is unable to resist acting to avoid it. Faden and Beauchamp (1986) filled the middle ground by distinguishing forms of manipulation. They described manipulation as the catch-all term for communication that is neither coercion nor persuasion; intentional and non-successful non-coercive influence altering the available choices of an individual, or a perception of those choices and influence that does not appeal to reason.
The essence of manipulation is having people unwittingly do what the manipulator intends for them to do. Faden and Beauchamp (1986) argued that ethical health risk communication is persuasion or, at worst, unintentional manipulation because it cannot be avoided.
Unintentional manipulation may occur through several avenues. Tversky and Kahneman (1981) demonstrated that, by framing information in particular ways (for example, in the health context, the probability of dying or living from a given procedure), the choices that people (including health care providers) make can be directed to a significant degree. Therefore, when "mere information" is presented, the presentation itself will include the danger of manipulative elements, and there is a need to recognize this dilemma and confront it. Informational manipulation occurs when the structure of perception of choices is altered by managing information to promote a desired action. Further, to varying degrees, nonsubstantive elements such as tone, manner, and order; word choice; time and setting; and the appearance, style, and charisma of the presenter can be forms of psychologic manipulation.
In his letter, Morgan has clarified his perspective and provided a communication example that does not appear to argue for manipulation. In the case of an acute emergency, Morgan would be satisfied with the following communication: "Madam, I'm Officer Jones …. There has been a terrible chemical accident, so we must get you and your family out of here right now. We'll explain more later." This communication does allow the woman to "formulate and share the imperative," share the officer's urgency for evacuation, and understand the reason, (i.e., "there has been a terrible chemical accident"). Perhaps if the message also included more context, "that the plume is about to engulf your home," a stronger argument could be made for the woman's ability to formulate the imperative. Further, the woman may be able to spread the risk message to her friends who may not be reached by the police officers. As we argued in our paper , this is a beneficial consequence of communication that fosters autonomy as opposed to manipulation. The message concludes with "we'll explain more later." This implies an ongoing relationship and dialogue with the woman to further foster her understanding.
In this example, however, the police officer may introduce "unintentional" manipulation. The police officer, in rushing from door to door telling people to evacuate, by his or her presence, tone, and excitement, will carry the message of the urgency of the evacuation. This will, to some degree, introduce elements of unintentional manipulation.
The communication example does not involve an attempt to manipulate the woman's behavior or defend this approach to prevent her from being harmed. It is consistent with the argument in our paper .
The author declares he has no conflict of interest.
Department of Community Health Sciences
University of Calgary Calgary, Alberta, Canada E-mail: tim.lambert@calgaryhealthregion.ca | 2018-07-18T20:51:37.779Z | 2003-01-01T00:00:00.000 | {
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135879347 | pes2o/s2orc | v3-fos-license | Physical and Mechanical Properties of Paricá Wood Species Treated with CCB Preservative
Wood of Paricá species (Schizolobium amazonicum) is highly susceptible to degradation by decay microorganis ms. Due to its increasing production in the northern region of Brazil, about 85 thousand hectares, and later use both for structural purposes and for the furniture industry, the need of extending life of Paricá wood applying chemical preservatives arises. One current alternative is the treatment by autoclave using CCB (Copper Chrome Boron). The aim of this study was to evaluate the influence of treatment with CCB in the properties: apparent density, parallel and perpendicular hardness to the grain, strength to shear and in the compression parallel to the grain of the Schizolobium amazonicum wood species, comparing the results with untreated wood. We observed that treatment with CCB did not change the properties of apparent density, shear strength, perpendicular hardness, strength and stiffness in compression parallel to the grain. For the hardness strength parallel to the grain was decreased in 28.9%.
Introduction
The growth of world demand for timber imp lies the search for new species of fast growth and silvicu ltural potential in this scenario, native species, generally, are advantageous in adaptability [1].
In 1948, through Forest Service State of São Paulo, were introduced to test the American species known in the sources as "yellow pine" which include P. palustris, P. echinata, P. elliottii and P. taeda. Among these, the last two highlighted for ease cultivation, rapid growth and intense reproduction in the South and Southeast of Brazil. Since then, a large number of species continued to be introduced and established in field experiments by government agencies and private enterprises, aiming commercial plantations [2].
According to ABRAF-Brazilian Association of Planted Forest Producers [3], in 2010, the area occupied by forest plantations of Eucalyptus and Pinus in Brazil totaled 6,510,693 ha, which 73% corresponding to the area of Eucalyptus and 27% to Pinus.
In Brazil, a native species that is attract ing increas ing interest in timber co mpanies and producers, main ly due to its rapid gro wth, is Schizolobium amazonicum, also known as Paricá. Belonging to the Legu minosae family, it is native fro m Bolivia, Colo mb ia, Costa Rica, Ecuador, Honduras, Mexico and Peru [4]. In Brazil, the species has a wide geographical distribution, being found not only in the South Region. According to ABRAF-Brazilian Association of Planted Forest Producers [3], the total area p lanted with Paricá only in the states of Pará, Maranhão and Tocantins in 2011 was approximately 85ha.
According to Souza[9], Paricá is a species that has low natural durability, being susceptible to fungus, termites, wood decay and insects. Therefore, a preservative treat ment is necessary to prolong the life of such timber.
Main treat ments for wood preservatives used in Brazil are the CCA (chro mated copper arsenate) and CCB (chro mated copper borate) [10]. CCA is the most commonly used water soluble preservative worldwide. It is usually emp loyed containing about 19% copper o xide [11].
CCA is widely used in the treat ment of wood that remain in contact with the ground being very effective to protect wood against insects (termites and borers), decay fungi and marine borers. However, environ mental concerns about the use of preservatives have been raised due to the dispersion of copper and arsenic into the environment before the complete fixation of the active ingredients with the possibility of contamination of soil and groundwater [10][11][12].
CCB preservative is an alternative product to CCA, with the difference using of the boron element instead arsenic. Its use has advantages such as low cost compared to newer products launched in the market; reduced leaching of copper and chromiu m, is environmentally friendly, does not increase the electrical conductivity of wood, is non-corrosive to metals such as carbon steel and alu minu m [11].
According to Pinheiro [13], chemical wood preservation is extremely important, as well as being proven and effective against bio deterioration. In some cases, can increase the values of the mechanical properties of wood. In this regard, Rocco Lahr et al. [14] studied the influence of preservation with CCB in hardness of wood (parallel and perpendicular to the grain) fro m p lanted forests, Pinus elliottii, obtaining gains in the properties investigated.
Given the potential for reforestation in the country and the growing plantation of Paricá wood in Brazil, studies involving the preservative treatment presenting relevant, can alter the physical and mechanical characteristics of the wood in question.
The aim of this research was to evaluate the influence of treatment with CCB in the properties: apparent density, parallel and perpendicular hardness to the grain, shear strength, strength and stiffness of parallel co mpression to the grain of the Paricá wood species.
Fro m each beam was removed one specimen for each one of the tests, thus obtaining: 24 specimens for hardness perpendicular and parallel to the grain, 24 specimens for shear test and 24 specimens for co mpression parallel to the grain. As the beams used had a limit in thickness of 3.5 cm, it became necessary to fit the specimens' dimensions for each test. For apparent density testing entire board was used. The methodology used in the tests to determine the physical and mechanical properties fo llo wed the ABNT NBR7190 [15] ANNEX B -Projects of wooden structures, the Brazilian Association of Technical Standards, recommendation. All beams were kept in a controlled environ ment, thereby maintaining 12% mo isture content. Amsler un iversal testing mach ine with a capacity of 25 tons was used to carry out mechanical tests, as shown in Figure 1.
Preservati ve Treatment
The CCB treat ment applied on wood through pressure in autoclave was conducted by the company PREMA SA, located in the city of Rio Claro / SP. The t reatment retention was 9.6 kg of active ingredient (m³).
Apparent Density
The specimen dimensions for this test were: 35 mm th ick × 100 mm wide × 600 mm h igh. Figure 2 shows the apparent density specimen. . Figure 2. Apparent density specimen
Shear Test
In this test the shear occurs in the direction parallel to the grain. The standard specimen dimensions for this test were: 35 mm thickness × 100 mm width × 60 mm height; with area dimensions of 35 mm × 50 mm to be sheared. Figure 3 shows the shear specimen. Species Treated with CCB Preservative
Hardness Parallel and Perpendicular to the Grain
This test was realized on all specimens' faces, resulting in strength values in parallel and perpendicular directions to the grain. The standard specimen dimensions for this test were: 35 mm thick × 100 mm wide × 100 mm high. Figure 4 shows the hardness specimen.
Compression Parallel to the Grain
The specimen dimensions for compression parallel to the grain testing [16][17][18][19][20][21][22] were: 35 mm thick × 100 mm wide × 150 mm high. Figure 5 shows the compression parallel to the grain specimen. The region measurement to be compressed was performed using deflectometer installed in the center of the specimen.
Statistical Analysis
The analysis of variance (ANOVA ) was used to investigate the influence of the treatment in the properties investigated. The significance level (α) was 5%, considering the null hypothesis (H 0 ) the equivalence between the means and the non-equivalence as the alternative hypothesis (H 1 ). P-value g reater than the significance level involves accepting H 0 , rejecting it otherwise. To validate the ANOVA model, Anderson-Darling and the Bartlett´s tests were used to verify the normality of the distribution and the ho mogeneity between variances, respectively, both at the 5% level of significance, considering the null hypotheses as the normality and of the distribution and the equivalence between variances. The null hypothesis is accepted if the P-value obtained in the tests is higher than the significance level, rejecting them otherwise. When significance of the factor was detected by ANOVA, Tukey test for g rouping of the averages was employed.
Apparent density
The Table 1 shows the results for apparent density tests. x m is the average values, S d is the standard deviations and Cv is the coefficient of variat ion. The values obtained for apparent density; with 12 % mo isture content for untreated and treated wood were respectively 383.04 kg/ m³ and 407.49 kg/m³. A NOVA shows that the average for wood Paricá untreated and treated density test are not statistically different fro m each other (P-value>0,05).
Thus, it can be assumed that the treatment did not change the Paricá density, differently of the results obtained from the research of Rocco Lahr et al. [14], this had an increase in apparent density by 27% in the treated wood co mparing to wood "in nature". Table 2 presents the shear tests results. 10 7 ANOVA revealed that the average values of the shear test parallel to the grain of Paricá with treat ment and without treatment are not statistically different fro m each other (P-value>0,05). Thus, it can be assumed that the treatment did not change the property of shear strength of Paricá wood specie.
Shear
Pinheiro [13] obtained, for the Pinus sp. Treated with CCB, shear strength values of 10% greater than the shear strength fro m the Pinus sp wood species untreated. Cv(%) 15 14 By ANOVA, it can be assumed that treatment with CCB did not change the hardness in the perpendicular direction to the grain (P-vale>0,05), this did not occur with the hardness in parallel direct ion to the grain of Paricá wood specie, present P-value less than the significance level set, showing a decrease of 28.9% for the wood treated. This fact is probably due to the occurrence of surface cracking in the treatment process under pressure.
Hardness Parallel and Perpendicul ar to the Grai n
Rocco Lahr et al. [14] obtained increases of 58% to 68% in hardness parallel and hardness perpendicular to the grain of Pinus elliotii wood specie. Table 3 presents the results for co mpression tests in parallel d irection to the grain. The strength and stiffness in compression parallel to grain of Paricá wood treated and untreated showed statistical equivalence between means (P-value>0,05). Thus, it can be assumed that the treatment did not change the strength and stiffness in co mpression parallel to grain of Paricá. Pinheiro [13] reached increments of 17% in co mpressive strength parallel to grain of Pinus sp. treated with CCA, and average gain of 55% for the wood treated with CCB.
Conclusions
In view of what is pointed out in the foregoing, we conclude that the impregnation of CCB in the Schizolobium amazonicum wood specie did not changed the properties apparent density, shear strength, hardness, strength and stiffness in comp ression parallel to the grain.
The hardness strength parallel to grain was decreased by 28.9%, this fact is probably due to the occurrence of surface cracking in the treat ment process under pressure. | 2019-04-28T13:09:10.303Z | 2013-08-01T00:00:00.000 | {
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255750063 | pes2o/s2orc | v3-fos-license | Focal Liver Lesions other than Hepatocellular Carcinoma in Cirrhosis: Diagnostic Challenges
Abstract Liver cirrhosis is associated with regenerative nodules and an increased risk of developing hepatocellular carcinoma (HCC). However, other benign and malignant liver lesions may also occur. Differentiating the other lesions from HCC is important for further therapeutic decisions. This review discusses the characteristics of non-HCC liver lesions in cirrhosis and their consequent appearance on contrast-enhanced ultrasonography (CEUS) with consideration of other imaging. Knowledge of this data would be helpful in avoiding misdiagnoses.
INTRODUCTION
Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with over 500,000 new cases diagnosed annually worldwide. [1,2] It is the second leading cause of cancer-related mortality in the world. [3] The yearly incidence of HCC among patients with clinical liver cirrhosis ranges from 0.5% to 11.0%. [4][5][6] Seventy-six percent of all solid lesions in 282 cases of liver cirrhosis corresponded to HCC in a German multicenter study. [7] In a contrast-enhanced ultrasonography (CEUS) study by Terzi et al. [8] 81% of 1006 solid lesions in surveillance in chronic liver disease corresponded to HCCs. [8] HCC has typical findings on CEUS, computed tomography (CT), and magnetic resonance imaging (MRI) due to the peculiarity of its vascularization, which make it possible to diagnose it noninvasively. According to the European Association for the Study of the liver (EASL) and the American Association for the Study of Liver Diseases (AASLD) guidelines, contrast-enhanced CT including the arterial and portal venous phase and MRI with liver-specific contrast medium should be used to diagnose/rule out HCC in cirrhosis. Other international guidelines also give equivalent importance to CEUS, [9][10][11][12][13][14][15][16][17][18][19] and overall, the diagnosis of HCC and other non-HCC lesions is primarily made by contrast-enhanced imaging.
In this context, all other focal liver lesions can also occur in liver cirrhosis with a frequency of about 20%. [7,8] Table 1 summarizes an overview of the frequency of HCC and non-HCC in liver cirrhosis.
Knowledge of the different types of benign and malignant lesions that can arise in the context of cirrhosis and about their ultrasound appearance on B-mode and CEUS is important to avoid misdiagnoses and, thus, the wrong therapeutic decision. This review focuses on this topic.
NOT EVERY LESION IN LIVER CIRRHOSIS IS AN HCC
Imaging diagnosis of HCC in liver cirrhosis is based on the identification of the typical characteristics, which differ according to imaging techniques or contrast agents and are as follows: arterial phase hyperenhancement (APHE) followed by washout in the portal venous or delayed phases on CT and MRI using extracellular contrast agents or gadobenate dimeglumine, APHE with washout in the portal venous phase on MRI using gadoxetic acid, and APHE with late-onset (> 60 s) washout of mild intensity on CEUS. [26] Arterial hyperenhancement is also shown by other FLL: hemangiomas, focal nodular hyperplasia (FNH)/FNH-like lesions, hepatocellular adenomas (HCAs), many dysplastic and very few regenerative nodules, cholangiocellular carcinoma (CCC), metastases, and lymphomas, whereas in CEUS, very specific diagnostic vascularization patterns of the vascular architecture in the arterial phase are revealed in detail. [9][10][11]17,19,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41] The probability that a new lesion smaller than 10 mm corresponds to HCC is low. [26,42,43] When a small hypervascular lesion is first detected on CT or MRI, it may be a true nodule, but it may also be a vascular phenomenon such as an arterioportal shunt. It has been published that 70%-90% of small (1-2 or < 2 cm) hypervascular foci that can only be seen on arterial phase (hyper arterial phase enhancement [HAPE]) imaging do not correspond to HCC. [44,45] Holland et al. [45] assessed small liver lesions < 20 mm on MRI, which exclusively showed hyperenhancement in the arterial phase on MRI.
In the liver explants, 93% of these lesions were benign.
The prevalence of HAPE-only lesions in patients with severe cirrhosis before transplantation was 35%. The majority of HAPE-only lesions (93%) had no correlative pathologic findings, were benign, and may have represented regenerative nodules or arterial-portal venous shunts. [45] In the study by Jang et al. [46] of 59 patients at high risk of HCC and having 1-2 cm nodules with APHE on CEUS, only 26 (44%) corresponded to HCC and 33 (56%) were benign (20 regenerative nodules [RNs]/dysplastic nodules [DNs], 11 hemangiomas, two focal fat sparring). Khalili et al. [47] found in 93 indeterminate 1-2 cm nodules on dynamic CT, MRI, and CEUS that the prevalence of HCC was low. Eighty-five percent of the FLL showed characteristics of benignity and the only significant predictors of malignancy were arterial phase hypervascularity and synchronous HCC elsewhere in the liver. Among a total of 138 nodules, 4% were hemangiomas. [47] In the study by Kim et al. [48] 23% of all newly diagnosed solid lesions measuring 10-20 mm on MRI corresponded to hemangiomas during surveillance for HCC. [48] CEUS might be effectively used for characterizing indeterminate lesions on CT and MRI or when these two are contraindicated. [49] If a small nodule does not show arterial hypervascularity on CEUS, it is unlikely to show typical features of hypervascular HCC on CT or MRI. [44] Forner et al. [22] differentiated 89 nodules detected by ultrasound surveillance in liver cirrhosis. Among them, 67.4% were HCC, 1.1% were CCC, and 31.5% were benign lesions (RN/DN 27.0%, hemangioma 3.4%, FNH 1.1%).
In a retrospective study by Compagnon et al. [50] of explanted livers following liver transplantation, 16.7% were false positive for pretransplant HCC diagnosed on imaging without biopsy. These were nine DNs, five RNs, one CCC, one hemangioma, and four were not lesions. All lesions were smaller than 30 mm. Sensitivity, specificity, positive predictive value, and negative predictive value for the preoperative clinical and radiologic diagnosis of HCC were 89%, 94.3%, 77%, and 93.3%, respectively. [50] In a study by Lee et al. [51] among 837 liver resection cases for presurgical imaging diagnosed as HCC without biopsy confirmation, 2.2% false positively diagnosed HCC cases were found. Among the false positives, 0.8% were benign FLL (hemangioma, inflammation, cortical adenoma, DN, angiomyolipoma, bile duct adenoma, and nonneoplastic liver parenchyma) and 1.3% were malignant FLL (cholangiocarcinoma, hepatoblastoma, lymphoepitheliomalike carcinoma, ovarian cystadenocarcinoma, and nasopharynx carcinoma metastasis). The false-positive rate in nodules ≤ 2 cm was 3.4%. Table 2 presents the false-positive diagnoses on imaging without CEUS and biopsy for HCC. diagnosis is made solely on the basis of hypervascularization in the arterial phase.
Cysts
In an autopsy study without liver cirrhosis, the prevalence of liver cysts was 15%. [52,53] An MRI study revealed no significant differences between the frequency of liver cysts in cirrhotic and noncirrhotic livers (17.5% and 13.8%, respectively) ( Table 4). [54] Dodd et al.'s [21] autopsy study of 508 liver cirrhosis explants discovered that small liver cysts occur with similar rates in healthy and cirrhotic livers. Large liver cysts are a rare finding in cirrhosis, and 13 out of 508 were peribiliary cysts. [21] Peribiliary cysts are retention cysts of the peribiliary glands, located in the periductal connective tissue. They typically present as small cysts on both sides of the portal vein, often with a hilar distribution, and can be confounded with dilated bile ducts; they, however, have no connection with biliary ducts (biliary cysts are one side of the portal vein). This asymptomatic condition has been reported under the labels "multiple cysts in the hepatic hilum", "hepatic cysts of periductal origin", and "multiple hepatic peribiliary cysts", [56] The first description was given by Nakanuma et al. [57] in 1984. Cirrhosis and portal hypertension are the most frequently associated conditions with peribiliary cysts. [55] The frequency of peribiliary cysts in liver explants was 1%. [55] Liver cysts are easy to diagnose on B-mode abdominal sonography, and usually, CEUS examination and other radiologic imaging are not necessary.
Hemangioma
Regarding the frequency of liver hemangiomas in liver cirrhosis, there are reports of a lower frequency [7,20,21] comparable to patients without liver cirrhosis. [54] In an autopsy study by Dodd et al. [21] hemangiomas were found in only 1.7% of all cirrhotic livers. [21] This is significantly lower than in non-selected autopsy studies without liver cirrhosis, which had a 20% incidence of hemangioma. [52,53] It appears that the process of cirrhosis (i.e., necrosis and fibrosis) obliterates existing hemangiomas. [21] Autopsy studies reveal that previous imaging (CT, MRI) had missed hemangiomas in liver cirrhosis. [21,58] Hemangiomas in liver cirrhosis usually become smaller and gradually develop fibrosis. This process already begins with liver fibrosis. [59] Nevertheless, some controversial data exists here. Brancatelli et al. [58] reported hemangioma sizes up to 10.0 cm in the cirrhotic liver. In patients with liver disease, the hemangiomas were more often solitary. [60] In a retrospective CT follow-up of hemangiomas in cirrhotic liver, Brancatelli et al. [58] demonstrated a decrease in size in 44% of cases. Retraction of the liver capsule was discovered in 31% of the patients. Vernuccio et al. [61] demonstrated in a case example that the process of sclerosis generally starts at the center and then extends to the entire lesion. Fibrotic degeneration may lead to retraction of the capsule or concavity of the entire lesion. These changes can reduce the size of the hemangiomas. This may explain why the hemangiomas in liver cirrhosis are smaller compared to a healthy liver. [21,58,59,61] The sclerosing process leads to a reduced contrast uptake to varying degrees.
On ultrasonography in the healthy liver with normal echogenicity, hemangiomas present as smoothly circumscribed, mostly homogeneous, hyperechogenic lesions and do not require contrast-enhanced sonography. [9,19,62] For this reason, typical hemangiomas are number-wise underrepresented in CEUS studies for the evaluation of liver lesions. [7] Depending on the liver echogenicity, hemangiomas may also appear isoechogenic or hypoechogenic. This means that other differential diagnoses come into consideration. Fortythree percent of the hemangiomas showed feeding vessels in duplex sonography and 7% showed homogeneous hyperenhancement. [62] A typical feature of hemangiomas in CEUS is a smooth contrast-receiving ring in the arterial phase with a peripheral, discontinuous nodular (syn.: globular) enhancement with progressive centripetal contrast ( Figure 1). The fill-in in the late phase can be complete or incomplete. (Partially) thrombosed hemangiomas do not fill up completely ( Figure 2). [9,19] Atypical CEUS features show small hemangiomas (< 15 mm) and larger hemangiomas. Hemangiomas below 10 mm, in particular, tend to lack the typical hemangioma contrast enhancement and are a challenge. [62] Small lesions, in particular, can fill up very fast and completely in the arterial phase. Hemangiomas with arteriovenous shunts Table 4: Cysts in liver cirrhosis: Similarities and differences with the noncirrhotic liver Common features with cysts in noncirrhotic liver Special features in the cirrhotic liver No significant differences between the frequencies [54] Large cysts are rare [21] Small liver cysts occur with similar rates [21] Peribiliary cysts (retention cysts of the peribiliary glands; the accumulation of fibrosis encases the glands, leading to an increase in size) [21,55] (high-flow or shunt hemangiomas) show a very rapid homogeneous hyperenhancement in the arterial phase and sometimes do not reveal the annular centripetal flow pattern. Here, differential diagnosis with other completely hyperenhanced lesions in the arterial phase is difficult (FNH, adenomas, HCCs, some CCCs and some metastases). [9,19] A possible explanation for the rapid enhancement of small hemangiomas is a hyperdynamic status with large arterial inflow, rapid tumor enhancement, and consequently large and rapid outflow. [62] But mostly these shunt hemangiomas are hyperenhanced in the portal venous and late phase, which is considered a benign criterion in the healthy liver. [29] Some hemangiomas are hypoenhanced in the late phase (especially when located superficially near the transducer head or after continuous insonation). [63] This makes it difficult to distinguish them from malignant liver lesions. Regressively altered hemangiomas may be completely sclerosed and are smoothly irradiated, but remain avascular and do not fill up at all. It is to be expected that the regressive changes that hepatic hemangiomas undergo in liver cirrhosis and the specific features of vascularization in liver cirrhosis are also reflected in the CEUS appearance of hemangiomas in liver cirrhosis. Since every arterially hypervascularized hepatic lesion in liver cirrhosis is suspicious for HCC and not every HCC shows hypoenhancement in the late phase, difficult differential diagnostic challenges can arise, especially in the case of shunt hemangiomas. The coincidence of HCC and hemangiomas in liver cirrhosis has been described. [62] Also, in patients with liver cirrhosis, 13% (2/15) hemangiomas corresponded to shunt hemangiomas. [62] The correct diagnosis of each individual focal lesion in cirrhosis is important for further therapeutic decisions. Misdiagnosis of a hemangioma as HCC can misinterpret a curative therapeutic option into a supposedly palliative situation. D'Onofrio et al. [23] studied 36 hemangiomas in liver cirrhosis, with a range of 0.6-3.5 cm. On B-mode ultrasound examination, 32 were hyperechoic, one was hyperechoic with a peripheral hypoechoic halo, two were hypoechoic, and one was hypoechoic with a peripheral hyperechoic halo. In Doppler ultrasound (US), all the hemangiomas displayed peripheral or intranodular venous vessels. Furthermore, in CEUS imaging, all the cases exhibited peripheral globular enhancement with centripetal fill-in in the arterial phase; in the late phase, two appeared isoechoic and 34 appeared homogeneously
Figure 1: An 80-year-old female presented with thrombosed hemangioma (in between markers) and liver cirrhosis after hepatitis C infection. She had normal AFP and no previous known tumor disease. Hypoechoic inhomogeneous liver lesion with echogenic rim on B-mode ultrasonography (A). No enhancement in the arterial phase, only suggested rim (B). At the beginning of the portal venous phase, the lesion is not enhanced, except for a few vascular pixels. These are seen in the marginal area and cannot be clearly assigned for differential diagnosis (C). In the late CEUS phase, the lesion remains completely avascular.
A retraction of the liver contour is seen above the lesion. On MRI, the lesion was assigned as a thrombosed hemangioma. Thrombosed hemangioma with capsular retraction is a finding that has been described on MRI for hemangiomas in liver cirrhosis. Capsular retraction can also be seen in tumors (D). CEUS: contrast-enhanced ultrasonography; MRI: magnetic resonance imaging; AFP: alpha-fetoprotein.
hyperechoic. [23] In the study by Jang et al. [46] 59 patients were monitored for HCC. In this study, 23% of all nodules with arterial hyperenhancement corresponded to hemangiomas on CEUS. All of them showed peripheral nodular enhancement and progressive fill-in. [44] In these studies, hemangiomas in liver cirrhosis in the arterial phase showed typical contrast behaviors for diagnosis. Diagnostic difficulties were not evident here.
In the study by Terzi et al. [8] of 1006 nodules in cirrhosis, a few high-flow hemangiomas fell in the LR-4 category. The authors concluded that this finding is not worrisome not only for its minimal numeric impact (< 1% of all lesions), but also mostly for the fact that MRI is highly accurate in establishing the diagnosis of hemangioma. [8] However, there are also a few experiences to the contrary. Smaller, rapidly enhancing hemangiomas appear as uniformly enhancing lesions in the arterial phase, and therefore, it can be difficult to differentiate them from small HCCs simply based on the contrast patterns, especially in the MRI performed with hepatobiliary contrast agents such as gadoxetate disodium. On images obtained with this agent, hemangiomas can exhibit pseudo washout in the transitional phase: the lesion appears hypointense relative to the surrounding parenchyma owing to the rapid uptake of gadoxetate disodium by the background parenchyma. This pseudo washout phenomenon is more gradual than true washout in malignant tumors. [64,65] Therefore, for the differential diagnosis, the pattern in the T2 nonenhanced phases is highly relevant. Brancatelli et al. [58] reported in the follow-up of hemangiomas in liver cirrhosis the loss of some typical features, such as nodular peripheral enhancement and isoattenuation to blood vessels. [58] Vernuccio et al. [61] reported that the fibrotic degeneration may lead to peripheral capsular retraction or concavity over the lesion and loss of the typical imaging features of hemangiomas, including T2 hyperintensity, nodular peripheral enhancement with centripetal filling, and the enhancement parallel to blood vessels, and that central fibrotic degeneration might result in central hypointensity in T2-weighted images and a lack of T2 shine-through compared to lesions occurring in healthy or mildly fibrotic livers. Unfortunately, there are no comparative statements on the CEUS patterns in hemangiomas not showing a typical appearance on MRI. Sclerosed hemangioma may appear as a hypoenhancing lesion or display arterial phase rim hyperenhancement. The decreased enhancement in the dynamic study correlates with the histologic degree of sclerosis. Galia et al. [66] noted that fibrotic hemangiomas are usually irregular in shape. Duran et al. [59] observed a smaller T2 shine-through effect for hemangiomas in liver cirrhosis with MRI, and this effect is less common in flash-filling hemangiomas than in other types. All other MRI parameters were similar compared to noncirrhotic patients. Hemangiomas are rarely surrounded by fibrotic tissue distorting the liver, paradoxically appearing hypovascular. To differentiate them from other lesions, especially hypovascular HCC, one should remember that these hemangiomas commonly exhibit irregular margins and marked hypoattenuation compared to the surrounding liver in noncontrast and postcontrast images. Of course, marked signal hyperintensity in T2-weighted MRI remains helpful. [59,67] The changes in hemangiomas in the cirrhotic liver are shown in Table 5.
FNH and FNH-like nodules
The FNH in the normal liver and the FNH-like nodule which only develops in the cirrhotic liver are morphologically identical. Whether it is an FNH or an FNH-like lesion in liver cirrhosis cannot be distinguished on imaging. FNH-like nodules are focal lesions occurring in liver cirrhosis and are morphologically and immunohistochemically very similar to classical FNH in an otherwise normal liver. [68][69][70][71] Histologic analyses revealed that the FNH-like nodules showed many unpaired arteries with thick-walled blood vessels, and this resulted in hypervascular enhancement that mimicked HCC on contrast-enhanced CT. [69] However, it is striking that FNH is diagnosed much less frequently on imaging in liver cirrhosis than in noncirrhotic liver. From this, it can be assumed that FNH also undergoes a morphological change in the cirrhotic liver or is more difficult to identify. In two autopsy studies [20,21] and the German multicenter study, [7] no cases of FNH in liver cirrhosis were described. This entity is the exception in liver cirrhosis and in the mirror of so far published papers, practically irrelevant. But FNH-like lesions should not result in the misdiagnosis of HCC. The risk of a falsenegative diagnosis is more relevant than that of a falsepositive diagnosis of HCC. However, the concern of this review is to consider the changes that non-HCC lesions undergo in the process of liver cirrhosis. Therefore, FNH and FNH-like lesions are also discussed and examples shown.
FNH-like nodules were diagnosed in 15% of 130 explanted cirrhotic livers. Of them, 75% were smaller than 10 mm. [71] It was hypothesized that the FNH-like nodules arise as a local hyperplastic response to vascular alterations in cirrhosis. The presence of esophageal varices and pretransplant treatment with chemoembolization were independently and significantly associated with the presence of FNH-like nodules. [71] Multiple FNH-like nodules were seen in 37% of cases. [71] No associations were found between FNH-like nodules, on the one hand, and low-grade DNs, high-grade DNs, and HCCs, on the other hand.
6.4% of FNH showed hypoenhancement in the late phase. This study also included patients with liver cirrhosis. It did not further differentiate which patients with FNH and hypoenhancement in the late phase were involved. [72] When FNH in liver cirrhosis in the arterial phase does not show the wheel spoke pattern and centrifugal enhancement flow pattern and the lesion is iso-or even hypoenhanced in the late phase, differentiation from HCC can be very difficult.
In the report by D'Onofrio et al. [23] among 128 patients with liver cirrhosis and 207 focal lesions, 101 were benign lesions and eight of them were FNH (mean diameter 3.45 ± 1.11 cm; range 2.5-6.0 cm). On B-modeultrasonography, these appeared hypoechoic in six cases, slightly and heterogeneously hyperechoic in one case, and heterogeneous in the remaining one case. On Doppler US, six displayed peripheral and intranodular arterial vessels. In CEUS, seven lesions enhanced during the arterial phase. In the late phase, six lesions appeared isoechoic and two appeared hypoechoic. The two hypoechoic lesions were properly characterized via MRI with a liver-specific contrast agent.
FNH-like nodules are rare and small. [71] Some of them were detected by radiologic imaging and misclassified as HCC, as described in the studies by Quaglia [73] and Libbrecht. [71] In both studies, only larger FNH-like nodules were detected. [71,73] Lee et al. [69] described that FNH-like nodules showed hypervascular enhancement in the arterial phase of the contrast-enhanced CT images, and this was followed by a washout pattern in the delayed phase, which is a feature generally considered to be highly suggestive of HCC in liver cirrhosis. They appeared as high-signalintensity masses on SPIO-enhanced MRI. In the study by Choi et al. [74] 33% (3/9) FNH-like lesions in liver cirrhosis or Budd Chiari syndrome were misinterpreted as HCC by radiologic imaging (CT, MRI). All of them showed nodules > 1 cm in diameter with arterial enhancement and portal/delayed washout on dynamic CT. [74] Among 62 patients with FNH-like lesion or FNH who underwent percutaneous needle biopsy, four patients (6.5%) were misdiagnosed as having HCC and two patients (3.2%) had inconclusive results by a first needle biopsy. [74] Histologic differentiation of FNH or FNH-like lesion from well-differentiated HCC by needle biopsy may be difficult due to a modest increase in cell density with an irregular trabecular pattern and unpaired arteries and a diffuse capillarization of the sinusoids. [71,74] Loh et al. [75] described an FNH-like lesion in a cirrhotic liver with radiologic features on CT and MRI that mimicked an intrahepatic cholangiocarcinoma (ICC) (Figure 3). [75] It is possible that with increasingly sensitive high-resolution modern imaging, more of these small FNH-like lesions could be detected in liver cirrhosis, but then also, they would be difficult to be differentiated ( Figure 4). [69,71,73] As some FNH lesions (2/8, 25%) in cirrhotic liver in the study of D'Onofrio et al. [23] also showed hypoenhancement in the late phase on CEUS, differential diagnosis with HCC is very difficult in these lesions. [23] Also, in radiologic imaging with CT and MRI, 33% of FNH/FNH-like lesions in liver cirrhosis were misinterpreted as HCC. [74] One should consider the possibility of an FNH-like nodule and, in case of doubt, perform histologic confirmation. However, as the data of Choi et al. [74] show, histology by needle aspiration also does not provide definitive confirmation in all cases. [74] Table 6 summarizes the specifics of FNH.
Hepatocellular adenoma
HCA is a tumor that is rarely diagnosed in the noncirrhotic liver. Four subtypes are distinguished: [76] inflammatory HCA (30%-50% of all HCAs), hepatocyte nuclear factor 1 alpha (HNF1α)-inactivated HCA (approx. 40% of cases), β-catenin-mutated HCA (5%-10% of cases), and unclassified HCA (5%-10% of cases). The β-catenin-mutated HCA has an increased risk of malignant transformation, and the HCA is > 5 cm in males. In the German multicenter study, HCA was diagnosed in one of the 216 patients with liver cirrhosis (0.3%). [7] In an autopsy study with 596 cases of liver cirrhosis, 43.6% had a liver lesion, among which 1.5% were adenomas, surprisingly more than hemangiomas (1.2%). [20] In CEUS, the typical behavior of HCA includes rapid centripetal filling in the arterial phase and persistent enhancement in the portal and delayed phases. None of the filling patterns in the arterial phase is specific to HCA, and they may also be observed in HCC or in hypervascularized metastasis. In the portal venous phase, they may show slight hypoenhancement, resulting in a difficult differential diagnosis from HCCs, whereas hypoenhancement in HCC Descriptions Peculiarities of FNH in liver cirrhosis compared to noncirrhotic liver FNH is very rare in comparison to noncirrhotic liver; [7,8,20,21] FNH-like nodules arise as a response to vascular alterations in cirrhosis [71] Differential diagnostic problems on imaging compared to HCC Arterial hyperenhancement with iso-and/or hypoenhancement in the late phase (75% respectively 25%); [23] Hypervascular enhancement with washout in the delayed phase on CE-CT [69,74] FNH: focal nodular hyperplasia; HCC: hepatocellular carcinoma; MRI: magnetic resonance imaging; CEUS: contrast-enhanced ultrasonography. typically starts after 60 s. [9,19,27,28,77] However, depending on the subtype, the behavior of HCA in the portal venous and late phases may differ. [78] Inflammatory HCA showed arterial hyperenhancement with centripetal filling, peripheral rims with persistent enhancement, and central washout in the late venous phase (sensitivity 64%; specificity 100%). In HNF1α-inactivated HCA, isoenhancement or moderate hyperenhancement with mixed filling in the arterial phase and isoenhancement in the portal and late portal venous phases were detected. Homogeneous hyperechogenicity on B-mode ultrasonography was the most specific pattern (sensitivity 88%; specificity 91%) and correlated with diffuse fat distribution on MR images. In the unclassified and β-catenin-activated HCA, CEUS showed features of benign hepatocellular tumors without specific features. [78] On MRI, HCAs are hyperintense or isointense on
Figure 4: A 38-year-old female presented with suspected FNH-like nodule, alcohol toxic cirrhosis (in between markers and shown by arrows). She had a history of continued alcohol abuse and had untreated hepatitis B and hepatitis C. She had undergone repeated hospitalizations with ascitic decompensation. Newly diagnosed hepatic lesion not present 6 months previously on B-mode ultrasonography (A). In CEUS, wheel spoke enhancement early arterial (B), corresponding temporal mapping also in parametric imaging (C). The lesion was enhanced in the arterial phase (D), isoenhanced or slightly hyperenhanced in the portal venous phase (E), and slightly hyperenhanced in the late phase (F). The lesion also remained iso-to slightly hyperenhanced in the further course of examination until 3 min p.i. MRI also showed an APHE hepatic lesion. The wheel spoke enhancement and persistent hyperenhancement on CEUS were suggestive of FNH.
However, a corresponding lesion was not pre-documented. Therefore, sonographically assisted biopsy with 4× percutaneous access was performed. This did not result in a definitive diagnosis -no HCC, but also no FNH. The CEUS findings were compatible with an FNH. Since the lesion was not known before, it could be in the overall context with an FNH-like nodule. The contrasting course in the CEUS argues against a regenerated node. On further hospitalization with ascitic decompensation, the lesion remained unchanged and the AFP was normal even after 6 months. FNH: focal nodular hyperplasia; CEUS: contrastenhanced ultrasonography; APHE: arterial phase hyperenhancement; HCC: hepatocellular carcinoma; MRI: magnetic resonance imaging; AFP: alpha-fetoprotein.
precontrast T2-weighted images and isointense or hypointense on T1-weighted images. HCA shows arterial enhancement and washout in the portal venous phase and hypointensity in the hepatobiliary phase. [79] There are few case reports of HCA in the cirrhotic liver, including a patient with liver cirrhosis in hepatitis B [79] and another with glycogen storage disease IV. [80] Seo et al. [79] reported an FLL in hepatitis B that appeared on MRI as a benign hepatocellular lesion such as FNH. Because of an increase in size in the follow-up, an ultrasound-guided biopsy was performed. Histologic findings revealed a benign hepatocellular nodule indicative of HCA. In addition, imaging showed liver cirrhosis. Alphafetoprotein (AFP) was normal. On follow-up, surgical resection was performed due to increase in size and a "nodule-in nodule" appearance on CT image. The histology revealed an HCC. This case demonstrates the difficulty of diagnosing HCA on imaging, especially in liver cirrhosis. [79] Calderaro et al. [81] described two patients with liver cirrhosis caused by metabolic syndrome and alcohol use (patient 1: female, alcohol intake, obesity [body mass index {BMI} = 30], type 2 diabetes; patient 2: male, alcohol intake, obesity [BMI = 30], and inflammatory HCA) and one patient with metabolic syndrome without alcohol use (patient 3: male, obesity [BMI = 37], type 2 diabetes) with severe steatosis hepatis/histologically liver cirrhosis and multiple inflammatory HCAs. Microscopic examination initially diagnosed FNH in patient 1 and an RN in patient 2. Only complete immunohistochemical staining and molecular analysis confirmed the diagnosis of inflammatory HCA. The authors concluded that if HCA is known to arise in nonfibrotic, noncirrhotic liver, their observations showed that inflammatory adenoma and inflammatory liver adenomatosis might also rarely develop in the setting of chronic liver disease and cirrhosis. Interestingly, all three patients had chronic liver disease related to well-established risk factors for inflammatory adenoma development in patients without fibrotic livers, namely, metabolic syndrome and alcohol intake. [81] HCAs are usually homogeneously hyperenhanced on CEUS in the arterial phase. They may show slight hypoenhancement in the portal venous phase. [9,19] Whether HCAs in liver cirrhosis show a different appearance in CEUS is unclear. This makes the differential diagnosis to HCC in liver cirrhosis difficult. Moreover, as the case report of Calderaro et al. [81] showed, needle biopsy is also not reliable in diagnosing HCA. The challenge in differential diagnosis for liver cirrhosis is primarily with HCC. According to data from Seitz et al. [7] the ratio of HCA to HCC in liver cirrhosis is 1:216. The incidence of HCA in liver cirrhosis is many times lower than HCC.
Metastases
There are a large number of autopsy studies [82][83][84][85][86] and metaanalyses of autopsy studies [86][87][88] showing that metastases of extrahepatic tumors are rarer in liver cirrhosis than in noncirrhotic livers. In a recent meta-analysis with biopsies, excisions, and autopsies from 1453 cirrhotic livers with liver masses, only 1.7% were metastases. [88] This applies to metastases of colorectal carcinomas as well as other tumors.
Several possible causes for the lower frequency of metastases in liver cirrhosis are discussed. A cirrhotic liver does not represent a breeding ground for the development of tumor cells because fibrosis and distortion of small hepatic capillaries, which occur in cirrhosis, known as sinusoid capillarization, represent a mechanical obstacle to the establishing of tumors. Portal venous flow is reduced by portal hypertension, and tumor seeding is impeded. Hepatofugal flow impedes the spread of metastasis to the liver. Other factors are Kupffer cell activation, increased concentrations of metalloproteinase inhibitors, and dysfunction of the lectins. [88][89][90] In the noncirrhotic liver, metastases can have a variable and unpredictable appearance in native B-mode ultrasonography: not only hyperechoic and isoechoic, but also hypoechoic lesions, different types of boundaries, a hypoechoic rim (halo sign). The lesion may contain necrotic avascular anechoic or hypoechoic areas. How metastases are to be differentiated in the cirrhotic liver depends on the degree of alteration and the RNs present, but in general, B-mode gray-scale US is often insufficient in small lesions to achieve a diagnosis of metastasis or even of malignancy. A typical feature in CEUS is hypoenhancement from the early portal venous to the late phase. Most metastases show early hypoenhancement from the portal venous phase. In the arterial phase, rim enhancement with irregular vessels is a typical feature. The extent of arterial contrast depends, however, on the primary tumor. For example, metastases of neuroendocrine carcinomas may be arterially hyperenhanced and show hypoenhancement only late. This results in diagnostic challenges with HCC. If hypoechoic lesions in a cirrhotic liver show a washout very early in the portal venous phase, metastases should be included in the differential diagnostic considerations, [9,19,72] following what has been described here just above.
Metastases are classified as LR-M (Liver Imaging with thrombi (B). There is also a small thrombus in the pars umbilicalis, which is hyperenhanced in the arterial phase in CEUS, and thus corresponds to a tumor thrombus (C). On CEUS, the hyperechogenic lesions in the right hepatic lobe are isoechogenic, slightly inhomogeneously enhanced in the arterial (D) and portal venous (E) phases. The adjacent thrombosed portal venous branch is hypoenhanced (F). In the late phase, the liver lesions are hypoenhanced (G). CEUS was used to perform ultrasonography-guided puncture of the liver lesions and the thrombosed portal vein branch. Histologically, a metastasis of the sigmoid carcinoma was found. There was no evidence of hepatocellular carcinoma. CEUS: contrast-enhanced ultrasonography; AFP: alpha-fetoprotein.
with liver cirrhosis had CCC. However, the pattern of CEUS HCC diagnosis at that time (in the year 2011) did not adopt the refinements first introduced by the European Federation of Societies for Ultrasound in Medicine and Biology guidelines in 2013 and subsequently by the LIRADS system in 2017 even in the group without liver cirrhosis, CCC was rare (only 3.3%). [7] In other studies, only 1.1% [22] and 1.3% [24] of all newly detected nodules in liver cirrhosis were histologically confirmed as CCC. In the study by Terzi et al. [8] 4% of all nodules in liver cirrhosis corresponded to a CCC. [8] On CEUS, cholangiocarcinoma shows arterial hyperenhancement, often a rim sign, and a rapid washout in the portal venous phase. Unlike HCC, the onset of washout occurs early in CCC, usually before 1 min, and the degree of hypoenhancement in the venous phase is more marked in CCC than in HCC. [8,[94][95][96] On CEUS, CCC shows the criteria of LR-M. Differential diagnoses are primarily metastases. In the study by Terzi et al. [8] the majority of CCC tended to segregate in the LR-M category, together with the mixed HCC-CCC tumors and metastases. Also, 12.5% (5/40) of the CCCs showed CEUS behavior of LR-3 and 10% (4/40 ) showed LR-4 behavior. The mean size was 1.6 cm in CEUS-LR-3 and 2.7 cm in LR-4; no case of misdiagnosis of LR-5 for CCC occurred. [8] Using the LR-M criteria to differentiate CCC from HCC, sensitivity, specificity, and accuracy were 97.3%, 87.7%, and 92.3%, respectively. [97] In the differentiation of ICC from LR-M HCC, the rim APHE is the most important feature. Rim APHE plus elevated CA19-9 and normal AFP are strong predictors of CCC rather than LR-M HCC. In differentiation of CCC and LR-M HCC, early washout and marked washout have limited value. [98] For differential diagnostic reasons, histologic confirmation of any suspected malignant lesion without the typical features of HCC (corresponding to the LR5 pattern in cirrhosis) usually should be performed.
Lymphoma
Primary hepatic lymphoma (PHL) manifests in the liver and perihepatic lymph nodes without any involvement of other organs or leukemic changes in the peripheral blood for at least 6 months after diagnosis. PHL is rare, less than 1% of all non-Hodgkin's lymphomas; most PHLs are B-cell lymphomas. [99] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of PHL. [100] PHL accounts for 0.4% of extranodal NHL and 0.016% of all NHL. [100,101] PHL has also been described in liver cirrhosis. [100,101] Primary hepatic extranodal marginal zone lymphoma of mucosaassociated lymphoid tissue (MALT) has been reported to be the second most common variant of lymphoma after DLBCL. Histologic liver assessment is required for the accurate diagnosis of primary hepatic extranodal marginal Reporting and Data System [LIRADS] malignant) in the CEUS-LIRADS algorithm. [12,14,40,[91][92][93] In contrast to HCC, metastases often have a rim sign and usually an early washout in the portal venous phase. [9,19,72] In the study by Terzi et al. [8] with 848 patients with liver cirrhosis and 1006 nodules, 87% were malignant, but only 0.25% were metastases. These are classified as LM-R according to CEUS-LIRADS. The two metastases showed arterial phase rim enhancement or APHE and an early washout. The CEUS pattern LR-M showed few HCC, CCC, HCC/ CC, and lymphoma. The authors concluded that the LR-M class requires histologic confirmation ( Figure 5). [8] The following conclusion could be drawn from this: if a solid lesion is detected in a cirrhotic liver, it is very likely to be HCC. However, this does not mean that patients with liver cirrhosis can never develop liver metastases.
Intrahepatic cholangiocarcinoma
CCC may be a mimicker of HCC in the setting of noninvasive diagnosis of HCC as both can show hypervascularity and washout on CEUS. After HCC, ICC is the second most common primary tumor in liver cirrhosis. The combined HCC/ICC is also possible and can only be differentiated histologically. The most important differential diagnoses of CCC are HCC and liver metastases or all liver tumors presenting as LR-M because of the imaging characteristics. [12,14,40,[91][92][93] ICC can appear on CEUS with different enhancement patterns in the arterial phase: peripheral irregular rimlike enhancement, heterogeneous hyperenhancement, homogeneous hyperenhancement, and heterogeneous hypoenhancement. [9,19] ICC is characterized by washout in the early portal venous phase and marked hypoenhancement in the late phase ( Figure 6). [9,19] On noncontrast CT, CCCs are usually seen as hypoattenuating focal liver masses with irregular margins. On MRI, CCCs are hypointense on T1-weighted images and moderately hyperintense on T2-weighted images, with the latter often associated with central hypointensity corresponding to areas of fibrosis. Typically, contrastenhanced images show rim enhancement during the arterial phase, followed by progressive and concentric enhancement after extracellular contrast administration due to the presence of marked fibrous stroma. [67] There has been a controversy on the use of CEUS because ICC can be misdiagnosed as HCC, and CEUS has been subsequently excluded from the diagnostic tests for HCC in the AASLD practice guidelines from 2011 onward. But CCC in liver cirrhosis is significantly rare compared to HCC. In the German multicenter study, 2.5% of patients variable, they may be hyperenhanced on arterial phase images and hypointense on hepatobiliary MR phase due to lack of hepatocytes. They show signal restriction on high-b value diffusion-weighted imaging. Nevertheless, differentiation from HCC can be difficult. [67,99] Fu et al. [104] describe the misdiagnosis of HCC in primary hepatic MALT lymphoma and hepatitis B. Under antiviral therapy, newly diagnosed FLL of 11 and 5 mm were held as HCC on MRI and PET-CT. Laparoscopic resection of the left lateral lobe was performed. Postoperative histology diagnosed hepatic MALT lymphoma. [105] Due to this lack of specific clinical, laboratory, and imaging features, definitive diagnosis of PHL requires liver biopsy compatible with lymphoma in the absence of extrahepatic disease. [100] The differential diagnosis of PHL includes HCC, especially in patients with preexisting hepatitis B or C infection and liver cirrhosis, which may be present in PHL. However, HCC is substantially more common than PHL. In the study by Terzi et al. [8] with 1006 FLL in liver cirrhosis, 82% HCC were compared to 0.2% lymphoma infiltration in the liver, although it is not known whether this was PHL or secondary lymphoma infiltration. The ratio of HCC:hepatic lymphoma is 410:1. [8] Both HCC and PHL may occur in patients who have cirrhosis with viral hepatitis.
The behavior of metastases, cholangiocarcinomas, and PHL in CEUS is summarized in Table 7.
DISCUSSION
Newly discovered focal liver lesions in liver cirrhosis are primarily suspicious for HCC. Diagnosis is based on radiologic imaging with contrast-enhanced MRI and CT, but CEUS is also accepted by many, despite not all, scientific societies. Typical appearance of HCC is arterial hyperenhancement with hypoenhancement in the late phase. CEUS is used for unclear radiologic findings [26,42] as well as when CT and MRI are contraindicated or not available. The probability of HCC and of malignancy, in general, increases with the size of the nodule. The zone lymphoma of MALT. [102] Preexisting liver disease, which may be etiologically important for the development of nongastric hepatic MALT lymphoma, is, in turn, a risk factor for the development of HCC. In a secondary liver manifestation of lymphoma, other organ and lymph node manifestations are already present. More often, non-Hodgkin's lymphoma affects the liver in advanced stages of a systemic disease. DLBCL is the most common lymphoma subtype in the western countries. [100] Most often, solitary liver lesions are seen in PHL (60%), but multiple lesions are also possible. [100,101] In contrast, multiple liver lesions are seen in about 90% of cases in secondary lymphoma involvement of the liver.
On B-mode ultrasonography, lymphoma lesions may be hypoechoic or almost nonechoic like cysts. But a hyperechoic center with a hypoechoic rim is also possible. [99] On CEUS, the lymphoma lesions show inhomogeneous hyperenhancement in the arterial phase and contrast agent washout in the portal and late phases. [103] Homogeneous hyperenhancement in the arterial phase followed by washout on CEUS is also described. Hypoenhancement on CEUS already occurs in the early portal venous phase. [102] Yamashita et al. [102] compared the appearance of hepatic extranodal marginal zone lymphoma of MALT from published case reports with HCC on CEUS, CT, MRI, and positron emission tomography (PET). The appearance is very similar, and a reliable differential diagnosis based on imaging is not possible. [102] However, the "vascular penetration sign" was described as a special characteristic. The hepatic lymphomas showed penetrating vessels in the arterial phase. This sign is sometimes also present in metastatic liver tumors and CCCs, but not in HCC. [102] In the study by Terzi et al. [8] 0.2% (2/1006) lesions in liver cirrhosis corresponded to lymphoma infiltrations (without specifying primary or secondary). These showed CEUS-LIRADS pattern of LR-M. Histologic assignment by ultrasound-guided needle biopsy is necessary, taking into account anamnestic data (Figure 7). [12] While the presentation of lymphomas on imaging is all other liver lesions, [9,19] but usually histologic confirmation is required in all these instances to achieve definitive, apart from hemangiomas.
Data on the frequency of benign focal liver lesions in the noncirrhotic conditions vary widely. This, however, depends on the type of imaging and information from autopsy studies. [7,20,21,52,53,106] Hemangiomas, in particular, undergo a morphological likelihood of HCC is low in FLL < 10 mm compared to nonmalignant lesions such as RN with or without dysplasia. [45] Although any newly diagnosed solid nodule in liver cirrhosis is primarily suspicious for HCC, small nodules or nodules that show only arterial hyperenhancement may still be nonmalignant. It is important to differentiate HCC from other arterially hypervascularized FLL such as hemangiomas, FNH or FNH-like lesion, HCA, metastases, CCC, and lymphoma. CEUS is excellent in the diagnosis of HCC, as well as in the differential diagnostic evaluation of Intrahepatic CCC is similarly rare as in the noncirrhotic liver. It should be emphasized that CCC can be reliably differentiated from HCC by CEUS. The most important distinguishing feature from HCC is the rapid distinctive washout, occurring within 60 s from contrast injection in cholangiocarcinoma. [7,94,95,111] Small lesions, in particular, can be a diagnostic challenge. In case of doubt, a biopsy is recommended by the EASL for all indeterminate lesions > 10 mm when various contrast-enhanced imaging modalities cannot assign the lesion. [26] In fact, HCC not reaching the typical imaging diagnostic criteria (i.e., CEUS-LIRADS LR5) does not have better prognosis than the typical ones. [112] Therefore, early diagnosis is not less important in these instances than in HCC with a typical enhancement pattern. However, it is also important to keep in mind that false-negative results have been reported in up to 30%, especially in small lesions. [71,74] In these cases, a decision must be made in the overall context with the aid of all diagnostic possibilities for short-term follow-up or for therapeutic intervention, including repeat biopsy.
CONCLUSIONS
Liver cirrhosis comprises necrosis, fibrosis, and regeneration. RNs, FNH-like nodules, DNs, and HCC frequently develop, and sometimes existing benign liver lesions may change through the process of fibrosis, inflammation, and vascular obliteration. Hemangiomas are more easily missed on imaging, and true FNH is a rarity. Differentiation of HCCs from FNH and adenoma can be difficult in these conditions. Metastases are also infrequent. The most important aim of diagnosis is to differentiate these liver lesions in liver cirrhosis from HCC in order to make the right therapeutic decisions. Due to the characteristics of the lesions in liver cirrhosis, this can be challenging for both imaging.
Declaration
Institutional review board gave approval and subjects granted permission for using their data/images. change in liver cirrhosis, where liver cirrhosis leads to obliteration of preexisting hemangiomas. [21] These changes start to occur early in the course of the chronic liver disease with liver fibrosis deposition, even before the development of liver cirrhosis, [59,107] and it is thought of as a dynamic process. Accordingly, hemangiomas in cirrhosis may show fibrotic changes, become smaller, and change shape. [58,59,61] In CEUS, the majority of hemangiomas showed a typical peripheral nodular enhancement. [23] Shunt hemangiomas, which are homogeneously enhanced in the arterial phase and show isoenhancement in the late phase, can be problematic in the differential diagnosis with HCC. [21] On CEUS, however, the majority of hemangiomas show a typical contrast course [23] comparable to hemangiomas in the noncirrhotic liver. [19,29] FNH is diagnosed much less frequently in liver cirrhosis than in the normal population. [7,21] The development of FNH-like nodules, despite rare, is specific to liver cirrhosis; these are morphologically identical to FNH, but exist only in liver cirrhosis. [68][69][70][71] They are often very small and only discovered on the liver explant. [71] The only significance of both types of liver lesions is in the differential diagnosis with HCC, especially if the typical wheel spoke-like vessel architecture cannot be differentiated in the early arterial phase and the lesions only appear arterially hyperenhanced.
In patients with cirrhotic liver, the FNH tends to show isoenhancement in the late phase, but 25% show even hypoenhancement. [23] A biopsy is usually performed. False positives for HCC in the percutaneous biopsy were exceptionally reported, particularly in case of FNH-like lesions. [74] HCA is a rare tumor in the healthy population. [7,52,53,106] Adenomas have also been exceptionally described in liver cirrhosis. [7,20,[79][80][81] In the noncirrhotic liver, many HCAs show mild hypoenhancement in the portal venous and delayed phases. [17,19,29,77] Thus, if an adenoma in liver cirrhosis shows isoenhancement or a mild washout with CEUS in the portal venous and late phases, it cannot be reliably differentiated from an HCC. Further evaluation by means of MRI is required. These characteristics on CEUS have a threefold probability of an HCC rather than an adenoma on a background of a cirrhotic liver. [7] Thus, an HCC must be assumed until proven otherwise in this scenario.
Liver metastases in hepatic cirrhosis are rarer than in noncirrhotic livers in autopsy studies. [86][87][88]108,109] Nevertheless, patients with cirrhosis can also develop liver metastases. On CEUS, the metastases are classified as LR-M. Differentiation from HCC is usually not difficult, [12,14,40,91] but nonetheless, biopsy is usually required to achieve a specific diagnosis of the malignant cellular type. [110] | 2023-01-13T14:09:05.606Z | 2022-12-01T00:00:00.000 | {
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6678533 | pes2o/s2orc | v3-fos-license | Quantifying the Decisional Satisfaction to Accept or Reject the Human Papillomavirus (HPV) Vaccine: A Preference for Cervical Cancer Prevention
Objective Only a portion of the US population is willing to consider HPV vaccination to date. The primary aim of this study is to determine the decisional satisfaction associated with HPV vaccination. Study Design This is a prospective survey conducted at an urban college where women 18–26 years old completed a decisional satisfaction survey about their HPV vaccine experience. Results Regardless of the decision to accept or reject HPV vaccination, the decisional satisfaction was very high (mean 5-item score = 21.2 (SD 3.8)). Women without HPV vaccination were decisionally neutral significantly more often than those already vaccinated; 22% were decisionally neutral for the option to accept HPV vaccination at that visit. Cervical cancer prevention was preferred significantly more often than genital wart prevention in all analyses. Conclusions Targeting those who are decisionally neutral about HPV vaccination may result in a higher uptake of HPV vaccination.
Introduction
Many women's health screenings including cancer prevention involve patient education, patient participation and shared decision making. In order to reach a high-quality decision, the benefits and harms of the alternative options must be clearly discussed. More importantly, the choice depends on how patients value benefits versus harms. The eventual goal is a strong match between the chosen option and the features that matter most to the informed patient [1].
Much of human papillomavirus (HPV) vaccination in the US has been presented through the eyes of public health with an overriding sense of community obligation to be vaccinated for whatever herd immunity may occur [2,3]. Healthy People 2020 aims for very high population vaccination rates [4], but recent surveys indicate only a portion of the population is willing to consider HPV vaccination [5]. While the need for understanding the social and decisional psychology of vaccine uptake is acknowledged [6], little work has studied these concepts in young women concerning their decision to be vaccinated against HPV infection, and whether the option of choosing between two different prophylactic HPV vaccines was important to them. Likewise, there has been little documentation of decisional conflict or decisional regret during or after the HPV vaccine decision making process, as there has been for influenza vaccination [7,8].
The primary aim of this paper is to quantify the satisfaction of the decision to receive at least one dose of HPV vaccine among young adult women. The secondary aim was to determine which demographic and medical history items influenced the value of cervical cancer prevention vs. genital wart prevention followed by choice of HPV vaccine.
Methods
This prospective study was approved by the University of Missouri Kansas City (UMKC) Social Sciences Adult Institutional Review Board (SSIRB) (#SS10-56X) and is part of a larger study on women's preferences for HPV vaccination.
All women, 18-26 years old, being seen at UMKC Student Health and Wellness, an urban college health service, were offered the opportunity to participate in the survey between January 2011 and August 2012. All members of the health care staff were educated on the purpose of the study; and provided scripts to introduce it at check-in. Women were only allowed to complete the survey once, and they self-reported demographic and medical history. For those who had received an HPV vaccine, we did not ask number or timing of doses. The study was open to both those with prior HPV vaccination and those who had not yet started the series. Women completed the survey prior to being seen by the health care provider, and could ask questions about HPV vaccination during the visit.
The cervical cancer prevention education information was constructed in a format similar to a decision aid [1]. It provided information on the benefits and harms associated with cervical cancer screening programs, with HPV vaccination alone (specifying the referenced differences between the two HPV vaccines), and with the combination of HPV vaccination and cervical cancer screening [9]. The decisional satisfaction scale was adapted for cervical cancer prevention from the original Holmes-Rovner model [10]. It is a unidirectional six item set of questions rated on a 5 point Likert scale (1 = strongly disagree, 5 = strongly agree). Higher scores indicate a higher satisfaction with the decision. Individual responses and a 5-item summary score were the outcomes of interest. The item assessing ''expected action'' was analyzed only for those who had not made a choice to receive at least one dose of HPV vaccine prior to the survey.
Three items were included in the survey modeled on the O'Connor's decisional conflict scale [11] that queried the perceived value of preventing different HPV-associated diseases: cervical cancer and genital warts. A final item presented a choice trading off between the two vaccines.
Statistics: Descriptive statistics included means testing with Student's t-test for significance testing. Chi-square comparison and Fisher's exact testing were used for ratios as appropriate and pvalues less than 0.05 were considered significant [12].
Results
Over five academic terms, 10,562 patients (male and female ages 16-62 years) were seen at the Student Health and Wellness clinic. Of these, 306 women without prior HPV vaccination and 299 vaccinated women agreed to participate in the survey. The average age did not differ between the two groups, but for the vaccinated women the average age of first intercourse was significantly lower and the average number of lifetime sexual partners was significantly higher (Table 1). Over 90% of the participants had never been pregnant. Significantly more white and fewer Asian women were vaccinated than not. Hormonal contraceptive use did not differ between vaccinated and unvaccinated groups, but those who were vaccinated used condoms significantly more than those not vaccinated.
Among the women 21 years and older, age-appropriate for cervical cancer screening, 24% of those without any doses of HPV vaccine had never had a Pap test, significantly higher than those with at least one dose of HPV vaccine. There were similar proportions of women with abnormal Pap tests and who underwent colposcopy in both the vaccinated and unvaccinated groups. Significantly more women with prior HPV infections had been vaccinated. A past history of genital warts or of other sexually transmitted infections (STIs) was equally distributed between the vaccinated and unvaccinated groups.
Satisfaction with Decision to Receive at Least One Dose of HPV Vaccine
The decisional satisfaction scores on the five item survey were high regardless of HPV vaccination status (Table 2) indicating that women were comfortable with their decision to date of accepting or rejecting HPV vaccination. For instance, satisfaction scores for ''This decision is best for me personally'' were equally high for both acceptors and rejecters of HPV vaccination. The decisional satisfaction scores from the other four qualities of the decision, while high for both groups, were significantly higher for those accepting rather than rejecting HPV vaccination indicating a high polarization towards vaccination among those choosing vaccination.
The ''expectation to be vaccinated at this visit'' choice showed low satisfaction scores for those not yet vaccinated (mean 2.1 (SD 1.0)), meaning that there was substantial disagreement with the intent to start HPV vaccination among those not yet vaccinated.
A decisional satisfaction score of 3 indicates neutrality and potential decisional conflict where certainty about choice does not yet exist. The highest decisional neutrality occurred among those without HPV vaccination regarding expectations of being vaccinated before leaving the office (22%). The lowest decisional neutrality occurred for the item ''I am informed about the cervical cancer prevention options'' indicating that only 9% of women regardless of HPV vaccine status were uncertain about their cervical cancer prevention options. Whereas a certain amount of decisional neutrality was evident among all women regardless of HPV vaccine status, women with no prior HPV vaccination had significantly greater decisional neutrality on the remaining four qualities of decisional satisfaction than those with at least one HPV vaccine dose.
Perceived Value of Prevention of Different HPV Associated Diseases
Women with diverse medical histories ranked the currently perceived value of preventing different HPV associated diseases differently (Table 3). Cervical cancer prevention was significantly more important than genital wart protection among all women overall (mean 4.5 (SD 0.8) vs. 4.3 (SD 1.0), P = 0.014). Where there were differences within medical histories, the women at least risk for cervical cancer valued cervical cancer prevention significantly more highly than genital wart prevention: women with a history of at least one dose of HPV vaccine; with no prior STIs; with at least one prior Pap test, but with no prior cytologic abnormalities; with no prior HPV infections and no prior colposcopies.
Women who experienced HPV related disease had higher preferences for cervical cancer and genital wart prevention than inexperienced women. For instance, women with at least one dose of HPV vaccine valued cervical cancer and genital wart prevention more than women with no vaccination (cervical cancer: mean 4.7 (SD 0.6) vs. mean 4.2 (SD 0.9), p,0.001); genital warts: mean 4.6 (SD 0.8) vs. mean 4.1 (SD 1.1) p,0.001). Similarly, women with a prior STI valued cervical cancer and genital wart prevention more than women without a prior STI. Among women 21 years and older, those with a prior Pap, with a prior abnormal Pap and a prior HPV infection valued cervical cancer and genital wart prevention more than those women without the respective medical histories. A past colposcopic experience only enhanced the women's value of cervical cancer prevention, not genital wart prevention.
When asked to make a choice between vaccines, significantly more women chose HPV2 over HPV4 (61% (325/537) vs. 39% (212/537), p,0.001, Table 4). This preference was significantly more pronounced among those 21 years and older, with no pregnancies, not using hormonal contraceptives but using condoms, not having received any HPV vaccine doses, with no prior Pap tests, no prior abnormal cytologies if a Pap test had been done, no prior HPV infections, no history of colposcopy and no history of other STIs. Among those women already having received at least one dose of HPV vaccine, significantly more women would have chosen HPV2 over HPV4 had they been given the choice initially (54% (145/267) vs. 46% (122/267), p,0.05).
Comment
Our work is the first to explore decisional satisfaction with HPV vaccination. Some have studied how normative messaging from peers, family and health professionals influence HPV vaccine choice [13,14]; others have studied the Health Belief Model where the decision to vaccinate is a balance between the perceived risk of disease severity including its health and social consequences as well as the benefits/harms of vaccination [15,16].
Recently discrete choice experimentation (DCE) has provided a health economics metric to understand how adults trade off risks and benefits among competing options for similar health care outcomes [17,18], such as HPV vaccination and Pap testing to prevent cervical cancer, in terms of willingness to pay or willingness to trade [19].
These theoretical models have resulted in using strong health care provider messaging for vaccination, promoting the commonness of HPV infections associated with multi-organ cancers, and emphasizing no out-of-pocket expenses as motivators to increase HPV vaccination rates. However, these strategies have not led to an increase in uptake, but rather a plateauing of those receiving HPV vaccinations [5].
Decisional satisfaction offers the perspective of determining which set of young women are still neutral about their HPV vaccine choice; and hence more likely to be open to more discussion about how the benefits and risks of HPV vaccination play into their value for cervical cancer prevention. While Healthy People 2020 anticipates 80% vaccination of the young adult women during their adolescence, the current trends support a large portion that will still have the opportunity to make a HPV vaccination decision as young adult women. Increased vaccination may be more likely if this decisionally neutral set of young women is targeted.
Women with a clear sense of what their personal value of HPV vaccination was (accept/reject) were highly satisfied with their decision. The women who have had at least one dose of HPV vaccine who expressed neutral satisfaction with their decision to be vaccinated may be indicating decisional regret about a decision made for them at an earlier time. Future research will use decisional conflict and decisional regret scales to attempt to understand these values [11,20]. In addition, we intend to study the decisional conflict surrounding continuing the second and third doses in a timely manner among young women who initiate HPV vaccination.
Our work shows that young women have greater value for cervical cancer prevention than for genital wart prevention. This was also seen in Oteng's work among adult women who were willing to trade genital wart protection for cervical cancer protection [19]. Translating these values into vaccine choices has depended on the level of information provided about the vaccines. While Oteng's work clearly showed the stronger preference for cervical cancer prevention, the attributes offered about the vaccines ignored the additional cervical cancer prevention associated with HPV2, leading to the more frequent choice of HPV4 than HPV 2 in that work. Our work presented the cross protection and duration of efficacy data available for both HPV4 and HPV2 in addition to the regulatory licensure claims. This information most likely facilitated the consistent alignment of preference for more cervical cancer prevention with the choice of HPV2 over HPV4. We should note, though, that having the two options for HPV infection prevention is valuable to young women, as there was not an absolute choice for one vaccine over the other.
Limitations: Our work is limited by several factors. In the time course of making a medical decision, the decision aid (or some degree of information about the medical issue) is presented followed by a survey of the satisfaction associated with the choice made. In our work half the population had already made the choice to receive at least one dose of HPV vaccine. This limitation offers a potential hypothesis-generating benefit, though, in that the decisional neutrality expressed might provide insight into decisional regret they may be experiencing up to five years after initial vaccination.
Within the decisional framework of HPV vaccination, we did not offer the choice of no HPV vaccination; therefore, we do not know how an option of no vaccination would change these results.
Separately, while our study population resembled most young adult HPV studies in that the number of lifetime sexual partners and age at first intercourse is historically similar [21][22][23][24][25], the balanced proportions of previously vaccinated vs. unvaccinated women, while occurring by chance, does not reflect the young adult female HPV vaccination rate [26]. Finally, the developmental capacity to make medical decisions is recognized to be more complicated in adults than in adolescents [27]. While these results are likely to be representative of young adults making their HPV related decisions, these results may not be applicable to adolescents or parents making decisions for prepubescent youth.
Conclusions
Targeting young adult females who are decisionally neutral about HPV vaccination may be a more direct method of increasing HPV vaccination rates in a targeted population than spending resources on those already highly satisfied with their decision not to vaccinate. | 2018-04-03T03:38:32.117Z | 2014-02-14T00:00:00.000 | {
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261768776 | pes2o/s2orc | v3-fos-license | EFL JUNIOR HIGH SCHOOL STUDENTS’ PERCEPTION TOWARDS USING YOUTUBE IN LEARNING PROCESS OF ENGLISH LISTENING SKILLS
: Choosing the right learning media can help students to get a better learning achievement. YouTube is known as the most visited video sharing and creating site in the world. This study aims to explore about students' perception towards using YouTube media in their learning process of English listening skills. Students tend to feel bored due to monotonous learning activities in the classroom without audio-visual media. This study also explored students’ perceptions on the types of videos they most p refer as a learning media in Junior high school. The subjects of this study are eighth grade students of one of junior high school in Karawang. This study used qualitative descriptive as the method. The data was collected from an online questionnaire through Google Form to make it easier to collect and analyze data. The result of this study showed that students have positive responses towards the English learning process in listening skills using YouTube. Students agree that using YouTube media as a learning tool in listening skills makes them more interested and motivated in deepening their English skills both for listening skills and speaking skills. They also recommended YouTube as an English learning media due to various video types that were provided in YouTube media.
INTRODUCTION
Learning a new language can be a complex process which requires the appropriate and suitable strategies and also huge amounts of time and effort.The most appealing and effective method of learning the language is always sought after by second language educators, who utilize a variety of English materials (Albiladi et al., 2018).In addition, using various types of strategies in learning, students' opinions or their perceptions must also be considered.As also in line with Albiladi et al., (2018) points out that it is essential to understanding and exploring students' opinions in order to inform the teacher whether the media should be used in the English learning process.Choosing enjoyable learning methods is an excellent choice because it will influence the learning performance or learning achievement.
In the learning process, using media is the most familiar tool to teach students.
Using media can help students to understand the material easier.As in line with (Van et al., 2021a) using media to help students learn English has a high potential for improving language abilities and promoting the learning process.Various media that can be used in EFL classroom such as websites, applications, games, movies, songs.Pamungkas & Adi (2020) also explained that in order for students to feel happy and comfortable, teachers should share the learning material using media so that students are easier to understand the material and deepen their skills.
Listening skill is one of crucial language skills that must be deepened to develop the competence of a second language (Derbal, 2022).Susiani et al., (2020) explained that Listening as a fundamental language ability is the basis to all good communication.
Language learners can learn word stress, pronunciation, vocabulary, and message comprehension through the communication process when they have the ability to appropriately receive and interpret messages.Listening skills in a second language require students to practice a lot not only in the classroom but also outside.Thus, the researcher chose listening skill for this study.Brown & Yule (1983) explained that listening comprehension is considered as an individual understanding of what the listener heard and may repeat the sound without a real comprehension.Listening skill is often considered to be the most challenging language skill to learn, yet it is still rare for teachers to teach about students' listening skills in the classroom.As mentioned by Hardiah (2019) Efl Junior High School Students' Perception Towards Using Youtube … Edusaintek: Jurnal Pendidikan, Sains dan Teknologi Vol.11 (1) 2024 | 353 that in the teaching and learning process, teaching listening skill is a skill that tends to be ignored.
In the listening classroom, students are usually only given audio containing conversations using English.Krivosheyeva et al., (2020) explained that in this kind of lesson, correct answers are emphasized, but the listening process is ignored.Teachers can use various media to encourage students' interest in practicing their listening skills.Using Audio-visuals such as movies or short videos through YouTube videos as a medium to practice students' listening ability can attract students' attention.As in line with Ahmed et al., (2021) stated that YouTube videos can help teachers to create engaging and innovative learning environments, be used to assess students' learning efficacy, improve students' listening comprehension, and allow students to recognize mimic and gesture in conversation.English-language videos filled with native speakers will give students a more real picture of their language.Atiyah & Izzah (2019) also mentioned that using audio-visuals can provide the ability to ensure authentic language (what they are communicating effectively).Audio-visual materials can give abstract ideas more substance, show action that is sped up or slowed down to make it simpler to perceive, show specifics of an object or process, and make learning more engaging so that it becomes enjoyable (Restami & Samsudin, 2023).
One of the most interesting media to develop listening skills is watching videos.
Using audio-visuals such as movie clips, short videos, television programs, and animations has risen significantly in language education these days.Not only able to hear the spoken language, the listeners are also able to see the animation, contextual situation, speakers' body language or gestures, and also speakers' expressions while watching the video (Ningtiyas et al., 2021).(Dhillon, 2015) stated that this media was chosen to improve students' listening skills about English conversations spoken directly by native speakers.According to Digital Information World (2019), YouTube is the second most popular website, where the users are able to view, download, create, and share YouTube videos.Students' critical thinking and motivation can be increased with the use of YouTube as the learning media Nofrika (2019) YouTube videos can assist teachers in creating exciting and innovative learning environments, as well as improving students' listening comprehension as stated by (Ahmed et al., 2021) Efl Junior High School Students' Perception Towards Using Youtube … Edusaintek: Jurnal Pendidikan, Sains dan Teknologi Vol.11 (1) 2024 | 354 Several studies that discussed learning using YouTube media such as Lestari (2019) about the use of Youtube vlog for students' listening skills.The research findings showed that students' listening skills of eighth grade junior high school are significantly improved through watching YouTube vlogs.Marbun et al., (2023) about the use of YouTube in students' listening skills, which showed that students learning through YouTube makes them more interested in following the learning process.Pratama et al., (2020) about the use of YouTube as a learning tool in teaching listening skills.The research findings showed that utilizing video in the classroom to teach listening has a huge impact on how easily students can comprehend the subject matter or the context of the lesson.Most of the studies are focused on the use of YouTube as a learning media for listening skills.The purpose of this study is to present the gap in exploring students' perception in listening classrooms through YouTube media by using surveys as the research method.
METHOD
In this study, the researcher used a descriptive qualitative method.According to Creswell (2014) Qualitative research is an inquiry process of understanding based on distinct methodological traditions of inquiry that explore a social or human problem.
Qualitative researchers collect data themselves through examining documents, observing behavior, or interviewing participants.This research was conducted in one of the junior high schools in Karawang.
The participants in this research were 10 students in the eighth grade of one of the junior high schools in Karawang.The participants were selected by using simple random sampling in order to get results from the various characteristics and abilities of students.
In order to fulfil the data needed, the online questionnaire was chosen to get the students' perception in the listening classroom through YouTube media.The questionnaire was made online through Google Form.The researcher shared the link of the questionnaire so the participants can fulfil the questionnaire easier.Besides, all of the questions in the questionnaire were translated to Indonesian language in order to avoid misunderstandings regarding the meaning of each question, so that the results obtained can be more valid.The instrument of data collection in this study used multiple choice and also Efl Junior High School Students' Perception Towards Using Youtube … Edusaintek: Jurnal Pendidikan, Sains dan Teknologi Vol.11 (1) 2024 | 355 paragraphs in order to get some perceptions of the participants, by using some questions that had been prepared by the researcher to ask several questions related to the topic directly to the students.
RESULT
This study used questionnaires as the instrument to collect the data.All of the questions are served in Bahasa Indonesia in order to make it easier for students to understand the questions and also to avoid student's misunderstanding about the questions.The questionnaire had been divided into 3 sections which are; students' perception towards YouTube Media, students' perception towards the teaching techniques, and students' perception of the learning experience in the listening classroom through YouTube media.The data from the questionnaire are the result from an online questionnaire made by the researcher in Google Form.
Students' Perception Towards YouTube Media
Several questions in this section are highlighted about students' perception towards YouTube media as a learning tool for learning English listening skills.In this session, it can be seen whether students have a good perception of YouTube media, especially for the English learning process, especially for listening skills.
Picture 1. Diagram of first section
Based on the diagram above, it is shown that all of the participants vote "agree" towards all of the questions in this section.In the first question (Q1) 100% of respondents agreed that using YouTube media as a learning tool is interesting.This is showed that students already have good perception towards the YouTube media.In the second question (Q2) 100% of respondents also voted "agree" to the statement "Using YouTube media in class makes me more interested in learning English".For the third question (Q3), all respondents also agreed that using YouTube media makes them feel more motivated to deepen their English listening and speaking skills.The diagram also showed that all of the respondents agreed in the fourth question (Q4) that YouTube media makes them feel more familiar with listening to English pronunciation.For the last question in this section (Q5) all of the respondents also agreed that YouTube media is easy to access.However, the diagram above showed that there are no respondents who choose a "disagree" option to all of the questions in this section.All of the respondents which means 100% of respondents are agree with the questions in this section which are made in a positive statement.
Students' Perception Towards the Learning Process Using YouTube
There are various types of videos with different content and length on YouTube that can also be used as a learning tool especially for the learning process of English listening skills.Their opinions towards the video selection for their learning process using YouTube are shown in the following chart.
Picture 2. Chart of video length selection
There are two types of video durations on YouTube which are short video and long video.For the short length video, it is usually below ten minutes.Meanwhile, the long length video is above ten minutes.Types of videos such as movies, vlog, animation, podcast, and language learning videos have various length duration.Learners are able to
Students' Perception of the Learning Experience using YouTube
A good experience will have a good impact on students' learning process to achieve their goals.Knowing students' perception on how students' feel about their experiences in learning English by using YouTube is an important thing to assess.Besides that, this is also to be able to find out what impact that students feel after learning English listening skills using YouTube media as a learning tool in their learning process.Their opinions of the learning experiences are shown in the following chart.The chart above shows that all of the respondents have a good perception towards their learning experience by using YouTube media.Various good feeling on students of experiencing learning English using an additional learning tool or media such as using YouTube media showed in this chart.The chart above, it shows that 40% of respondents said that they feel more enthusiastic when learning English listening using YouTube media.The other respondents which are 30% of respondents also said that they feel more entertained and do not feel bored when learning English by using YouTube media.
Besides that, there are another 30% of respondents even said that they feel more motivated to learn English, especially to deepen their English skills by using YouTube media.
Picture 6. Chart of aspects that students feel are getting better Some positive impact that students feel is also shown in the chart above.There are various aspects that are mentioned by respondents in this section.The aspects that tend to be further improved by learning English listening skills using YouTube media are stated by the respondents are listening skills and speaking skills.There are 40% of respondents said that using YouTube media as a learning tool is improving their listening skills.30% of respondents thought that learning English using YouTube media tends to learn the right pronunciation of English words.Meanwhile, the other 30% of respondents said that they learn new vocabulary when they are experiencing learning English using YouTube media.
In addition to getting more valid data, there are questions in the online questionnaire which are made in the form of open-ended questions.These questions are made with the highlight of students' experience towards learning English listening using YouTube media.Due to this section being an open-ended question, students are able to share their learning experiences and also be able to express their opinions freely about their learning process in the learning English listening through YouTube media.
The first question is about "what impact do you feel both while learning English listening through YouTube media and after learning English listening through YouTube media?".The answer of the students towards these questions, are: "Waktu belajar bahasa inggris pakai media YouTube jadi lebih seru dan ga bosan.
Lebih mudah juga untuk dipahami dan jadi tau juga cara pengucapannya" From the answers above, it is shown that most of students are have a similar opinion towards the impact that they feel both while learning English listening through YouTube media and after learning English listening through YouTube media.Most students stated that they are enjoy learning English listening through YouTube media.
They also feel more interested in following the learning activity.Besides that, students also feel that learning English listening through YouTube media makes them not feel bored while studying, because if they only use textbook or just audio, they usually tend to feel sleepy more easily because they are bored with the learning activities that are less interesting.
Efl Junior High School Students' Perception Towards Using Youtube … Edusaintek: Jurnal Pendidikan, Sains dan Teknologi Vol.11 (1) 2024 | 361 The impact that they feel after learning English listening through YouTube media is also shown in those answers.Most of students also have a similar answer at this point.
Most of them stated that learning English listening through YouTube makes them easier to understand the learning material.Besides that, they also stated that learning English listening through YouTube media makes them know how to pronounce words that are unfamiliar to them.
The second question is about "Do you recommend the use of YouTube media in the English learning process, especially for listening skills?".Below are the students' answers towards this question: "Aku merekomendasikan belajar bahasa inggris menggunakan YouTube, karena lebih seru dan banyak juga pilihan video atau channelnya" "Ya, merekomendasikan.Karena lebih menarik untuk dipakai belajar sekaligus hiburan, dan mudah juga untuk diakses kapanpun.Bagus juga buat membiasakan mendengarkan bahasa inggris dan belajar pengucapannya" "Iya, merekomendasikan.Karena di YouTube juga banyak video atau channel yang khusus buat belajar bahasa inggris, jadi bagus buat latihan listening ataupun kosakata dan pengucapannya" The answers above also showed that most students have similar points in their answers.First thing is that all of them recommend using YouTube as a medium for learning English.Second is that most students stated that it is a good way to use YouTube as the learning medium for learning English.They stated that YouTube media is easy to access every time, so it is a flexible media or website to access.Besides that, other reasons students recommend the use of YouTube as a learning media to learning English also stated by them such as YouTube has many videos with different channels and also different contents so they are able to choose the video they want to watch for learning freely.Most students also stated that learning English through YouTube is good because it can make them more accustomed to listening to English conversations, so it is good to practice their listening skills.Besides that, learning new vocabulary and knowing how to pronounce it is also stated by students.
DISCUSSION
Using learning media such as YouTube in the learning process of English listening skills has good responses from students.This result is also in line with Cahyana (2020) that revealed positive responses about the implementation of YouTube in learning English which includes listening development and motivation.Based on the result in the section of students' perception towards YouTube media, it is shown that 100% of respondents who are second grade of junior high school students agreed that using YouTube media as a learning tool in learning English makes them more interested and motivated.As in line with Van et al., (2021b) that it has a strong potential in quickly promoting the process of learning English and enhancing learners' language skills in employing technology in English learning.
In the section of students' perception towards the learning process by using YouTube, it is shown about the students' video selection both from the video length and video types.The result showed that most of them chose long videos which were above ten minutes.For the video types, students have various tendencies in choosing video as a learning tool in their learning process.The order of the most choices starts from the animated video.This result is in line with Nurdiawati (2019) that explained students' listening skills can be enhanced by using YouTube animated video as a teaching and learning tool, also encouraging them in learning.Besides, there are other types of videos that have also been chosen by students' which are movie and language learning in the second place, then finally followed by video vlogs and podcasts.
There are also various types of activities that can be used in English learning using YouTube for listening skills.For example, there are fill-in the gaps, summarizing and paraphrasing, answering questions, and listening comprehension.These activities can be used after students finished watching YouTube videos as a learning material.However, students' have more tendency in the learning activity using YouTube media, even though they have various choices.
Students' experiences in learning English using YouTube have also shown a positive impact.The result above, shows that most of the students are feeling enthusiastic, entertained, and even feel motivated when learning English using YouTube media, especially in deepening their English skills.This is in line with the findings of Anggrarini & Faturokhman (2021) stated the fact that the explanation on YouTube is regarded as being more clear and detailed encourages pupils to study.The chart also shows that there are various English skills that students' feel tend to be much better when learning using YouTube media.Most of them stated that their listening skills have improved, while the others stated that they can learn pronunciation and also new vocabulary.This result is in line with Qomariyah et al., (2021) explained that the learning atmosphere in the classroom is more engaging, inspiring, creative, and cooperative by using YouTube videos.
Students' opinions about the impact that they feel both while learning English listening through YouTube media and after learning English listening through YouTube media also showed a positive response from students.It can be seen from the answers of students that while learning English listening using YouTube as the learning media, most of them are tend to feel enjoy and more interested in following the learning activity.Most students have stated that they are tend to feel bored and even feel sleepy if learning only using textbooks or just audio.Meanwhile, while learning English listening through YouTube they feel more excited and entertained so that they do not feel bored or sleepy.
This finding is in line with Pratama et al., (2020) showed that participants considered learning through YouTube to be entertaining, educational, crucial for enhancing language abilities, and more flexible in communicating.This point also supported by Muslihah et al., (2022) explained that there are instances during the educational process when students become bored with their learning, which can reduce their comprehension and result in lower accomplishment.Besides that, all of students also recommend to learning English for listening by using YouTube.Various reasons which are stated by students are that YouTube is easy to access every time, has many videos with different channels and contents, and also good for practicing both listening skills or getting used to listening to English conversations and also for practicing speaking skills such as learning new vocabularies and learning how to pronounce the words.This finding is similar to Nofrika, (2019) explained that students are improved their English skills including listening skills and speaking skills such as pronunciation, list of vocabulary, and also grammar.
CONCLUSION
Using learning media in the goals to help the learners to be easier in understanding the learning material, also in creating a better interest in learning it has a high potential especially in promoting and encouraging the English learning process.A younger learner, including the students of junior high school, needs additional learning media instead of just using a textbook in order to gain better interest and motivation in the English learning process.Based on the result, it can be concluded that YouTube media can be used as a learning tool in the English learning process.Students' have positive perceptions in using YouTube media in the process of learning English.Students tend to feel more enthusiastic, entertained, and even feel more motivated to deepen their English skills.By using YouTube as a learning medium, students can choose various types of videos with various lengths of video.
There are animation videos which are chosen most by students, also movie, language learning video, vlog, and podcast.Besides, there are also some aspects that students feel better or even improve by learning English using YouTube, which are listening skills with the highest percentage and then followed by speaking skills such as learning new vocabularies and also learning pronunciation, especially for some words that students do not know.In addition, the focus analysis of this study with the keywords "YouTube media", "learning process", and "listening skills".It reveals that, in terms of the study focus on the English learning process using YouTube media, keywords are crucial issues that have been, are being, and still need to be investigated.
ACKNOWLEDGEMENT
This study was never possible without the great support and contribution of my advisor Yogi Setia Samsi S.S., M.Hum., who has motivated me to write an academic paper also who has tutored me about research methodologies in advance and assessed the drafts with detail-oriented.
School Students' Perception Towards Using Youtube … Edusaintek: Jurnal Pendidikan, Sains dan Teknologi Vol.11 (1) 2024 | 356 School Students' Perception Towards Using Youtube … Edusaintek: Jurnal Pendidikan, Sains dan Teknologi Vol.11 (1) 2024 | 357choose any videos with a certain duration.The chart above shows that 60% of respondents choose long length video which are the duration is above ten minutes.Meanwhile, the other 40% of respondents choose short length video which are the duration is below ten minutes as their video selection for their English listening skills learning process using YouTube media.The large number of students who chose videos with long durations showed about their enthusiasm in participating in the learning activities using YouTube media.Picture 3. Chart of video types selectionThe videos that are divided on YouTube come in a variety length duration.Besides that, there are also various types of videos with different content and of course those various contents can also be used as English listening learning materials.Every student must have different interests when choosing types of videos to learn English.Students are able to choose their favourite content to use as their learning material.The chart above shows that respondents have various tendencies in selecting the type of video for their learning process based on their own interest.Most of the respondents which are 40% of them, choose animation as their favourite type of video.Besides, there are 20% of respondents choose movies, 20% of respondents choose language learning video, 10% of respondents choose podcast, and the other 10% of respondents choose podcast as their favourite type of video for the learning process in English listening skills.Chart of activity types selection There are various types of activities used after watching YouTube videos as a learning medium.The chart above showed types of activities that students chose for the learning process in English listening using YouTube media.The highest percentage which is 40%, respondents choose answering questions as the activity after watching YouTube in the learning process.The second most popular choice is listening for comprehension with 30% of respondents.Besides that, another 20% of respondents choose summarizing and paraphrasing as the activity after watching YouTube videos as a learning medium.Meanwhile 10% of respondents choose fill-in the gaps. | 2023-09-14T15:09:21.308Z | 2023-09-04T00:00:00.000 | {
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54203608 | pes2o/s2orc | v3-fos-license | Strain Measurement With Nanometre Resolution By Transmission Electron Microscopy
Strain is routinely used in state-of-the-art semiconductor devices in order to improve their electrical performance. Here we present experimental strain measurements obtained by different transmission electron microscopy (TEM) based techniques. Dark field electron holography, nanobeam electron diffraction (NBED) and high angle annular dark field scanning electron microscopy (HAADF STEM) are demonstrated. In this paper we demonstrate the spatial resolution and sensitivity of these different techniques on a simple calibration specimen where the accuracry of the measurement can easily be assessed.
Abstract. Strain is routinely used in state-of-the-art semiconductor devices in order to improve their electrical performance. Here we present experimental strain measurements obtained by different transmission electron microscopy (TEM) based techniques. Dark field electron holography, nanobeam electron diffraction (NBED) and high angle annular dark field scanning electron microscopy (HAADF STEM) are demonstrated. In this paper we demonstrate the spatial resolution and sensitivity of these different techniques on a simple calibration specimen where the accuracry of the measurement can easily be assessed.
Introduction.
Although the effects of introducing strain in semiconductor devices is well known from an electrical point of view, little is known about the distribution of strain. Indeed, it is only in the last few years that methods that can measure the strain with the required level of spatial resolution have been developed. Dark field electron holography [1], NBED [2] and HAADF STEM [3] are different TEM based techniques that have been developed in order to solve this problem. A calibration specimen was designed so that the different strain mapping techniques could be tested and compared to accurate simulations that would account for the relaxation of the thin TEM specimen. The calibration specimen that was examined was grown by reduced pressure chemical vapour deposition and comprised from top to bottom, a 150-nm-thick capping layer, then four 10nm-thick SiGe layers with Ge concentrations of 45%, 38%, 31% and 20%, each separated by 30nm of silicon on a silicon substrate. As the growth is epitaxial, the lattice spacing in the in-plane (xdirection) does not change. However, in the growth (z-direction) the lattice parameter is expanded relative to the unstrained substrate due to the presence of Ge and it is this relative expansion which is measured. The dark holography experiments were performed using a probe corrected FEI Titan operated at 200 kV, the precession NBED (PED) and the HAADF STEM experiments were performed using a double corrected FEI Titan operated at 200 kV. All of the data was processed using software written at CEA. Figure 1 shows the process flow for dark field electron holography. An electron biprism is used to interfere an electron wave that has passed through a strained region of interest with an electron wave that has passed through an unstrained reference, such as the substrate. By using an objective aperture to select a diffracted beam corresponding to the lattice places of interest, a phase image can be calculated by Geometrical Phase Analysis (GPA) that corresponds to a displacement field for the chosen set of lattice parameters from which a strain map can be calculated. Dark field holography is well adapted for the semiconductor industry as 2D strain maps can rapidly be produced with a field of view of up to a micron and a spatial resolution of 4 nm. We have benefited here from the stability of the Titan TEM to acquire electron holograms for up to one minute to provide strain maps with a sensitivity of +/-0.02 % [4]. Figure 2 shows the process for nanobeam electron diffraction. A near-parallel electron beam is used to provide diffraction patterns from which the local strain can be determined from the shifts in the diffraction spots relative to a reference pattern. By using the three condenser lens system in the FEI Titan TEM we can now provide an electron beam with a convergence angle of 0.2 mrads and a FWHM of 6 nm for 200 kV electrons. The electron beam is scanned across the region of interest in STEM mode and the patterns are automatically processed using an in-house software to provide strain profiles. This technique is extremely easy to perform and 1D strain profiles with a sensitivity of 0.06 % has now been demonstrated with a spatial resolution of 3 nm [5]. The main problem with NBED is that the probe is not perfectly parallel, thus the diffraction pattern is composed of discs containing dynamical effects leading to incorrect measurements of the strain. Recently we have shown that by precessing the beam, evenly illuminated diffraction spots can be recorded which leads to accurate strain maps. In addition, a more convergent beam can be used leading to an improved spatial resolution or around 2 nm and a sensitivity of 0.02 % [6]. Figure 3 shows results obtained using precession diffraction, more than 1500 diffraction patterns have been acquired in order to calculate this strain map (a) a pseudo HAADF image is formed from the diffraction patterns showing the region examined and (b) a strain map for the {004} direction. Figure 3(c) shows a strain profile extracted from an averaged area of 10 nm compared to simulations demonstrating the excellent signal to noise obtained. The results from the precession are slightly less than the simulations as a thinner specimen was examined than for the dark holography results.
Geometrical Phase Analysis
Strain maps have been obtained from high resolution transmission electron microscopy images for many years, however the interpretation of TEM images is complicated. The contrast is dependent on specimen thickness and focus, thus the results can be misleading unless a very small field of view is examined which is not always suitable for semiconductors. The excellent stability of the latest generation electron microscopes means that strain maps can now be obtained directly from HAADF STEM images [7]. Figure 4 shows the process for forming strain maps by HAADF STEM. A HAADF STEM Image is acquired and a FFT is applied in order to obtain information about the components of the lattice in the direction of strain that is being examined. Here we select the information from two different and orthagonal {111} spots as their intensity is relatively high and then two different phase images are obtained from which strain maps for the growth and in plane directions can similtaniously be obtained. In order to minimise the artefacts from the scanning, the scan direction is chosen to be in the same drection as the strain field being measured. Vertical artefacts can be seen in the strain map due to electrical interference in the scan which is not observable in the raw HAADF images. Spatial resolutions of 1 nm can be obtained using this technique which is relevant for many different types of specimens such as quantum dots and state of the art semiconductor devices which are currently in development [8]. Comparison of the techniques.
In the last few years a great deal of attention has been made on the subject of strain mapping using transmission electron microscopy and encouraging progress has been made. Dark field electron holography is an excellent technique that provides strain maps quickly with minimal data processing. However, a perfect crystal is required with a reference which is exacly aligned with the region of interest. An additional problem with dark holography is that a perfect specimen is required to prevent unwanted diffraction effects. NBED has been used mainly by the semiconductor industry as it is both easily accessible and straightforward to perform. NBED can give inaccurate results and tends only to supply strain profiles rather than maps. The application of precession to NBED now removes many of these artefacts and recent improvements in data handling means that maps can now be easily acquired, although this can be time consuming. The strain map shown in Figure 3 took one hour to acquire and an additional hour to process after the microscope was set up This can be compared to dark holography which takes less than a minute to acquire and process the data. The advantage of PED is that it is very versitile and allows strain maps to be acquired of almost any type of material. HAADF STEM is a very useful strain mapping technique which is used in the semiconductor industry as the dimensions of devices can now be less than 10 nm, however the signal to noise limits the technique and this is used often to see if a device is "strained or not". HAADF STEM strain mapping is very insensitive to "bad specimen preparation" and is thus a robust technique. However, a very stable electron microscope is required to have acceptable signal to noise in the calculated strain maps.
Summary
There are many different methods of measureing strain with nm-scale resulution using transmission electron microscopy. These methods are used routinely in the semiconductor industry to provide important information about the processes that are used in building prototype devices. All of these different approaches have advantages and disadvantages. Figure 5 shows a summary of the different techniques in March 2014. However, the continuous developments driven by improvements in hardware, software and specimen preparation mean that the story of strain mapping in the TEM is far from over. | 2018-12-03T04:35:41.357Z | 2014-08-01T00:00:00.000 | {
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257384726 | pes2o/s2orc | v3-fos-license | Functional Disability and Symptomatic Slow-Acting Drugs for Osteoarthritis in Adults with Periodontitis
Osteoarthritis (OA) patients have decreased functional ability and restricted access to healthcare facilities and are on a spectrum of medications. These can impact their oral health. This study aims to investigate the association between periodontal disease and OA disease parameters, specifically the functional disability and the medications taken. This was a cross-sectional study on OA participants recruited from the Hospital Canselor Tuanku Mukhriz. Periodontal health parameters were obtained from an oral examination of the participants. A Health Assessment Questionnaire (HAQ) was administered to ascertain the functional status of the participants. Out of the 130 participants recruited, 71 (54.6%) had periodontitis. There was a correlation between the teeth count with OA severity, where participants with a greater Kellgren–Lawrence score had less teeth (rs = 0.204, p = 0.025). Participants with a greater degree of functional limitation also had less teeth (rs = −0.181, p = 0.039) and a higher clinical attachment loss (rs = 0.239, p = 0.006). There were no associations found between the symptomatic slow-acting drugs in OA and periodontal health parameters. In conclusion, there was a high proportion of periodontitis in patients with OA. Functional disability was associated with measures of periodontal health. It is suggested that clinicians treating OA patients consider the need for a referral for dental care when managing this group of patients.
Introduction
Osteoarthritis (OA) is traditionally described as a 'wear and tear', mechanically driven disease, that is, a consequence of the aging process affecting mostly older adults [1]. It is a disorder involving movable joints, characterized by cell stress and extracellular matrix degradation initiated by micro-and macro-injury. This process stimulates maladaptive repair responses, including pro-inflammatory pathways of the innate immunity [2,3]. More contemporary views of OA argued that the inflammation contributes to OA synovitis and its pathology [4]. OA is now viewed as a complex disease, with inflammatory mediators released by cartilage, bone and synovium and its progression governed by a set of multifactorial components [5]. The role of inflammation in this new paradigm highlights inflammatory signals, including cytokines, surface-expressed pattern recognition receptors, complement factors, pathogen-associated molecular patterns and damage-associated molecular patterns that can lead to the degradation of the cartilage matrix in OA [5].
Periodontal disease (PD) is a disease that affects the supporting structures of the teeth. It is characterized by microbially associated, host-mediated inflammation that results in loss of periodontal attachment [6]. Periodontitis is an immune-inflammatory infection that can cause low-grade systemic inflammation, which may influence the development of systemic comorbidities. The periodontitis-associated systemic inflammation can cause hematogenous dissemination of periodontal bacteria and inflammatory mediators from periodontal tissues to the bloodstream [7,8]. This can later spread to other parts of the body. Despite the similarities between the role of inflammation in the pathogenesis of OA and PD, limited studies have investigated the association between the two diseases and its related factors.
Chung and co-workers studied the data of 7969 adults from the Korea National Health and Nutrition Examination Survey (KNHANES) during 2010-2014. They reported that severe OA and periodontitis were associated with each other in a subgroup analysis involving female patients [9]. Kim et al. also reported that periodontitis was associated with the presence and severity of radiographic knee OA in their Korean nationwide study, also using data from the KNHANES [10]. Other studies on periodontal disease and OA were case-control studies where OA participants were recruited as controls. These studies include those from United States veterans, reporting 26.4% of OA participants with periodontitis [11], and Vietnam, where 28% of OA participants had periodontitis [12].
OA can involve the joints of the upper extremities, causing functional limitations of the hand. In a study on OA participants, radiographic changes due to OA were found to be associated with reduced grip and pinch strength [13]. This can limit the ability of the participants to utilize oral hygiene aids and remove plaque efficiently [14]. Apart from that, patients with OA can have mobility limitations that affect their ability to access the dental clinic for routine dental care [14][15][16]. The accessibility of dental services and level of patient care, as well as transport availability, were suggested as factors that affect the use of dental services by patients with arthritis [16].
Oral medications used to treat OA can broadly be categorized into analgesics, including paracetamol; non-steroidal anti-inflammatory drugs (NSAIDs); and symptomatic slow acting drugs for OA (SYSADOAs), including glucosamine, chondroitin sulphate, diacerein and avocado and soybean unsaponifiable (ASU). There is evidence that NSAIDs can cause reduced bleeding and a reduction in the rate of bone resorption in observational and intervention studies [17,18]. Diacerein has been hypothesized to be a potential therapeutic drug for periodontitis due to its anti-inflammatory activities that selectively inhibits signal transduction affecting cytokine profiles and ameliorating disease breakdown [19]. In an animal study investigating the effects of diacerein in the management of ligature-induced experimental periodontitis in rats, significant decreases in the IL-1ß level in the test group suggest that diacerein may play a therapeutic role as a potent anti-inflammatory drug in the management of periodontitis [20].
ASU is routinely prescribed in OA and has been suggested as an adjunct for the treatment of periodontitis. ASU added to gingival fibroblasts in culture showed the potential to prevent the deleterious effects of IL-1ß in periodontitis [21]. Animal studies in rats also showed that ASU prescribed as an adjunct to conventional mechanical debridement has subtle beneficial effects on periodontal repair [22]. However, a clinical study found that ASU did not have a favorable effect in the treatment of chronic periodontitis [23].
Current evidence shows that glucosamine exhibits anti-inflammatory effects by reducing the levels of pro-inflammatory factors, such as tumor necrosis factor-alpha, interleukin-1 and interleukin-6 [24]. There is, however, limited data on the effects of glucosamine and chondroitin sulphate on periodontitis. A randomized controlled trial investigated the effects of Arthocare Forte containing chondroitin (400 mg) and glucosamine sulphates (500 mg) administered to patients with tooth mobility over a period of 5 years [25]. The participants underwent non-surgical periodontal therapy, and half were treated with Arthocare Forte. They found that the medication speeds up the regenerative capacity and the stability of the periodontium compared to the control group.
Generally, there is limited evidence on the effects of medications, such as the SYSADOAs and NSAIDs used to treat OA, on the oral health parameters and periodontal disease. Further studies are needed in this area to assist in understanding how these medications affect specific oral health parameters and whether there is potential for these medications to be used in the treatment of periodontitis.
It was hypothesized that: (a) the prevalence and severity of PD is higher in participants with OA compared to the general population; (b) participants with functional limitations have a greater PD severity, and (c) there is a difference in the PD among participants taking the different SYSADOAs. Hence, this study aimed to investigate: (a) the prevalence and severity of PD among patients with OA compared to the general population; (b) the association between PD parameters and OA parameters, including functional limitations, and (c) the differences in PD among participants taking different SYSADOAs.
Materials and Methods
The participants were recruited from the Osteoarthritis Clinic under the Orthopaedic Department in Hospital Canselor Tuanku Mukhriz, Malaysia, using convenience sampling. Consecutive patients attending the OA Clinic who met the study criteria were invited to participate in the study. The flowchart of the recruitment process is shown in Figure 1.
Arthocare Forte. They found that the medication speeds up the regenerative capacity and the stability of the periodontium compared to the control group.
Generally, there is limited evidence on the effects of medications, such as the SYS-ADOAs and NSAIDs used to treat OA, on the oral health parameters and periodontal disease. Further studies are needed in this area to assist in understanding how these medications affect specific oral health parameters and whether there is potential for these medications to be used in the treatment of periodontitis.
It was hypothesized that: (a) the prevalence and severity of PD is higher in participants with OA compared to the general population; (b) participants with functional limitations have a greater PD severity, and (c) there is a difference in the PD among participants taking the different SYSADOAs. Hence, this study aimed to investigate: (a) the prevalence and severity of PD among patients with OA compared to the general population; (b) the association between PD parameters and OA parameters, including functional limitations, and (c) the differences in PD among participants taking different SYSADOAs.
Materials and Methods
The participants were recruited from the Osteoarthritis Clinic under the Orthopaedic Department in Hospital Canselor Tuanku Mukhriz, Malaysia, using convenience sampling. Consecutive patients attending the OA Clinic who met the study criteria were invited to participate in the study. The flowchart of the recruitment process is shown in Figure 1. The inclusion criteria were: (i) OA patients, as confirmed by the ACR Classification, (ii) above the age of 18 years old, (iii) dentate and (iv) able to give verbal and written consent. The exclusion criteria were: (1) patients who were completely unable to read, write or understand the Malay or English Language; (2) coexistence of other autoimmune diseases; (3) uncontrolled systemic disease or malignancy; (4) patients who were pregnant or planning to become pregnant; (5) any current or previous history of periodontal treatment, including root surface debridement/ periodontal surgery; and (6) any previous or current use of phenytoin or cyclosporin. The inclusion criteria were: (i) OA patients, as confirmed by the ACR Classification, (ii) above the age of 18 years old, (iii) dentate and (iv) able to give verbal and written consent. The exclusion criteria were: (1) patients who were completely unable to read, write or understand the Malay or English Language; (2) coexistence of other autoimmune diseases; (3) uncontrolled systemic disease or malignancy; (4) patients who were pregnant or planning to become pregnant; (5) any current or previous history of periodontal treatment, including root surface debridement/ periodontal surgery; and (6) any previous or current use of phenytoin or cyclosporin.
This study was conducted as part of research assessing oral health in patients with joint diseases [26]. Ethical approval for the study was obtained from the Ethical Board of the Universiti Kebangsaan Malaysia (UKM/PPI/111/8/JEP-2017-553), and the study conformed to the provisions of the Declaration of Helsinki (as revised in Brazil 2013). All participants of the study gave informed consent, and the anonymity of the participants has been preserved in the conduct and reporting of this study.
Oral Examination
The assessment of the oral and periodontal health used parameters such as the number of remaining teeth, the Plaque Index [27], the Gingival Index [28], the probing pocket depth (periodontitis) and clinical attachment loss (CAL). For the Plaque Index, the original O'Leary protocol used a disclosing solution (such as Bismarck Brown Iodine Stain). However, the disclosing solution was not used in our study. A dichotomous scoring system of (present/absent) was used. Six sites per tooth, namely, the mesiobuccal, midbuccal, distobuccal, mesiopalatal, midpalatal and distopalatal sites, were assessed for both the Plaque Index and the Gingival Index. The periodontal charting was carried out using the University of North Carolina-15 (UNC15) probe. The clinical attachment loss (CAL) was then calculated as a sum of the pocket depth and recession.
Diagnosis of periodontitis was made based on the criteria as outlined by Papapanou et al. [29]. They are as follows: (1) interdental CAL is detectable at ≥2 non-adjacent teeth, or (2) buccal or oral CAL ≥3 mm with pocketing ≥3 mm is detectable at ≥2 teeth, but the observed CAL cannot be ascribed to non-periodontitis-related causes. The periodontal findings were also later recoded to the Community Periodontal Index (CPI) [30] to allow for comparison with the national data available for Malaysia [31]. Oral examination was conducted by a single examiner (NMNA). Prior to the initiation of the study, NMNA was calibrated against a gold-standard periodontist (NM) to ensure inter-and intra-examiner reliability.
Patients who were diagnosed with any dental disease were given a follow-up appointment or advised to seek care at any dental clinic if they were not able to attend for the appointment given.
Measures of Functional Limitations
The Malaysian version of the Health Assessment Questionnaire (HAQ) [32] was administered to ascertain the functional status of the subject. The authors have permission to use this instrument from the copyright holders. The HAQ is a questionnaire with eight sections (Dressing and Grooming, Arising, Eating, Walking, Hygiene, Reach, Grip and Activities) that is validated to assess the functional limitations in participants with musculoskeletal diseases. The HAQ also included vertical, 100 mm, patient global and pain visual analogue scales. Administration of the questionnaire was done by a single researcher prior to collection of the oral health and OA parameters to minimize risk of bias.
Osteoarthritis Parameters
The patient's medical notes were accessed to extract information regarding the disease duration, type of OA and prescribed medications. The prescription of any medications were noted, and the type of SYSADOAs and painkiller taken were specifically extracted from the hospital patient system.
The participants' radiographs were read by a musculoskeletal radiologist (FMF) to determine the Kellgren-Lawrence score [33]. Prior to reading the radiographs, FMF was calibrated to another independent musculoskeletal radiologist. The radiographs were each assigned a grade from 0 to 4. The grades correlated to the increasing severity of OA, with Grade 0 signifying no presence of OA and Grade 4 signifying severe OA. For participants suffering a combination of OA where more than one type of joint is affected, the Kellgren-Lawrence score was taken from the joint that was most severely affected. Examiner FMF was blinded to the clinical findings of the participants. The extracted information was checked to ensure that it was dated no longer than three months in duration from the clinical examination.
Statistical Analysis
Statistical analysis of variables was performed with IBM SPSS version 19.0 (IBM Co., Armonk, NY, USA). Univariate comparisons were made using the Chi-squared test. The correlations between periodontal indices and OA disease characteristics were analyzed by Pearson or Spearman correlation coefficients to test for the association between the variables. The Mann-Whitney U test was applied for analysis of independent nonparametric variables. All p values are two-sided, and p values less than 0.05 were considered statistically significant.
Demographic Data
The flowchart of the steps of subject recruitment and data collection is reported in Figure 1. The demographic characteristics and periodontitis diagnosis of the study participants are shown in Table 1. The participants were mostly older adults with a mean age of 61.5 (±9.3). Out of the 130 participants with OA, 98 (75.4%) had knee OA, 7 (5.4%) had hip OA and 6 (4.6%) had hand OA, while 19 (14.6%) had a combination type of OA.
Proportion of Osteoarthritis Participants with Periodontitis
From the 130 participants recruited, 71 (54.6%) had periodontitis. Generally, the OA participants had a higher proportion of periodontitis compared to the Malaysian NOHSA findings of 48.5% [34]. The severity of periodontitis suffered by the OA participants was also greater, with more participants having a CPI of 4 compared to the NOHSA findings. This is shown in Figure 2.
.Proportion of Osteoarthritis Participants with Periodontitis
From the 130 participants recruited, 71 (54.6%) had periodontitis. Generally, the OA participants had a higher proportion of periodontitis compared to the Malaysian NOHSA findings of 48.5% [34]. The severity of periodontitis suffered by the OA participants was also greater, with more participants having a CPI of 4 compared to the NOHSA findings. This is shown in Figure 2.
Osteoarthritis Parameters with Periodontal Health Parameters
The mean ± standard deviation for the OA parameters measured are as follows: OA disease duration: 6.08 ± 6.40; Kellgren-Lawrence Score: 2.95 ± 1.01; HAQ Score: 0.40 ± 0.43; Pain Score: 5.04 ± 2.96; and Global Health Score 7.37 ± 1.80. The distribution of the PD parameters and their correlation with the OA parameters are shown in Table 2. There was a correlation using the Spearman Rho test between the Kellgren-Lawrence score of the participants with teeth count, where participants with a greater Kellgren-Lawrence score had less teeth (rs = 0.204, p = 0.025, two-tailed, N = 121). There was no other correlation between any parameters of PD (plaque index, gingival index, probing pocket depth and clinical attachment loss) with the Kellgren-Lawrence score.
Osteoarthritis Parameters with Periodontal Health Parameters
The mean ± standard deviation for the OA parameters measured are as follows: OA disease duration: 6.08 ± 6.40; Kellgren-Lawrence Score: 2.95 ± 1.01; HAQ Score: 0.40 ± 0.43; Pain Score: 5.04 ± 2.96; and Global Health Score 7.37 ± 1.80. The distribution of the PD parameters and their correlation with the OA parameters are shown in Table 2. There was a correlation using the Spearman Rho test between the Kellgren-Lawrence score of the participants with teeth count, where participants with a greater Kellgren-Lawrence score had less teeth (rs = 0.204, p = 0.025, two-tailed, N = 121). There was no other correlation between any parameters of PD (plaque index, gingival index, probing pocket depth and clinical attachment loss) with the Kellgren-Lawrence score. The overall HAQ score was correlated with the teeth count (rs = −0.181, p = 0.039), average PPD (rs = 0.209, p = 0.017) and CAL (rs = 0.239, p = 0.006). Participants with a greater degree of functional limitation, as shown by the HAQ score, had worst periodontal health, with less teeth, a higher PPD and a higher CAL. The global health score was inversely correlated with the plaque index (rs = −0.178, p = 0.043), where participants with a higher global health score had less plaque.
The specific sections of the HAQ were further investigated against the oral health parameters to ascertain which type of functional limitations was correlated with which oral health parameter. Out of the eight HAQ sections, only 'Arising', 'Eating' and 'Walking' were correlated with specific oral health parameters. Participants reporting a limitation in 'Arising' and 'Eating' had deeper CAL (rs = 0.206, p = 0.019; rs = 0.211 p = 0.016) and PPD (rs = 0.245, p = 0.005; rs = 0.278 p = 0.001). Both CAL and PPD are parameters related to periodontal health, indicating that participants with a limitation in 'Arising' and 'Eating' had worst periodontal health. A limitation in 'Walking' was correlated with all 5 oral health parameters investigated, namely, teeth count (rs = −0.233, p = 0.008), plaque index (rs = 0.195, p = 0.026), gingival index (rs = 0.177, p = 0.045), average PPD (rs = 0.209, p = 0.017) and average CAL (rs = 0.265, p = 0.002). All five oral health parameters are measures of the overall oral health, indicating that participants with walking limitations have worst oral and periodontal health. Table 3 shows the type of SYSADOAs taken by the participants. Most of the participants were not taking any SYSADOAs (53.8%), while those who were taking SYSADOAs were mostly prescribed either Glucosamine (13.1%) or Piascledine (27.7%). There was no difference in the periodontal health parameters between participants taking different SYSADOA medications and when compared to those not on any medications. There was also no difference in the oral health parameters between the participants taking NSAIDs and those who were not.
Discussion
Osteoarthritis is a complex condition affecting the whole joint, where an association with systemic inflammation could also be present [35]. OA is now viewed as a complex biological response connecting biomechanics, inflammation and the immune system, rather than a purely mechanical disease due to the wear and tear of the cartilage in the joints [5]. The prevalence of periodontitis among patients with OA in this study is higher at 54.6% when compared to the national average of 48.5% [31]. Other reports on the prevalence of periodontitis in OA participants reported a relatively lower prevalence of 39.4% [10], 26% [36], 26.4% [11] and 28% [12]. This increased prevalence can be attributed to the limitations in function, as well as the increase in the overall systemic inflammation experienced by participants with OA. Other factors include the poorer general health of the subjects, as well as the older age of the participants.
The results showed that participants who reported functional limitations in the HAQ had a more severe form of periodontitis with less teeth, a higher PPD and a greater CAL. Participants with a limitation in 'Arising' and 'Walking' had the worst periodontal health. This can be attributed to the mobility limitations that affect the OA participants' ability to access the dental clinic for routine dental care [14][15][16]. Patients with arthritis can be limited in their access to dental services and may also have transportation restrictions, which are factors affecting the use of dental services by this group of patients [16]. In view of the increased severity of periodontal disease in this group of patients, dental services should be made more accessible, such as by offering home visits and having the location of the dental clinics be more accessible, such as on the ground floor or with elevators or ramps to better facilitate the dental visits of these OA patients. Dental healthcare personnel can also visit musculoskeletal clinics to provide care at regular intervals.
The correlation between a limitation in 'Eating' and the severity of periodontitis, as measured by CAL and PPD, suggests that OA patients with nutrition issues may be more predisposed to PD. Bone formation, healing of the periodontium and periodontal regeneration are affected by various vitamins, minerals and trace elements, such as vitamin C, vitamin D, magnesium and calcium [37,38]. It is possible that the limitation in eating in this population contributes to the increased prevalence and severity of PD in patients with OA [37][38][39]. The assessment of diet during dental visits can allow the clinician to better address the factors related to the periodontal disease among patients with OA. Referral to a dietitian can also be a strategy for the improvement of both the oral health and the general health of this group of patients.
Restrictions in the ability to grip objects can lead to poorer oral hygiene, worsening of the periodontal health clinical parameters and poorer oral health. However, there was no correlation between limitations in 'Grip' with any oral health parameters in this study. This can be attributed to the relatively low number of subjects with hand OA, which was 14.6%. The tool used to assess functional limitations in this study, which was the HAQ, were also not designed to specifically assess hand function. This can explain the lack of correlation between 'Grip' limitations and the oral health parameters.
This study found an association between the severity of OA and the number of teeth in the mouth, where participants with a more severe form of OA had less teeth. The association can be an indicator that the two diseases may be related. However, it is also possible that the association is due to the participant's age, where the mean age of the participants in this study is 61.5 years. With increasing age, participants will tend to have a greater severity of OA and will also have less teeth, as the estimate of tooth loss is 0.2 teeth per year [40]. Tooth loss can lead to the reduced nutritional value of the food intake, as well as a reduction in social participation that can eventually lead to social isolation and depression. Preventive measures to limit tooth loss, as well as the provision of a prothesis to replace teeth, can be invaluable for these patients.
Medications taken for OA termed SYSADOAs have been reported to have beneficial effects on the periodontium. Diacerein has anti-inflammatory activities that affects the cytokine profiles [19], while ASU cultured with gingival fibroblasts showed potential to prevent the deleterious effects of IL-1ß in periodontitis [21], and supplements containing chondroitin and glucosamine sulphate have been reported to speed up the regenerative capacity and stability of the periodontium [25]. However, there was no significant difference of the periodontal health parameters between the participants taking the different medications for OA in this study. This can be due to the cross-sectional nature of this study, where the longitudinal effects of these medications cannot be accurately detected, as well as the relatively small numbers of participants taking the different types of SYSADOAs. In this group of participants, not all of the SYSADOAs are available in the clinics, and some of them had to be purchased privately. This may impact the type of medication taken for the OA, depending on the socio-economic background of the participants.
The limitations of this study include the methods of measurement for the functional disability. This study utilized a self-reported assessment of function and disability as assessed by the HAQ. Other methods of assessment may be questionnaires that specifically assess the hand function, such as the Functional Index for Hand Arthropathies (FIHOA), or a performance-based test, such as the Arthritis Hand Function Test (AHFT). A more objective measurement of hand function will give a better picture of the ability of the subjects to perform regular oral hygiene care. The older age of the participants in this study may also be a confounding factor affecting the periodontal health, as well as the functional limitations, of the participants. Other limitations are that factors such as the duration, compliance and dose of the medications taken were not analyzed. These may play a role in the relationship between the medications and PD and should ideally be analyzed in the future.
This study is one of very few studies reporting on the proportion of OA subjects with periodontitis. The findings of this study can be useful for dental practitioners and policymakers to have a better understanding of the factors and clinical parameters related to the oral and periodontal health of OA patients. It can also be used to design a more rigorous study investigating factors linking OA and periodontitis in the form of cohort studies or randomized controlled trials.
Conclusions
There is a high proportion of periodontitis among patients with musculoskeletal disease, namely, OA. Functional disability among OA patients was associated with clinical measures of periodontal health. Within the limitations of this study, SYSADOAs taken by the participants were not associated with the diagnosis, stage, grade and periodontal health parameters in OA participants. It is suggested that clinicians treating OA patients consider the need for a referral for dental care when managing this group of patients. | 2023-03-08T16:03:49.685Z | 2023-03-01T00:00:00.000 | {
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228945675 | pes2o/s2orc | v3-fos-license | Phylogenetic Position of Geosmithia spp. (Hypocreales) Living in Juniperus spp. Forests (Cupressaceae) with Bark Beetles of Phloeosinus spp. (Scolytinae) from the Northeast of Mexico
: Geosmithia members are mitosporic filamentous fungi commonly recorded and isolated from bark beetles of the Scolytinae subfamily and their respective host’s species. This genus includes 18 species formally described and 38 phylogenetic species recorded in several localities from Africa, Asia, Australia, Europe, and North and South America, where they exhibit frequent associations with phloeophagous and wood-boring bark beetles. Among phloephagous bark beetle species, specifically, in members of the genus Phloeosinus Chapuis, almost 10% of Geosmithia strains have been isolated. By its physiographic elements and high bark beetle and conifer species richness, Mexico is a potential region to host a high diversity of Geosmithia species and potential new species. In the present study, we systematically sampled and isolated, cultured, and molecularly identified members of the Geosmithia species associated with Phloeosinus spp. and their Juniperus spp. host trees at the north of Sierra Madre Oriental, at Nuevo Leon State, Mexico. Phylogenetic analyses based on 378 internal transcribed spacer region (ITS) sequences supported the presence of strains from Geosmithia langdonii-Geosmithia sp. 32 clade associated with Phloeosinus serratus vector and with Juniperus coahuilensis (JC) host, and the presence of strains from Geosmithia sp. 21- Geosmithia xerotolerans clade with Phloeosinus deleoni and Juniperus flaccida (JF) in this geographical region. The genetic and morphological di ff erences found in our strains with respect to those previously described in the species from both clades ( Geosmithia langdonii - Geosmithia sp. 32 and Geosmithia sp. 21- G . xerotolerans ) suggest that both Geosmithia lineages from Nuevo Leon correspond to two potential new species in the genus.
Introduction
Associations among fungi and bark beetles constitute one of the most successful ecological adaptations that promoted complex and dynamic interactions in this insect group [1]. Most of these Forests 2020, 11, 1142 3 of 19 in this region is highly diverse based on extensive systematic samplings from several "vector" bark beetles and host plant species. Numerous valid documented and undescribed phylogenetic Geosmithia species have been discovered through sampling from Western and Southeastern USA [19,29].
Species discovery is still a major endeavor of the field of taxonomy. In some taxa, it is calculated that approximately half of the new species are discovered from samplings of only a few specimens and localities. Despite the fact that this practice provides incomplete distribution and morphological data, species discovery from a few specimens/localities provides the necessary information to help taxonomists know it and relative taxa [37].
Because of its physiographic elements and high bark beetle and conifer species richness, Mexico is a region expected to host a high diversity of Geosmithia and potential new fungal species; in spite of this, there are no records of these symbiotic associations in the country. Therefore, we conducted a survey to study bark beetles of the genus Phloeosinus and its host plants in Nuevo Leon state, center of Sierra Madre Oriental (SMOr) Mexico, to explore Geosmithia diversity and determine its possible association with-bark beetles and their plant hosts.
The Sierra Madre Oriental (SMOr) is a mountain system considered a biotic unit in different regionalization proposals [38]. Different biomes are distributed within it, which in turn are home to a high level of biological diversity [39,40]. At least three Phloeosinus species inhabit the north of this region, P. baumanni Hopkins, P. deleoni Blackman, and P. tacubayae Hopkins, two of them endemic to the country (https://www.barkbeetles.info/index.php). Seven Juniperus species are also distributed, namely J. angosturana R. P. Adams, J. coahuilensis (Martínez) H. Gaussen ex R. P. Adams, J. depeanna Steud, J. flaccida Schltdl, J. pinchotii Sudw, J. saltillensis T. M. Hall, and J. zanonni. R. P. Adams [40], on host species that have previously been demonstrated to harbor a high frequency of Geosmithia species in other latitudes [19,30]. This ratifies the importance of studying Geosmithia diversity in Mexico.
The goal of the present study is to explore the diversity of Geosmithia associated with Phloeosinus bark beetles with Juniper host preferences in the north of the SMOr, at Nuevo León State, Mexico. Through isolation, culture, morphological and molecular techniques, we associate fungal strains with the recognized phylogenetic groups in the genus and compare morphological attributes of isolated strains with those shown in the described species, to evaluate the presence of potential species in Geosmithia in this unexplored region.
Sampling
Potential vector bark beetles and their tree hosts were collected from June to December 2019 from two areas from Nuevo Leon State, SMO, located in the northeast of Mexico ( Figure 1). One of them is located 27 km northwest of the municipality of Galeana, 400 m from kilometer 145 of the Matehuala-Monterrey highway, in an area of undisturbed open vegetation, with semi-arid xerophytic scrub dominated by the Juniperus coahuilensis (JC)species in an arboreal state (Figure 2a,b), while the second one is 4 km away from Iturbide municipality, 100 m from the Iturbide-school forest of the Universidad Autónoma de Nuevo León highway, in an area of semi-arid pine forest and transition of between Pinus species and Juniperus flaccida (JF), where the latter dominates in an arboreal state (Figure 2h,i).
In each area, healthy trees of the Juniper species were selected, and we deployed freshly cut branches of the targeted tree as a lure for bark beetles. The cut branches were 80-100 cm long × 10-15 cm diameter and were laid on the floor near to the tree they were obtained from, for environmental exposure for approximately 1-2 months (Figure 2c,i). The branches were monitored weekly to assess the occurrence of colonizing beetles, which can be recognized by the presence of a sawdust-like substance, called frass, created by bark beetles colonizing, which is accumulated in tree crevices and may have fallen to the floor gallery, resembling very fine, cream-brown coffee ground material at the floor, together with branches ( Figure 2d,e,k,l). Those branches with colonization signals were collected and transferred to the laboratory of Entomology of Facultad de Ciencias Forestales, Universidad Nacional Autónoma de Nuevo León, Linares Nuevo León state for its protection and examination. In total, 11 cut branches were sampled, 4 of them corresponding to J. coahuilensis from Galeana municipality and the remainder to J. flaccida from Iturbide. Sampling is displayed in Table 1. Table 1. Table 1. In each area, healthy trees of the Juniper species were selected, and we deployed freshly cut branches of the targeted tree as a lure for bark beetles. The cut branches were 80-100 cm long × 10-15 cm diameter and were laid on the floor near to the tree they were obtained from, for environmental exposure for approximately 1-2 months (Figure 2c,i). The branches were monitored weekly to assess Trunks were debarked to expose the wood, galleries, and beetles (Figure 2f,j). The bark was removed in both the vertical plane and the entire circumference. In each gallery system, some bark beetles were removed with tweezers, stored in 70% alcohol for identification and in Petri dishes for fungal isolation, without mixing insects from different gallery systems. Bark beetle adults were identified by external morphological characters using the taxonomic key of Wood [31].
The isolation of putative Geosmithia spp. was realized directly from gallery systems and bark beetle adults of the Phloeosinus species. Of each trunk colonized, at least one gallery system and the respective insects from it were sampled. The fungus was scraped from the gallery surface if growth of mycelium was observed on it (Figure 2g,m). For the bark beetles, the collection was done by vortexing a pull of whole beetles (10 specimens) in a 1.5-mL tube containing 1 mL of sterile wash solution (0.02% Tween 80 solution in water) for 1 min. The fungal scraping and 100 mL of wash solution of insect bodies were inoculated onto Petri dishes with Malt Extract Agar (MEA2, BD Difco) Czapek yeast autolysate agar (CYA) [23] supplemented with trace elements (0.001% ZnSO 4 -7H 2 O and 0.0005% CuSO 4 -5H 2 O, and Panela Medium Agar (PMA) [41]. Parafilm-sealed Petri dishes were inverted in plastic containers, incubated in the dark at 28 • C, and examined daily for 14 days.
Cultural and Morphological Characteristics
The identification and morphological characterization of the Geosmithia isolated followed the protocol of Pitt [23]. Pure cultures of Geosmithia spp. were obtained by using a sterilized mycology handle to take some sample and reseed in other MEA2, CYA, and PMA plates, which were incubated at 25 • C for 7-14 d with an examination at 24 h intervals until the emergence of Geosmithia fruiting structures. Additionally, a duplicate slide culture with CYA media was realized to observe the reproductive structures of the Geosmithia as described Harris [42].
To observe the reproduction structures with scanning electron microscopy (SEM) and phase-contrast microscopy (PCM), a slide culture technique for fungi was performed following the techniques described by Aylmore and Todd [43]. For each Geosmithia culture, three slides were prepared, one of them for PCM and the remainder for SEM. In brief, square blocks (5 mm per side) of the CYA medium were cut; blocks on the slides were inoculated on four sides of the CYA square with mycelial fragments; an agar cube was covered with a coverslip on the upper surface and incubated for 48 h. The cover glass was removed from the slide culture when hyphae and production of spores were observed over the surface of the glass. Fungal structures were observed using a lactophenol cotton blue stain on a clean microscope slide. For electronic microscopy, the glasses with hyphae and spores adhered were dehydrated and critical point dried with CO 2 , mounted, and coated with a mixture of gold-palladium and subsequently observed by SEM in a Hitachi model SUI510 scanning electron microscope.
Conidiophore and ontogenesis of conidia were observed in plate cultures according to Cole et al. [44] and incubated in daylight for the best development of conidiophore roughness. Micromorphology was studied on seven-day-old colonies grown on MEA and CYA, and the conidiophores were taken from margins and near colony centers, as well as from areas that differed in their texture. A total of 20 randomly selected conidia of each strain were measured. The substrate mycelium from the colony margins was studied for the presence of substrate conidia. Mounts were prepared in Melzer's reagent and 20% lactic acid with 0.05 g cotton blue.
DNA Extraction, Amplification, and Sequencing
Genomic DNA of Geosmithia isolates was extracted from pure cultures by following the protocol of Hernandez-García et al. [45]-the DNA genomic was stored at −20 • C until use. Extractions were performed from pure isolated fungi coming from each debarked gallery system, corresponding to both isolates from scraped galleries and insects contained in they ("wash solution of insect bodies"). A region that ranged from 300 to 500 bp for the internal transcribed spacer region (ITS) was amplified with primers ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) [46]. The PCR amplifications were performed in a TC-5000 thermocycler (Techne, Staffordshire, UK) using a total reaction volume of 25 µL, which contained 50-100 ng DNA template, 1X reaction buffer, 2.0 mM MgCl 2 , 0.4 µM each primer, 0.4 mM dNTPs, and 1 U Taq polymerase (Invitrogen Life Technologies, Sao Paulo, BR). The reaction conditions were the following: initial denaturation at 94 • C for 5 min, 30 cycles at 94 • C for 1 min, 55 • C for 1 min, 72 • C for 1 min, and a final extension at 72 • C for 5 min. Amplification products were purified and sequenced in the Laboratory of Genomic Sequencing of Biodiversity and Health, Instituto de Biología, Universidad Nacional Autónoma de México, Mexico.
To evaluate the phylogenetic position of the obtained Geosmithia sequences with respect to Geosmithia spp. previously associated with bark beetles, a series of phylogenetic analyses (PA) by Maximum likelihood (ML) were performed. Thus, 366 sequences of the ITSs corresponding to 18 species formally described and 38 undescribed phylogenetic species of Geosmithia spp. available in public databases from several studies were used in our analyses [15,19,21,25,29,49]. Each sequence was considered a molecular operational taxonomic unity (MOTU). In the first phylogenetic analysis (PA1) to estimate the relationship of target sequences concerning the "five" Geosmithia complexes previously recognized [11], a data set of 378 Geosmithia sequences was included, to Acremonium alternatum Link (AY566992.1) and Emericellopsis pallida Beliakova (NR_145052.1) as outgroups. Subsequent phylogenies were reconstructed to locate Geosmithia strains isolated within the complexes. In these analyses, only the sequences from the closest clades to the target sequences displayed in PA1 were included, the most distant MOTU's with respect to the most inclusive monophyletic clade were used as an outgroup.
The best nucleotide substitution model for each analysis was determined in jModelTest 2.1.10 [50] and selected based on the lowest Akaike Information Criterion (AIC) value. Maximum likelihood (ML) phylogenetic analyses were conducted by using IQTREE v 1.6.12 [51] with recommended partition parameters. To assess the tree topology, Bootstrap support in IQTree was calculated using the ultrafast option [52]. The tree was visualized and edited by using FigTree 1.4.4 [53], and modified using Inkscape (https://inkscape.org/en/). The genetic distances of Nei, between and within target Gesomithia sequences, and for the closest, MOTUs were calculated in MEGA 10.1 [54].
Results
Bark beetles were attracted to six of 11 cut branches used as lures and traps, two belonging to J. coahuilensis from the Galeana municipality and four belonging to J. flaccida from the Iturbide municipality ( Figure 1). Based on morphological attributes, two species of Phloeosinus were identified on these hosts, Phloeosinus deleoni Blackman in J. flaccida, and P. serratus in J. coahuilensis. From these samples, 12 gallery systems (gs) and their respective bark beetles were studied; four gs of P. serratus and eight of P. deleoni. In all gallery systems of both species, the growth of mycelium was observed as a thin layer of withe velvety powder covering the walls of the gallery system, which was conspicuously evident on the pupal chambers of the gallery systems (Figure 2g,m).
Presumptive molecular identification of the 24 isolates based on ITS sequences using blastn NCBI (https://blast.ncbi.nlm.nih.gov/), supported that all obtained sequences correspond to Geosmithia genus. Sequences of isolates from J. flaccida-P. deleoni (gs = 8; insects = 8) showed around 95.7% identity and 99% of query coverage with Geosmithia sp. CCF3355 isolated of Phloeosinus punctatus LeConte from Juniperus occidentalis Hook. Sequences isolated from J. coahuilensis-P. serratus (gs = 4; insects = 4) showed around 98.7% identity and 98% of query coverage with Geosmithia langdonii U91 associated to Bostrichidae sp. from host feeding on Baccharis pilularis DC, both reported in California, USA. Sequences edition and alignment show that from the 12 Geosmithia sequences associated with J. flaccida-P. deleoni and from eight recover from J. coahuilensis-P. serratus that six and two corresponded to similar haplotypes, respectively; they were not included in the phylogenetic analysis. For PA1, the aligned ITS dataset included 366 sequences plus 12 target sequences around 500 bp (n = 378).
Phylogenetic Analysis
The first maximum likelihood phylogenetic analysis (PA1) based in six Geosmithia sequences isolated from J. flaccida-P. deleoni, six from J. coahuilensis-P. serratus and including 378 ITS sequences of 18 species formally described and 38 undescribed species of Geosmithia, recovered two big clades previously shown in others studies ( [11,19,29]; Figure 3a). Group 1 is composed mainly by, G. pallida Figure 3b). In these clades, the "five" well-defined groups corresponding to the Geosmithia complexes were observed as previously recognized [11]. All target sequences from J. flaccida-P. deleoni, were located with a bootstrap value of 98% within a clade integrated mostly by the sequences of Geosmithia sp. 21 and the only available sequence of the recently described specie G. xerotolerans [25]. All Gesomithia sequences from J. coahuilensis-P. serratus were included within the clade integrated by of Geosmithia langdonii Kolarik, Kubatova, Pazoutova and Geosmithia sp. 32 with a bootstrap value of 67% (Figure 3b).
Morphological Characterization
Strains obtained from J. flaccida-P. deleoni (Figure 6a-f). Conidiophores on MEA arising from the surface hyphae, hyaline aerial mycelium; stipes determinate, more frequently indeterminate, verrucolose, septate, in some cases arising from peg foot or initials suggesting foot-cells (Figure 6cf); terminal penicilli more frequently terverticillate, and in few cases quaterverticillate, or even more Figure 5. Phylogenetic relationships within of strains from Geosmithia sp. 21-G. xerotolerans clade from Galeana, Nuevo León and its most closely related species based on the ITS sequences. The phylogenetic tree was obtained by Maximum Likelihood from 49 Geosmithia sequences. Emericellopsis pallida and Acremonium alternarum were selected as outgroups. Acronyms Pdel and Jfla corresponding to strains isolated from Phloeosinus deleoni and Juniperus flaccida, respectively. The asterisks on the tree indicate the sections of the branches that were cut out, for representational purposes. The straight line with the asterisk under the tree indicates the length of the section that was cut, which was the same in all cases.
Discussion
In the present study, a systematic sampling using branch sections of Juniper species as a lure for bark beetles of the genus Phloeosinus allowed us to explore the diversity of Geosmithia fungal species in Nuevo León state, center of Sierra Madre Oriental (SMO), northeast Mexico. This is the first study that documents the symbiotic relationship of this fungus genus associated with its bark beetle vectors and host trees (vector galleries), in this country. Phylogenetic analysis based on internal transcribed spacer region (ITS) sequences supported the presence of strains associated with Geosmithia langdonii-Geosmithia sp. 32 and Geosmithia sp. 21-Geosmithia xerotolerans clades in this geographical region. The characterization of colonies and conidiophores of these strains showed conspicuous morphological differences respect to those previously reported in the described species within their respective clades. Together, morphological and genetic differences found in Mexican Geosmithia suggest that both strains from Nuevo León could correspond to undescribed species in the genus.
Identity of Geosmithia Strains
In most Geosmithia members, ITS allow species-level identification, as such, it has been used as a "DNA barcode" to document the diversity of this taxon in different geographical regions around the world [29]. The recognition of monophyletic clusters using this marker, together with morphological attributes, is used as a criterion to recognize species in this genus [13,19,27,29]. In the present study, the molecular assignation (BLAST) and the ITS based phylogenies of 12 Geosmithia Nuevo León strains corresponded to two different lineages within this genus (Figures 3-5). Isolates collected in Galeana from the adult body of Phloeosinus serratus and its respective gallery system on Juniperus coahuilensis (Figure 2a,b) were clustered within G. langdonii -Geosmithia. sp. 32 clade ( Figure 3); all strains collected in Iturbide from the adult body of P. deleoni and its gallery system on J. flaccida (Figure 2h,i) were clustered within the Geosmithia sp. 21-G. xerotolerans clade (Figure 3) Phylogenetic analysis showed that sequences of both Geosmithia strains from Nuevo León, were monophyletic within their respective lineages ("G. langdonii-Geosmithia sp. 32" and "Geosmithia sp.
Discussion
In the present study, a systematic sampling using branch sections of Juniper species as a lure for bark beetles of the genus Phloeosinus allowed us to explore the diversity of Geosmithia fungal species in Nuevo León state, center of Sierra Madre Oriental (SMO), northeast Mexico. This is the first study that documents the symbiotic relationship of this fungus genus associated with its bark beetle vectors and host trees (vector galleries), in this country. Phylogenetic analysis based on internal transcribed spacer region (ITS) sequences supported the presence of strains associated with Geosmithia langdonii-Geosmithia sp. 32 and Geosmithia sp. 21-Geosmithia xerotolerans clades in this geographical region. The characterization of colonies and conidiophores of these strains showed conspicuous morphological differences respect to those previously reported in the described species within their respective clades. Together, morphological and genetic differences found in Mexican Geosmithia suggest that both strains from Nuevo León could correspond to undescribed species in the genus.
Identity of Geosmithia Strains
In most Geosmithia members, ITS allow species-level identification, as such, it has been used as a "DNA barcode" to document the diversity of this taxon in different geographical regions around the world [29]. The recognition of monophyletic clusters using this marker, together with morphological attributes, is used as a criterion to recognize species in this genus [13,19,27,29]. In the present study, the molecular assignation (BLAST) and the ITS based phylogenies of 12 Geosmithia Nuevo León strains corresponded to two different lineages within this genus (Figures 3-5). Isolates collected in Galeana from the adult body of Phloeosinus serratus and its respective gallery system on Juniperus coahuilensis (Figure 2a,b) were clustered within G. langdonii -Geosmithia. sp. 32 clade (Figure 3); all strains collected in Iturbide from the adult body of P. deleoni and its gallery system on J. flaccida (Figure 2h,i) were clustered within the Geosmithia sp. 21-G. xerotolerans clade (Figure 3) Phylogenetic analysis showed that sequences of both Geosmithia strains from Nuevo León, were monophyletic within their respective lineages ("G. langdonii-Geosmithia sp. 32" and "Geosmithia sp. 21-G. xerotolerans" clusters; Figures 4 and 5), and the average genetic distances among target sequences from Nuevo León concerning conspecific reference sequences within each group (G. langdonii-Geosmithia sp. 32 until up to 3.1%; Geosmithia sp. 21-Geosmithia xerotolerans until up to 5.1%) were higher than those calculated among conspecific reference sequences and other closeness Geosmithia members previously reported in GenBank. The monophyletic group within of G. langdonii-Geosmithia sp. 32 clade from Galeana displayed 2.0% of divergence than to the closer sequence of G. langdonii (HF546250.1; strain U91) from the Phloeosinus thujae vector and Thuja occidentalis host from California, USA ( Figure 4). The monophyletic group within of Geosmithia sp. 21-G. xerotolerans clade from Iturbide displayed a 3.8% divergence and was closer to Geosmithia sp. 21 (AM421053.1; strain MK592) from Hypoborus ficus vector on Ficus carica host from Aquitánie, France ( [19,29]; Figure 5).
The genetic differences observed among target sequences from Nuevo León within both clades and those previously reported are similar to the 2.2% divergence in the ITS sequence data displayed among other Geosmithia phylogenetic species and higher than 1.2% divergence in other species in the related genus Penicillium Link [55].
The genetic and morphological differences found in our strains with respect to those previously described in the species from both clades Geosmithia langdonii-Geosmithia sp. 32 and Geosmithia sp. 21-G. xerotolerans suggest that both Geosmithia lineages from Nuevo León could correspond to undescribed species in the genus; however, these results should be taken with caution. In the case of the clade G. langdonii-Geosmithia sp. 32 is necessary because the species included in it are indistinguishable using the ITS, and they can only be identified using other molecular markers such as TEF1 or TUB2 [19]. In the case of our strains clustered with Geosmithia sp. 21-G. xerotolerans, a more comprehensive morphological comparison was not possible because the Geosmithia sp. 21 has not been formally described or assigned to other of Geosmithia species yet; our phylogenetic analysis suggest that previous strains Geosmithia sp. 21 most probably can correspond to Geosmithia xerotolerans [25]. This species was recently described based on morphological and molecular information, however the phylogenetic analysis that supported its description did not include molecular data of Geosmithia sp. 21, and thus did not consider the relatedness between these species. More iterative taxonomy studies need to be done including more molecular markers and isolated from other localities to evaluate the genetic and morphological variation of Geosmithia Mexican strains and its closer species to determinate the status of Mexican strains.
Geographic, Bark Beetle Vector, and Host Tree Records
Several diversity studies have recorded new localities, vectors, and host species associated with strains of G. langdonii-Geosmithia sp. 32 and Geosmithia sp. 21-G. xerotolerans clades, which led to them being recognized as generalist fungal [19], because they inhabit Palearctic and Nearctic regions and have been isolated from bark or ambrosia beetles (adults and galleries) as well endophyte on the same tree in a wide geographical range [14,15,19,29]. In the case of G. xerotolerans, it has only been recovered from the surface of a darkened house wall taken in Els Pallaresos, Tarragona province, Spain [25].
Our study extends the presence of these globally distributed clades in North America and provides the first records of this genus in Mexico ( Figure 4). The new records of Geosmithia from Nuevo León, Mexico indicates that the distribution of both clades in America is substantially wider than previously reported, running through the west side of the Rocky Mountains (California, Colorado states), southeast of the USA (Florida, only Geosmithia sp. 21) to the North of Sierra Madre Oriental, Mexico. These records, together with those outside of America, support the distribution of G. langdonii-Geosmithia sp. 32 clade in temperate sub-Mediterranean (Slovakia, Czech Republic, and Bulgaria) and Mediterranean Europe (Portugal, Turkey; [15]), as well as from the western states of the USA (California, Colorado states; Kolarik et al., [19] and Northeast, Mexico; in Geosmithia sp. 21-G. xerotolerans clade, in temperate sub-Mediterranean and Mediterranean Europe (Azerbaijan, Croatia, France, Israel, Jordan, Italy, Slovenia, Spain, Syria, and Turkey), as well as in the western states of the USA (California, Colorado states) and southeastern (Florida [28] and Northeast, Mexico).
Strains of G. langdonii, Geosmithia sp. 32 and Geosmithia sp. 21 have been isolated from different families of Coleoptera vectors frequently associated with Scolytinae bark beetles [15,17,19]. Their specificity patterns and those of other conspecifics are congruent across different geographical regions, displaying a regular association with phloem-feeding bark beetles in a wide host spectrum [15,56]. Our Geosmithia strains correspond to this general pattern because both G. langdonii and Geosmithia sp. 21 were associated with the phloephagous bark beetle species, Phloeosinus deleoni and P. serratus, respectively, constituting new records of vector species. Including those vectors species recorded previously, strains from G. langdonii-Geosmithia sp. 32 clade have been isolated from at least 17 species of beetles corresponding to three families (Bostrichidae, Cerambicidae, and Curculionidae), from which 15 are Scolytids (Supplementary Table S1): Strains from Geosmithia sp. 21-G. xerotolerans clade have been isolated from at least 25 beetle species corresponding to three families (Bostrichidae, Cerambicidae, and Curculionidae), most of them Scolytinae (Supplementary Table S1).
The wide spectrum of vector species of G. langdonii, Geosmithia sp. 32 and Geosmithia sp. 21 is coupled with a high diversity of host plants corresponding to different families [19,29]. The Mexican strains from Juniperus coahuilensis and J. flaccida also increase the host species recorded of fungal species in both clades. In the strains from G. langdonii-Geosmithia sp. 32 clade, the host spectrum quantified at least 17 species through its geographical distribution (Supplementary Table S1), classified within seven plant families (Anacardiaceae, Asteraceae, Cupressaceae, Euforbeaceae, Fagaceae, Pinaceae, and Ulmacea). The strains from Geosmithia sp. 21-G. xerotolerans clade have been recorded from at least 17 host species, classified within five families (Cupressaceae, Fabaceae, Moraceae, Rosaceae, Oleaceae, and Pinaceae).
Geosmithia Diversity
The community structure of Geosmithia species in landscapes is driven principally by the diversity of both bark beetles and their host plant as well as their interactions [19,29]. On small ecological scales, previous data have supported that neighboring populations of the same vector species can transmit relative similar Geosmithia assemblages in the same or different host species [18]. As mentioned above, both sampling areas (Galeana and Iturbide municipalities) are in the north of the physiographic province Sierra Madre Oriental. Thus, they present similar environmental conditions, landscapes, climate, and seasonal rain regimes [19,29]. Given these common characteristics, their geographic proximity, and because in both areas, only one dominant arboreal species was found (J. coahuilensis and J. flaccida, respectively), each one associated with a unique bark beetle species (P. serratus and P. deleoni), a similar Geosmithia species composition pattern between them and low diversity in each were expected.
Our sampling, with multiple repetitions of cut branches as a lure of bark beetles, supports a low diversity of Geosmithia, with only one fungal species per geographical area, as reported by Kolarik [16,19], strains associated with G. xerotolerans-Geosmithia sp. 21 clade from "Iturbide" and strains associated with G. langdonii-Geosmithia sp. 32 clade from "Galeana", which indicates that the genus Phloeosinus harbors a low diversity of fungi, as was observed in other members of genus [16,19]. Both fungal species were recovered across multiple sampling sites in several tree branches and gallery systems (adults and tunnels) supporting a non-incidental association.
Entomochory in Geosmithia Strains
Despite that dispersion of Geosmithia species can be performed by different mediums as wind or water, the establishment of its communities has been explained by the vertical dispersion with vector insects [14][15][16][17]22,29,57] because species are isolated from the adult body and gallery systems. Geosmithia species from Nuevo León were isolated from the insect surface and its respective galleries. Particularly, conidia were located in pupal chambers (Figure 2), sites where metamorphosis occurs and the adults have direct contact with the spores, just before their emergence, which could promote a more efficient transmission and ensures horizontal transfer. To support this hypothesis, we found that 100% of the beetles and gallery samples of both bark beetle species in Nuevo León were coupled with Geosmithia; however, more studies are necessary to analyze the fungal growth within gallery systems and its role in beetle dispersion.
Although we sampledsome tree branches in each region, both fungal species were not found to co-exist, and each region presented a unique Geosmithia "species", associated with a particular plant composition and vector species; the sampling area at Galeana corresponded to semi-arid xerophytic scrub dominated by the J. coahuilensis species; in Iturbide, vegetation corresponded to semi-arid pine forest dominated by J. flaccida. These results indicate that alpha diversity in Geosmithia communities is low in small geographical scales that present few potential vectors and hosts, but that beta diversity is higher between landscapes that display different and particular species composition of hosts and vectors.
Even though the Geosmithia species developed stable symbiotic relationships with different bark beetle species and resemble ophiostomatoid fungi in their host and vector affinities and life strategy evolution, the ecological role of Geosmithia species in beetle galleries is unclear. Some recent studies suggest that the frequency of isolation of Geosmithia in Phloeosinus species indicates a closer symbiotic relationship among them. Phloeosinus Chapuis is constituted by more than 60 taxonomically valid species, 29 of them live on the American continent [31], of which 10 out of 29 (≈35%) had been sampled to search Geosmithia, displaying an incidence of 100% with almost one Geosmithia member isolated per bark beetle species [19,29]; such is the case of P. cupressi, P. sequiae, P. canadensis, and P. punctatus in which the same Geosmithia sp. 21 and G. langdonii were isolated, the last only form P. cupressi and P. sequoiae.
Conclusions
Our results document the presence of strains from Geosmithia langdonii-Geosmithia sp., 32 and Geosmithia sp. 21-G xerotolerans clades in Mexico, supporting their distribution in North America from the Rocky Mountains, as well as southeastern sections of the USA (only Geosmithia sp. 21) to North of Sierra Madre Oriental, Mexico. In North Mexico, these fungal strains were associated with the phloem-feeding bark beetle vectors Phloeosinus serratus and P. deleoni, and showed the capacity of developing in the gallery systems of insects on the host species Juniperus coahuilensis and Juniperus flaccida, respectively. Each fungal strain inhabits a particular forest community and displays a specific association with vector insects and host plants. Genetic and morphological data suggest that both Mexican Geosmithia strains correspond to potential new species. | 2020-10-29T09:02:26.331Z | 2020-10-28T00:00:00.000 | {
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245078106 | pes2o/s2orc | v3-fos-license | Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA): Technical Updates and Pathological Yield
Since the endobronchial ultrasound bronchoscope was introduced to clinical practice, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has become the procedure of choice to sample hilar and mediastinal adenopathy. Multiple studies have been conducted in the last two decades to look at the different technical aspects of the procedure and their effects on the final cytopathological yield. In addition, newer modes of ultrasound scanning and newer tools with the potential to optimize the selection and sampling of the target lymph node have been introduced. These have the potential to reduce the number of passes, reduce the procedure time, and increase the diagnostic yield, especially in rare tumors and benign diseases. Herein, we review the latest updates related to the technical aspects of EBUS-TBNA and their effects on the final cytopathological yield in malignant and benign diseases.
Introduction
Over the last two decades, there have been major developments in our ability to sample mediastinal and peribronchial structures. We have come a long way from the initial use of transbronchial needle aspiration (TBNA) for sampling through a rigid bronchoscope in 1949, to Wang et al. introducing TBNA through flexible bronchoscopy in 1983. Endobronchial ultrasound (EBUS) with real-time visualization of mediastinal structures was first employed in 1992 [1]. As pulmonologists gain more experience in performing EBUS-TBNA, the ability to diagnose pathological involvement of mediastinal structures has significantly improved. The American College of Chest Physicians (ACCP) guidelines for non-small cell lung cancer (NSCLC) report that EBUS-TBNA has a 91% sensitivity in establishing a diagnosis as compared with 81% for cervical mediastinoscopy [2]. Given EBUS-TBNA's minimally invasive nature and high sensitivity, it has become the procedure of choice to stage the mediastinum [3].
Multiple studies have evaluated different components of the EBUS-TBNA procedure to optimize the pathological yield and eliminate unnecessary steps while reducing the time and complications. Newer ultrasound modes and many bronchoscopic tools have been introduced to optimize the selection of the lymph node and the collection of cytological and histological material.
In this article, we aim to review the latest updates related to the technical performance of EBUS-TBNA and describe how emergent data can affect its diagnostic yield in lung EBUS-TBNA is usually performed in the endoscopy suite under moderate sedation (MS), deep sedation, or general anesthesia (GA) [4]. The choice of anesthesia strategy is largely driven by institutional policy and operator preference. In a prospective randomized study, Casal et al. compared the diagnostic yield of EBUS-TBNA performed under MS with GA. There was no difference in the diagnostic yield, the number of lymph nodes (LN) sampled, the number of passes per LN, or the rate of major complications. It should be noted that 6% of patients assigned to the MS group did not tolerate sedation, and the EBUS-TBNA had to be done under GA [5].
Needle Size
The optimal needle size has been a subject of interest for interventional pulmonologists since the introduction of EBUS-TBNA. The factors that need to be considered are the ability to acquire satisfactory specimens to establish the diagnosis without increasing the side effects. The current literature supports the use of 21 G or 22 G needles, as no difference in specimen adequacy or diagnostic yield was found between the two sizes [6].
When compared with the 22 G needle in sampling the same LN, the 25 G needle had a similar diagnostic yield for malignancy. The histology specimens containing malignant cells and the number of malignant cells were significantly higher in the 22 G compared with the 25 G needle, and no difference in complications was seen [7,8]. Other studies comparing aspirate done with 25 G and 22 G needles showed comparable specimen adequacy and diagnostic accuracy [9]. Similarly, there was no difference in specimen adequacy and diagnostic yield when the 25 G needle was compared with the 21 G needle [10].
Moreover, when the 22 G was compared with the larger 19 G needle in sampling the same LN in an alternating manner, there was no improvement in the overall diagnostic yield. However, more bloody passes and lower sample adequacy were observed with the 19 G needle aspirates [11]. Similarly, in a randomized controlled trial of 78 patients, Dooms et al. showed that despite having a larger tissue aspirate, the specimen was bloodier with the 19 G needle, with an overall similar diagnostic yield and specimen quality compared with the 22 G needle [12].
In a prospective analysis of 83 EBUS-TBNA samples obtained from 47 patients, sampling of the same LN with 19 G and 21 G needles showed more cellular material based on the cell area in the cell block obtained with the 19 G compared with the 21 G (7.34 vs. 5.23 mm 2 , p = 0.02) [13]. In a prospective randomized controlled trial that included 107 patients, Wolters et al. showed that aspirates using the 19 G needle contained significantly more tissue and tumor cells compared with the 22 G needle [14]. However, it remains unclear whether this difference affects the molecular analyses and PD-L1 staining of these specimens.
Conversely, in a retrospective single-center study, Jones et al. found a higher proportion of lymphoma (9%, 5%, and 0%) and benign disease (89%, 70%, and 38%) in LN sampled with the 19 G, 21 G, and 22 G, respectively. The 19 G needle was observed to be superior to both 21 G and 22 G in subclassifying malignant diseases, with lower rates of NSCLC-NOS (non-small cell lung cancer-not otherwise specified), and it reduced the need for invasive mediastinoscopy [15].
Although studies comparing different needle sizes did not show any statistically significant differences in diagnostic yield, we cannot exclude that a small difference may exist, especially as some of these studies were underpowered to detect a difference, had a retrospective design, and may not have tested different needles on the same lymph nodes (Table 1).
Use of Suction and Stylet
EBUS-TBNA has traditionally been performed with the needle advanced and with a stylet occluding the needle until the lymph node is accessed under EBUS guidance. A 20 cm suction is then applied, and aspiration is done by moving the needle in the lymph node 10 to 20 times. The addition of suction has not been shown to improve the diagnostic yield or sample adequacy when compared with lower suction of 10 cm or no suction at all [16][17][18].
In a randomized controlled trial, Lin et al. evaluated the diagnostic yield of malignancy and the specimen adequacy of using suction and a stylet, suction with no stylet, and stylet with no suction. Each LN was sampled with the three methods using a 22 G needle. There were no significant differences among the groups in specimen adequacy rate or diagnostic yield of malignancy, although using suction increased the tissue-core acquisition rate compared with the no suction group [19].
Fanning
Fanning is a technique employed by endoscopists to influence the diagnostic yield of a procedure. It consists of sampling multiple areas within a lymph node in each pass by altering the angle of the needle with each subsequent agitation during a pass. This was shown to be superior in EUS-FNA of pancreatic lesions [20], but no such data are present for EBUS-TBNA. However, preliminary data involving lymph nodes in ex vivo calf lungs have shown that fanning methods collected larger samples as compared with no fanning [21].
Core Needle
Core biopsy specimens can also be obtained via EBUS. These samples involve the use of a Franseen tip 22 G fine needle biopsy (FNB) device equipped with three cutting edges (Acquire ® 22 G FNB needle, Boston Scientific Co., Natick, MA, USA) ( Figure 1A-C). In a study evaluating the diagnostic yield of FNB in EUS compared with a historical control using the Expect ® 22 G FNA needle (Boston Scientific Co., Natick, MA, USA), FNB had better histological samples in fewer attempts [22]. In a retrospective analysis of the first 100 patients undergoing EBUS with FNB, Balwan et al. showed that core biopsy was seen in 87% of patients, the pathological diagnosis was established in 97%, and the diagnostic yield for granulomatous lymphadenopathy was obtained in 95.6%. No patient-related adverse events were noted [23].
The ProCore ® needle from Cook Medical (Bloomington, IN, USA) is designed to provide a core of histological tissue in contrast to the cytological specimens from standard fine needle aspirations. It comes in two sizes, 22 G and 25 G. It has a reverse bevel that aims to collect a core histological sample by shearing material from the lesion during retrograde motion ( Figure 1D) [24].
In a retrospective study comparing 110 patients who had an EBUS using a 22 G needle with 125 patients who had an EBUS using the ProCore ® needle, the EBUS core biopsy had a higher sensitivity than standard EBUS-TBNA (92% vs. 77%, p = 0.001). Additional sampling methods such as mediastinoscopy and CT-guided FNA were obtained in 30% of patients who underwent standard EBUS-TBNA versus 15% of those who had EBUS core biopsy (p = 0.006) [25]. However, in a prospective trial, Dhooria et al. found no difference in the diagnostic yield of patients with intrathoracic lymphadenopathy with suspected sarcoidosis when these patients were randomized to EBUS-TBNA with the ProCore needle versus the standard 22 G TBNA needle [26].
Mini-Forceps Biopsy
A histological sampling of the lymph node can be done via mini-biopsy forceps, which is introduced through the initial hole made by the TBNA needle ( nodes larger than 2.5 cm. Sampling was done with a 22 G needle, 19 G needle, and a 1.15 mm (FB-56D-I; Olympus Ltd., Japan) mini-forceps with a cup opening of 7.3 mm. A diagnosis was obtained in 36%, 49%, and 88% of the cases while using the 22 G needle, 19 G needle, and the mini-forceps, respectively. The mini-forceps diagnostic yield compared with the needle was the highest in patients with sarcoidosis (88% vs. 36%, p = 0.001) and lymphoma (81% vs. 35%, p = 0.038). No complications occurred [27]. Herth et al. evaluated the role of transbronchial forceps biopsy (TBFB) in 75 patients without known or suspected NSCLC. Specimens were acquired from subcarinal lymph nodes larger than 2.5 cm. Sampling was done with a 22 G needle, 19 G needle, and a 1.15 mm (FB-56D-I; Olympus Ltd., Japan) mini-forceps with a cup opening of 7.3 mm. A diagnosis was obtained in 36%, 49%, and 88% of the cases while using the 22 G needle, 19 G needle, and the mini-forceps, respectively. The mini-forceps diagnostic yield compared with the needle was the highest in patients with sarcoidosis (88% vs. 36%, p = 0.001) and lymphoma (81% vs. 35%, p = 0.038). No complications occurred [27].
Similarly, Chrissian et al. showed that combining EBUS-TBNA with EBUS-TBFB in a population of 50 patients with a low likelihood of NSCLC resulted in a higher diagnostic yield of 97% compared with either modality alone (81% for EBUS-TBNA and 91% for EBUS-TBFB), with no additional complications [28].
In a retrospective study of 91 patients who had a forceps biopsy with EBUS-TBFB after a non-diagnostic rapid on-site evaluation (ROSE), no difference was seen in the overall diagnostic yield of TBNA versus TBFB. Out of the non-diagnostic TBNA samples on rapid on side evaluation (ROSE) and cell block, subsequent TBFB sampling resulted in additional pathological diagnosis in 16% of the cases; 67% of these were non-caseating granulomas. No complications were reported [29].
Currently available mini-forceps include the Olympus mini-forceps (FB-56D-I; Olympus Ltd., Tokyo, Japan) with an outer diameter of 1.15 mm and a cup opening of 7.3 mm [27], the Boston Scientific "SpyBite biopsy" Forceps (model: M00546270, Natick, MA, USA) with an outer diameter of 1 mm [29], and the CoreDx™ Pulmonary Mini-Forceps by Boston Scientific with an outer diameter of 0.96 mm and a 4.3 mm jaw opening ( Figure 2). The specimen obtained by EBUS-TBFB should be handled as the histology specimen and placed in formalin for fixation or in saline if culture is required [31].
In summary, EBUS-TBFB appears to be complimentary to EBUS-TBNA and can be used when additional tissue is needed for molecular marker studies and in the diagnosis of lesions when the initial sampling with EBUS-TBNA is inadequate or non-diagnostic.
Lymph Node Cryobiopsy under EBUS Guidance
A histological sampling of the lymph node has been reported with the use of a 1.1 mm cryobiopsy probe placed in the same hole created by the TBNA with a 3 sec freeze time before pulling the probe out. The specimen is thawed in saline and fixed in formalin [32]. In a prospective study of 197 patients undergoing EBUS-TBNA and EBUS cryobiopsy for mediastinal lesions of at least 1 cm, cryobiopsy had higher sensitivity than TBNA in rare tumors (91% vs. 25%; p = 0.001) and benign disorders (81% vs. 53%; p = 0.04). The diagnostic yield was similar in malignant lymphadenopathy. Two cases of pneumothorax and one case of pneumomediastinum were reported [33].
EBUS Elastography
Pathological processes make the tissue harder compared with the normal surrounding structure. Elastography is a recent modality that allows the calculation and visualization of tissue elasticity during EBUS. Data are converted into an RGB (red, green, and blue) color image where hard tissue is shown in blue, medium tissue in green, and soft tissue in red [34]. The images are then superimposed onto the standard grayscale B-mode ultrasound scan [35]. Lesions can be classified as type I, predominantly non-blue; type II, partly blue; and type III, predominantly blue ( Figure 3). Types I and III (but not type II) were shown to be highly accurate in predicting benign or malignant disease, respectively [34,36,37].
Quantitative elastography data can also be produced by measuring the strain ratio (SR) of the lesion compared with the normal surrounding tissue. Malignant lymph nodes have a higher SR. An SR > 2.5 had a 100% sensitivity for predicting malignant lymph nodes [34].
A meta-analysis of 17 studies for differentiating benign versus malignant adenopathy found a pooled sensitivity of 0.90 (95% confidence interval (CI), 0.84-0.94) and specificity of 0.78 (95% CI, 0.74-0.81) [38], suggesting that this modality could be important in real time differentiation of benign versus malignant lymphadenopathy.
Rapid On-Site Evaluation
ROSE of EBUS-TBNA samples allows for the rapid evaluation of the adequacy of the sample and a preliminary diagnosis before the specimen is completely evaluated. ROSE helps in assessing the adequacy of the sample, determining the need for additional molecular testing, and potentially decreasing the number of procedural sites. Multiple studies have been performed to compare the utility of ROSE for EBUS-TBNA samples [39][40][41][42]. Griffin et al. demonstrated, in a retrospective study, that there was no difference in diagnostic yield or number of sampled sites with the use of ROSE [39]. In a prospective small study including 81 patients who underwent EBUS with and without ROSE, Cardoso et al. found that the use of ROSE resulted in a higher rate of adequate samples and diagnostic accuracy, although the difference did not reach statistical significance [42]. In a larger randomized prospective trial of 236 patients undergoing EBUS-TBNA, the diagnostic yield in the ROSE group was significantly higher (90% vs. 81%, p = 0.003) and the rate of a suspicious specimen on cytology and non-diagnostic specimen in pathology was significantly lower compared with the non-ROSE group [43]. Another randomized controlled trial of 108 patients showed that ROSE use resulted in a lower puncture number with no increase in the procedure time. Even though the overall diagnostic yield was higher in the ROSE group (85% vs. 75%), it did not reach statistical significance [44].
Overall, ROSE is a helpful tool to optimize the preparation of the specimen and evaluate the adequacy of lymph node sampling. It may help in increasing the diagnostic yield and avoiding repeated procedures (for additional desired testing) without affecting the total time of the procedure [45].
Adequacy of Samples Obtained during EBUS
Currently, there are no defined standardized criteria to determine the adequacy of a sample obtained with EBUS. In general, a sample is considered adequate when a diagnosis is made, such as granuloma or malignancy even in the absence of lymphoid tissue, or if sufficient benign lymphoid tissue is present [45]. Nayak et al. defined an adequate sample as any smear that contains more than 5 fields with at least 100 lymphocytes per low-power field (×100) in a smear PLUS less than 2 groups of bronchial cells per low-power field (×100), or the presence of germinal center fragments, irrespective of the above-mentioned criteria [46]. In addition, any smear with positive results such as malignancy or granuloma was considered adequate. Based on the above, each site can be assigned one of the following categories: non-diagnostic, negative for the disease, granulomatous, suspicious for malignancy, or positive for malignancy [46]. In 2016, Choi et al. suggested using a core tissue length of at least 2 cm, presence of malignant cells, anthracotic pigment, or a lymphocyte density of more than 40 per 10 high-power fields (at ×40 magnification) as criteria for an adequate specimen [47].
Cell Block and Molecular Testing
Cell block is a technique to preserve gross pathological specimens using paraffin blocks for histopathological analysis. It allows for the evaluation of cytological architecture and immunochemical staining, thereby allowing for better characterization of the malignancy [48]. Cell block analysis can increase the yield of EBUS-TBNA by 7% and can generate data for genetic analysis in patients with adenocarcinoma [49]. In 2008, Lee et al. found that a maximum of three passes per lymph node station resulted in a maximal yield for cytopathologic diagnosis [50].
The identification of predictive malignant cell biomarkers in NSCLC has enabled targeted therapies to be utilized for better patient outcomes [51]. Testing for tumor markers such as EGFR, KRAS, and ALK has hence become the standard of care [52]. Once a diagnosis of carcinoma is obtained, extra passes for a cell block should be done for additional studies [45]. In a retrospective study from 2013 of 85 patients who underwent EBUS-TBNA, Yarmus et al. showed that a minimum of four passes per lymph node station is required to provide an adequate amount of specimen when advanced molecular marker analysis is limited to EGFR, KRAS sequencing, and ALK fluorescence in situ hybridization [53]. With the increased availability of additional targetable biomarkers to drive treatment decisions, it remains unclear what is the optimal number of passes that should be obtained [45].
EBUS-TBNA can provide adequate DNA sampling for next-generation sequencing (NGS). The amount of DNA needed for this modality depends on the NGS technique used. Cho et al. found the average total DNA amount from EBUS sampling to be 1971 ng with a range of 100 ng to 10,340 ng [54].
In general, the adequacy of EBUS-TBNA for molecular analysis depends on the sample size, cellularity, tumor cell fraction in the samples, the presence of contaminants such as blood or bronchial cells, and the sensitivity of the molecular testing platform [45,55]. Trisolini et al. evaluated the role of ROSE in molecular profiling in NSCLC and found that complete genotyping was achieved in 90% of the ROSE arm compared with 80% of the non-ROSE arm [56]. Although this difference did not reach statistical significance, it may be clinically relevant. A close collaboration between the molecular lab and the cytopathologist is required to determine sample adequacy for molecular testing.
Overall, the recent literature shows that lymph node sampling via EBUS-TBNA can provide enough material at least 92% of the time for a complete genomic test, which included several biomarkers by NGS and nCounter [57], and more than 94% of the time for immunohistochemical testing for PD-L1 [57,58].
Lymphoma
EBUS-TBNA is a relatively safe procedure for the evaluation of mediastinal/hilar lymphadenopathy, with a reported diagnostic yield of up to 90% [59]. That yield is lower for lymphoma. Studies looking at the yield of EBUS-TBNA for lymphoma have included very low numbers of patients.
In one of the largest studies, which included 75 patients with a final diagnosis of lymphoma, EBUS-TBNA was able to establish a diagnosis in 84% of the patients and was able to subtype lymphoma in 67% of de novo cases and in 81% of the relapsed cases [60]. The lowest yield was in patients with Hodgkin's lymphoma compared with non-Hodgkin's lymphoma [60], and in newly diagnosed compared with recurrent lymphoma [60,61]. Moonim et al. prospectively reviewed 100 cases of suspected lymphoma sampled by EBUS-TBNA. A final diagnosis was achieved in 88% of the de novo lymphoma cases and 100% of the relapsed cases. The reported diagnostic accuracy was 91% with the lowest sensitivity (79%) reported for Hodgkin's lymphoma [62]. Dayan et al. reported similar results with a diagnostic accuracy of 92% [63].
While EBUS-TBNA can be the first diagnostic modality, EBUS-TBFB and EBUS core biopsies might be able to provide larger histopathological tissue to help in subtyping lymphoma [27,28]. In one meta-analysis of 443 patients, adding mini-forceps biopsy to EBUS-TBNA increased the diagnostic yield significantly from 30% to 86% (p = 0.03) [30].
Flow cytometry is of particular importance in the immunological phenotyping of lymphomas [64]. Since diagnosis and subtyping are essential to provide treatment for patients with lymphoma, negative results should not exclude lymphoma [61]. Surgical excision remains the gold standard [65].
Sarcoidosis
EBUS-TBNA is the first choice for pathological confirmation of sarcoidosis [66]. It has a pooled sensitivity of more than 80% for diagnosing sarcoidosis [67], significantly higher than transbronchial lung biopsy (TBB) or endobronchial biopsy (EBB) alone. The combination of EBUS-TBNA with TBB and EBB results in a significant increase in the diagnostic yield (90%) for the diagnosis of stages I and II sarcoidosis [68][69][70]. In addition to improving the diagnostic yield, EBUS-TBNA with rapid on-site evaluation may alleviate the need to perform unnecessary TBB [71].
In a prospective study of 109 patients who underwent EBUS-TBNA for suspected stages I and II sarcoidosis, the cumulative yields for detecting non-caseating granulomas through the first, second, third, fourth, fifth, and sixth passes for the main target lesion were 63%, 75%, 82%, 85%, 86%, and 88%, respectively. The increase was statistically significant up to pass #4 [72]. A higher yield was associated with sampling nodes with a short axis of more than 1 cm, and with stage I compared with stage II sarcoidosis [73]. The number of nodes sampled appears to increase the yield in some studies [74], but not in others [73].
Procedurally, the most common endosonographic findings from sarcoidosis lymph nodes include the presence of a homogeneous texture, oval shape, a conglomeration of lymph nodes [75,76], distinct margins [75,76], and increased non-hilar perfusion [76]. The presence of the necrosis sign and absence of the clustered formation were independent factors predictive of tuberculous nodes as opposed to sarcoidosis [76].
There was no difference in the diagnostic yield in relation to needle size (22 G vs. 25 G [9], or 21 G vs. 22 G [77]) in patients suspected of having sarcoidosis. Similarly, the number of agitations of the EBUS needle (10 vs. 20) did not influence the diagnostic yield or the specimen adequacy in this population [78]. However, adding mini-forceps biopsy to EBUS-TBNA increased the diagnostic yield significantly from 58% to 93% (p < 0.00001) in one meta-analysis of 443 patients [30].
Conclusions
EBUS-TBNA remains the first-line minimally invasive test to evaluate mediastinal and hilar adenopathy. Since its introduction, the procedure has been refined to eliminate unnecessary steps and reduce the procedure time while optimizing the diagnostic yield. In areas where no difference between the different techniques was found, larger high-quality randomized controlled trials are recommended. In addition to providing nodal staging, EBUS-TBNA allows the acquisition of molecular markers that are essential in guiding the choice of therapy in patients with non-small cell lung cancer. It also provides an excellent diagnostic yield in stages I and II sarcoidosis. Newer tools such as core needles and miniforceps are now available, and they appear to increase the histopathological specimen size and possibly the diagnostic yield in patients with lymphoma and benign diseases, therefore reducing the need for more invasive interventions such as mediastinoscopy. • Core needle biopsy had higher overall sensitivity compared with standard EBUS-TBNA.
•
Additional biopsy tests such as mediastinoscopy and CT-guided FNA were obtained in fewer patients who underwent EBUS core biopsy.
Number of passes • Prospective study (n = 102 patients) [50] • A maximum of three passes per lymph node station results in a maximal yield for cytopathologic diagnosis.
Conflicts of Interest:
The authors declare no conflict of interest. | 2021-12-12T16:31:55.998Z | 2021-12-01T00:00:00.000 | {
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229279617 | pes2o/s2orc | v3-fos-license | The periodontal status of removable appliances vs fixed appliances
Abstract Background: Although several researchers have analyzed the dental identity of patients experience with corrective methods using fixed and removable appliances, the consequences stay debatable. This meta-analysis intended to verify whether the periodontal status of removable appliances is similar to that of the conventional fixed appliances. Methods: Relevant literature was retrieved from the database of Cochrane library, PubMed, EMBASE, and CNKI until December 2019, without time or language restrictions. Comparative clinical studies assessing periodontal conditions between removable appliances and fixed appliances were included for analysis. The data was analyzed using the Stata 12.0 software. Results: A total of 13 articles involving 598 subjects were selected for this meta-analysis. We found that the plaque index (PLI) identity of the removable appliances group was significantly lower compared to the fixed appliances group at 3 months (OR = −0.57, 95% CI: −0.98 to −0.16, P = .006) and 6 months (OR = −1.10, 95% CI: −1.60 to −0.61, P = .000). The gingival index (GI) of the removable appliances group was lower at 6 months (OR = −1.14, 95% CI: −1.95 to −0.34, P = .005), but the difference was not statistically significant at 3 months (OR = −0.20, 95% CI: −0.50 to 0.10, P = .185) when compared with that of the fixed appliances group. The sulcus probing depth (SPD) of the removable appliances group was lower compared to the fixed appliances group at 3 months (OR = −0.26, 95% CI: −0.52 to −0.01, P = .047) and 6 months (OR = −0.42, 95% CI: −0.83 to −0.01, P = .045). The shape of the funnel plot was symmetrical, indicating no obvious publication bias in the Begg test (P = .174); the Egger test also indicated no obvious publication bias (P = .1). Conclusion: Our meta-analysis demonstrated that malocclusion patients treated with the removable appliances demonstrated a better periodontal status as compared with those treated with fixed orthodontic appliances. However, the analyses of more numbers of clinical trials are warranted to confirm this conclusion.
Introduction
In the present age, the advancements in the design and manufacturing of dental motion materials using computer has encouraged the demand for optimized requirements in orthodontic treatment technology. In 1946, Kesling first proposed the concept of moving orthodontic appliances to move misplaced teeth. [1] However, in the last decade, the concentrated cell method has also been a preferred treatment as it covers a range of malocclusion types. [2] However, several researchers have successfully demonstrated how the present appliances can correct and treat almost all diseases, ranging from mild to severe malocclusion, with better periodontal status. [3][4][5] Despite the known effectiveness of conventional methods practiced across the world, the shortcomings associated with these methods cannot be overlooked. For instance, the conventional methods in dentistry are inconvenient and even painful, often posing difficulty in cleaning. Patients are required to be cautious with the stent and are required to regularly clean the plaque collected around the wire to improve the oxidation-reduction potential. Previous studies have reported that the use of fixed orthotics can stimulate the growth of subgingival plaques, which trigger adverse reactions and increase the discomfort of patients. [6][7][8] Therefore, the use of an alternate removable orthodontic device is expected to facilitate convenience and better healing for patients requiring urgent interventions. [8][9][10] In the recent years, a large number of studies have been reported on times health identity of patients treated with concentrating and removable appliances. [11][12][13][14][15][16][17][18][19][20][21][22][23] However, the inference derived from these papers remains controversial. Therefore, clinicians can only rely on their clinical experience and the low-quality evidence reported in the literature when formulating treatment plans. Accordingly, considering the situation, we hypothesized that the periodontal status of patients treated with removable appliance was better than that of patients treated with fixed appliances, and employed a meta-analysis to confirm our hypothesis.
Search strategy
For this meta-analysis, articles were sourced from the databases of EMBASE, Cochrane library, Medline, PubMed, CNKI, and Wanfang without time or language restrictions. All relevant studies published through to December 2019 were included. In addition, we conducted manual retrieval in the research process, mainly using the research results in the references. Relevant studies were identified using the following key terms: "removable aligners", "removable thermoplastic aligners", "clear appliances", "invisalign", "periodontal index", "periodontics", and "periodontium". Because this analyses was based on previously published studies, so there was no require for ethical approval and patient consent.
Inclusion criteria
This review included prospective cohort studies or randomized controlled trials (RCTs) that compared the periodontal status in patients treated with fixed appliances versus removable appliances. The subjects were patients diagnosed with malocclusion and who received orthodontic treatment for the same, who showed good oral health, no obvious periodontal disease, no systemic disease, no long-term history of taking antibiotics, among others. We focused on removable or fixed orthopedic appliances as the means of intervention.
Exclusion criteria
Case reports, review articles, and animal studies were excluded. Moreover, original articles whose reference literature could not be used after contact with the author were excluded.
Observation index
Plaque index (PLI), gingival index (GI), and the sulcus probing depth (SPD) were recorded in this study. The outcomes for the PLI, GI, and SPD at 3 and 6 months were assessed in this meta-analysis.
Data collection and analysis
Two investigators formed the data research object. In case of a conflict between the reports of the 2 investigators, further inspection of the measurements was made until finalization of the results. In case no agreement could be reached, a professional scholar was invited to resolve the issue. The data extracted from the references included the following: publication date, author name, country of the study, method of treatment, number of 2 methods, age, gender, patient recruitment time, the measurement period, and result measurements of different literatures.
Quality assessments
The quality of all research was assessed with reference to the Newcastle-Ottawa Scale (NOS). The research evaluation criteria were mainly divided into 3 aspects: measurement results, comparability, and queue selectivity. These aspects were further categorized into the number of stars, in a descending order, with grade A = 7-10 stars, grade B = 4-6 stars, and grade C = <3 stars. [24] During this process, in case of a conflict, negotiation was made to resolve the dispute. As per the description given in Table 1, all references in the meta-analysis belonged to grade A. Therefore, it can be concluded that this study involved the analysis of high-quality literature.
Statistical analysis
The data from the individual studies were pooled and analyzed using the Stata 12.0 software (Stata Corporation, College Station, Texas). I 2 test and Chi-Squared-based test were applied to analyze the heterogeneity among the included articles. The range of heterogeneity was as follows: extreme = 75% to 100%; large = 50% to 75%; moderate = 25% to 50%; and low = < 25%. The fixed-effects model was generally used to evaluate the research content because I 2 was <50%. A random effect model was used whenever the value was >50%. After obtaining the results of combined odds ratio (OR) and 95% confidence interval (CI), the Z test was employed for data analysis, with P < .05 considered as statistically significant. Any publication bias was assessed by using the Begg test and the Egger test. Sensitivity analysis was applied to analyze large heterogeneity studies and to find the source of heterogeneity.
Characteristics of studies
According to the above-mentioned retrieval methods, 192 relevant studies were selected for the analysis. After skimming the titles, abstracts, and reviewing the full-text content, 179 studies were excluded due to the lack of available data or the non-RCT nature of the study, among other reasons. Finally, 13 studies involving 598 patients met the inclusion criteria. [11][12][13][14][15][16][17][18][19][20][21][22][23] Among which, 297 patients were treated with removable appliances and 301 patients with fixed appliances. The flow diagram of the study selection procedure is presented in Fig. 1. The basic information of each included literature is shown in Table 2.
The status of GI
A total of 4 studies evaluated the GI of 2 appliances after 3 months of treatment, and 8 studies evaluated the GI of 2 appliances after 6 months of treatment. The results of heterogeneity test were as follows: P GI3 = .783, I 2 = .0% and P GI6 = .000, I 2 = 91.8%, respectively. These results demonstrated no statistical significance in GI between the removable and fixed appliances groups at 3 months (OR = À0.20, 95% CI: À0.50 to 0.10, P = .185). However, patients treated with removable appliances showed significantly lower GI status at 6 months (random effects model OR = À1.14, 95% CI: À1.95 to À0.34, P = .005), as shown in Figs. 4 and 5.
The status of SPD
Six researches evaluated the SPD of 2 appliances after 3 months of treatment, and 8 researches evaluated the SPD of 2 appliances
Sensitivity analysis
The sensitivity analysis was conducted after removing each of the included articles one by one. However, the results demonstrated no significant change in the results of the combined effect, which implied that the result of the meta-analysis was stable.
Publication bias
Begg test and Egger test were conducted to assess the publication bias (Fig. 8). Symmetry of the funnel plots implied no obvious publication bias (P = .174), and the results of Egger test also demonstrated no publication bias (P = .1).
Discussion
The removable appliances appeared as creative orthopedic appliances in the late 1990 s. [25] The conventional orthodontic appliances were based on brackets and wires for orthodontic tooth movements. These are aesthetically pleasing, comfortable, simple, predictable, and portable devices. Because of the influence about times disease, Ristic et al [26] put forward the GI cell slowly upgrade at 4 weeks and 3 months when taking the concentrate tool, then reached its peak at 6 months. Meanwhile, some scholars have demonstrated the the progress of gravity could reach its highest value after 5 to 6 months of use. [23,26,27] From now on, gravity is regularly remained in the rank during the treatment period. Therefore, we partly researched the times situation in the first 6 months. Our results revealed that the GI, PLI, and SPD indexes were significantly reduced with removable orthotic devices as compared with that with conventional fixed orthotic devices (P < .05). These statistics thus signify that removable appliances are more beneficial for a healthy periodontal status. The probable reasons supporting the superiority of removable appliances are as follows: patients with removable appliances can take the appliance out of their mouth and clean it. In addition, patients can remove the appliance at the time of cleaning their teeth, which is convenient. A removable appliance helps in better flossing and hence in maintaining better oral hygiene. Removable appliances covering most of the crown area can control the force exerted on that area. Removable appliances help make the teeth www.md-journal.com move closer as an overall movement while preventing the destruction of the periodontal tissues due to the migration of the supragingival plaque to the subgingival tissues. Several studies have evaluated the influences of orthodontic appliances on the periodontal health. For instance, Miethke and Vogt [17] reported that, at the baseline level and at 3 distinguishing development time steps, patients arranged with fixed appliances were at significantly greater PLI risks than those arranged with removable appliances. However, they discovered no statistically significant difference in the SPD between the groups of patients treated with fixed appliances versus those fixed with removable appliances. Abbate et al [27] performed a similar initial orthodontic treatment on 50 adolescents aged 10 to 18 years and found that the adolescents wearing removable appliances had a higher periodontal status than adults using fixed appliances after the same treatment course. However, Alstad and Zachrisson [28] found no significant difference in the PLI or GI between these 2 treatment approaches. Despite the extensive use of fixed and removable appliances, there seems to be a lack of evidence supporting any specific appliance as being more beneficial for the periodontal health. Bollen et al [29] and Van et al [30] reviewed the literature and inferred that orthodontic measurement by itself does not upgrade the risk of periodontal pathologies. However, several studies have reported that the choice of oral hygiene procedures have a profound effect on the periodontal health of orthodontic patients. [31] Notably, as per a recent observation, morbidity due to periodontitis increases with the age of the patient. Many adult patients realize the importance of dental health and begin to apply orthodontic treatment for straightening their teeth. [32] Orthodontic therapy may often lead to periodontal diseases, because the use of orthodontic appliances during the treatment may affect the oral hygiene procedures and lead to the accumulation of microbes in the mouth. However, some researchers argue that periodontal diseases are only partially related to orthodontic treatment. They state that orthodontic appliances can interfere with oral hygiene procedures to produce bacteria and induce their proliferation. [26,[33][34][35][36][37] Some clinical and experimental trials have demonstrated that, despite maintaining good oral hygiene in patients, the use of orthodontic appliances can cause inflammation and lead to periodontal damage if the inflammation is not completely controlled, and that the attachment disappears with the accelerated development of periodontal damage. [38,39] Several previous studies have reported that fixed orthodontics serve as a greenhouse for plaque to build, which can lead to the development of inflammatory manifestations, such as gingival swelling or bleeding. [40][41][42] Currently, several studies have compared different orthodontic appliances with removable appliances and found the performance of removable appliances much superior. This is because removable appliances have been found to contribute significantly in building up oral hygiene by inhibiting the accumulation of dental plaque. [17,42,43] In terms of clinical presentation, the therapy of removable appliances is more secure for periodontium when compared with the therapy of fixed appliances. [44] Notably, removable appliances can help maintain the oral hygiene and thereby reduce the amount of plaque retentive surfaces. Considering these points, it can be concluded that removable appliances are a great orthodontic treatment appliance for patients with poor periodontal health.
However, some scholars believe that because patients must wore removable appliances for more than 20 hours a day, if patients failed to clean their mouth in time, food residue may stay in the gap between appliances and gingival mucosa, and prevent the self-cleaning of saliva in the patient's mouth. And because the appliance is an integrated appliance, covering the gingiva in a large area may caused gingival compression and injury, or some patients may not mastered the correct method to remove and wear the appliance during the correction period which may leaded to injury to the gingival tissue too. Therefore, they believe that the removable appliances is more harmful to the periodontal health of patients than the fixed appliances. [13,45] Thus, strong evidence is still needed to support our hypothesis.
This meta-analysis has some limitations. First, the available research data were limited to those from China, Germany, and Italy only, and hence the conclusion may not be applicable to other countries. Second, without analyzing a large number of studies, it is difficult to conduct a comprehensive and detailed study, and some studies with a small sample size could not provide sufficient statistical power to identify the actual association. Third, the index measurements of the position and the quantity of teeth were coincidental. Some study assessed the full mouth teeth, while others measured only certain teeth, which may have resulted in a bias during the implementation. Moreover, the types of malocclusion included were not corresponding, which may have enhanced the presence of confounding factors.
Conclusion
This meta-analysis demonstrated that the periodontal status of patients treated with removable appliance was much superior to that with the conventional fixed appliances. Owing to the limitation in terms of both quality and quantity of the involved studies, we suggest that the inference of this review be verified further using more number of RCTs. | 2020-12-17T05:07:58.020Z | 2020-12-11T00:00:00.000 | {
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249244227 | pes2o/s2orc | v3-fos-license | Cause of Death During Renal Cell Carcinoma Survivorship: A Contemporary, Population-Based Analysis
Background As the survival rates of patients with renal cell carcinoma (RCC) continue to increase, noncancer causes of death cannot be ignored. The cause-specific mortality in patients with RCC is not well understood. Objective Our study aimed to explore the mortality patterns of contemporary RCC survivors. Methods We performed a retrospective cohort study involving patients with RCC from the Surveillance, Epidemiology, and End Results (SEER) database. We used standardized mortality ratios (SMRs) to compare the death rates in patients with RCC with those in the general population. Results A total of 106,118 patients with RCC, including 39,630 who died (27%), were included in our study. Overall, compared with the general US population, noncancer SMRs were increased 1.25-fold (95% confidence intervals [CI], 1.22 to 1.27; observed, 11,235), 1.19-fold (95% CI, 1.14 to 1.24; observed, 2,014), and 2.24-fold (95% CI, 2.11 to 2.38; observed, 1,110) for stage I/II, III, and IV RCC, respectively. The proportion of noncancer causes of death increased with the extension of survival time. A total of 4,273 men with stage I/II disease (23.13%) died of RCC; however, patients who died from other causes were 3.2 times more likely to die from RCC (n = 14,203 [76.87%]). Heart disease was the most common noncancer cause of death (n = 3,718 [20.12%]; SMR, 1.23; 95% CI, 1.19–1.27). In patients with stage III disease, 3,912 (25.98%) died from RCC, and 2,014 (13.37%) died from noncancer causes. Most patients (94.99%) with stage IV RCC died within 5 years of initial diagnosis. Although RCC was the leading cause of death (n = 12,310 [84.65%]), patients with stage IV RCC also had a higher risk of noncancer death than the general population (2.24; 95% CI, 2.11–2.38). Conclusions Non-RCC death causes account for more than 3/4 of RCC survivors among patients with stage I/II disease. Patients with stage IV are most likely to die of RCC; however, there is an increased risk of dying from septicemia, and suicide cannot be ignored. These data provide the latest and most comprehensive assessment of the causes of death in patients with RCC.
INTRODUCTION
Renal cell carcinoma (RCC) is one of the top 10 most prevalent cancers and accounts for 4% of all new malignancies in the United States (U.S.) (1, 2). Approximately 1.7% of people are diagnosed with kidney cancer at some point in their lives (1, 2). As treatment has improved, the death rate from kidney cancer has decreased. It is estimated that there will be 793,530 cancer survivors in the U.S. by 2030 (1-3). Therefore, understanding the real causes of death could help prioritize death risk during survivorship and may provide a roadmap for reducing the mortality burden after RCC.
The causes of death from prostate cancer, colon cancer, testicular cancer, and other cancers have been well described (4)(5)(6)(7)(8)(9). Several studies have described the causes of death in patients with RCC. However, these studies were not compared with the risk in the general population, either based on small sample studies, or limited to secondary primary tumors, and the classification of the cause of death is not detailed enough (10)(11)(12). To address these limitations, we assessed contemporary, population-based data on the causes of death during RCC survivorship. We present our results based on patient characteristics and the AJCC 6th stage, and the risk of death from each cause was compared with that of the standard population.
Data Source
This was a retrospective, observational, cohort study. We used data from the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) 18 registries, November 2020 submission (2000 to 2018) for SMRs, which covers approximately 34.6% of the U.S. population.
Patients
We included all patients diagnosed with RCC as their first malignant tumor between January 1, 2004, and December 31, 2015. Patients with an unknown follow-up time, vital status, and staging information (I, II, III, or IV) were excluded. We excluded patients diagnosed through autopsy or death certificates only. Figure 1 shows the inclusion and exclusion criteria of the present study.
Outcome Assessments
The outcome variable of interest was overall survival after RCC diagnosis. The SEER cause of death code was based on the International Statistical Classification of Diseases and Related Health Problems 10th, 1999 (ICD-10).
Ethics Statement
We were granted permission from the National Cancer Institute USA to access the SEER dataset for research purposes only (reference number: 20025-Nov2020). All data from the SEER database were de-identified, and the extracted data did not require informed consent.
Statistical Analyses
We used standardized mortality ratios (SMRs), defined as the observed number of deaths divided by the expected number. The expected numbers of deaths were calculated based on age-and sex-specific mortality rates in a standard population. The followup time began from the date of first diagnosis to the date of death, loss to follow-up (date of the last visit), or December 2018, whichever came first. The 95% confidence intervals (CIs) for SMRs were estimated using exact methods. All SMRs were generated using the SEER*Stat version 8.3.9.2. Table 2). Heart disease was the most common noncancer cause of death (n = 3,718 [20.12% of all-cause deaths]), and the proportion increased with the extension of survival time (Figure 2A). The most common causes of non-RCC cancer deaths were respiratory system and digestive system cancers (n = 803 [4.35%] for respiratory system cancer and n = 815 [4.41%] for digestive system cancer). Over the whole follow-up period, the risk of death was greater than that in the general population (SMR, 1.54; 95% CI, 1.52-1.56), with the highest risk observed within the first year after RCC diagnosis (SMR, 1.81; 95% CI, 1.74-1.89), the risk levels gradually became stable (SMR, 1.51 for 1 to <5 years; SMR, 1.52 for 5 to <10 years; SMR, 1.51 for ≥10 years). The risk of almost all deaths increased except for breast tumors (SMR, 0.38; 95% CI, 0.28-0.5) and Alzheimer's disease (SMR, 0.84; 95% CI, 0.75-0.93) ( Figure 2B). Over the entire follow-up period, the mortality rate was greater than that of the general population (SMR, 2.93; 95% CI, 2.86-3.00). The SMR was the highest in the first year of diagnosis (SMR, 4.54; 95% CI, 4.29-4.81), and the risk levels gradually declined (SMR, 3.15, 1 to <5 years; SMR, 2.30 for 5 to <10 years; SMR, 1.88 for ≥10 years). Figure 2C). Heart disease was the most common noncancer cause of death (n = 328 [2.23%]). In contrast to other stages, stage IV patients also had a higher risk of suicide (SMR, 3.46; 95% CI, (2.17-5.24). -S7). Patients who have never been married have a higher risk of death than those who are married and in other marital statuses, regardless of whether it is a cancer or a noncancer factor (Tables S8-S10). Noncancer causes of death were lower in patients who underwent surgery than in those who did not (Tables S11 and S12). In contrast, patients who received chemotherapy or radiotherapy showed an increase in noncancer causes of death (Tables S13-S16). There was no significant difference in SMR between the different years of diagnosis (Tables S17-S19). The prognosis of patients with other histological types was the worst, and the prognosis of patients with chromophobe histological type was the best (SMR, 1.08; 95% CI, 1.01-1. 16
DISCUSSION
In the U.S., there are more than 320,000 RCC survivors, and the number is still increasing. It is of vital importance, during survivorship, to optimize healthcare management. We stratified our results according to patient characteristics and stage. In the patient cohort with stage I/II RCC, non-RCC causes of death were 3.32-fold more frequent (76.87% vs. 23.13%, respectively). In patients with stage III/IV disease, 59.17% (n = 3,912) and 84.65% (n = 12,310) of the patients died from RCC, respectively, and the risk of death due to most non-RCC causes was still higher than that in the general population. These results mirror those of previous studies (11).
Our study demonstrates the causes of death in RCC patients with different stages and characteristics, which may help clinicians. For example, the prevention and control of noncancer causes and screening for non-kidney cancer can be emphasized.
In patients with RCC, the most common noncancer death was heart disease. In our study, patients with stage IV disease had a higher SMR in heart disease than those with stage I/II and III (SMR, 1.92; 95% CI, 1.72-2.14 for stage IV; SMR, 1.23; 95% CI, 1.14-1.32 for stage III; SMR, 1.23; 95% CI, 1.19-1.27 for stage I/II). In the past decade, several tyrosine kinases and vascular endothelial growth factor inhibitors have been used for first-line therapies in patients with metastatic RCC (13,14). These drugs greatly improved the survival rate of patients with metastatic RCC. However, cardiotoxicity cannot be ignored (15)(16)(17).
Hemocytopenia due to antineoplastic therapy is a common occurrence in clinic (18)(19)(20)(21). Neutropenia is independently associated with septicemia (22,23). In addition, the process of tumor metastasis may damage the immune system, resulting in a higher infection risk in cancer patients (24). In this study, patients with stage IV RCC had a markedly higher risk of death from septicemia, especially those who died within a year (SMR, 3.95; 95% CI, 3.05-5.04 for infections; SMR, 2.20; 95% CI, 1.83-2.62 for pneumonia and influenza). In our study, patients with stage IV disease were at a higher risk of suicide. This outcome is similar to that of Guo et al. (25).
In this study, we found that patients who underwent direct cancer surgery had a lower SMR than those who did not. It seems possible that these results are due to patients undergoing surgery having fewer comorbidity and better health conditions than those who did not.
Patients who were never married or in other marital statuses had a higher risk of all-cause death, kidney cancer-specific death, and other noncancer deaths than married patients. The fact that marriage provides social support may partly explain this finding (14,26,27).
However, this study has some limitations. First, our study design was retrospective, which inevitably resulted in a selection bias. We also performed our best to reduce bias. We used strict screening criteria to reduce the selection bias and use SMR to control for age, sex, and ethnic differences, rather than direct mortality to reduce confusion bias. Second, the SEER database lacks important information on treatment strategies and comorbid states, which may cause bias. Third, most of our participants were white, and whether our conclusion can be extended to other races, it still needs to be further verified. Finally, there may be potential misclassification of the cause of death in the SEER database. However, previous studies have shown that this variable is accurate in most situations (28).
CONCLUSIONS
In summary, this study provides the latest and most comprehensive assessment of the causes of death in patients with RCC. Causes of death varied according to patient demographics. Non-RCC causes of death account for more than 3/4 of RCC survivors among patients with stage I/II disease. Patients with stage IV are most likely to die of RCC; however, there is an increased risk of dying from septicemia, and suicide cannot be ignored. Therefore, attention should be paid not only to antineoplastic therapy, but also to the occurrence of other risks.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. | 2022-06-02T13:30:11.744Z | 2022-06-02T00:00:00.000 | {
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259194208 | pes2o/s2orc | v3-fos-license | Von Gierke Disease (Glycogen Storage Disease Type I) and Life-Threatening Abdominal Aortic Aneurysm: A Case Report of an Extremely Rare Condition
Von Gierke disease, also known as glycogen storage disease type I, co-existent with an abdominal aortic aneurysm (AAA), is an extremely rare combination of diseases that requires challenging therapeutic measures. We present, for the first time in literature, the case of a 62-year-old female with von Gierke disease who required open surgical repair of an AAA with challenging neck anatomy outside of instructions for use of endovascular repair. Even though the surgical risks for life-threatening complications, such as pancreatitis, metabolic acidosis, and kidney failure, were high, the 6-month postoperative course was uneventful. Despite the invasiveness of the treatment, surgery to treat the AAA was safe and effective. Further data is needed to draw robust conclusions about the treatment of choice for those patients with diseases in co-existence with AAAs.
INTRODUCTION
Glycogen storage disease type I (GSDI), better known as von Gierke disease, is a rare hereditary disease that affects one in 100,000 of the population. It consists of a deficiency of enzymes that play a significant role in glycogen metabolism [1].
This syndrome manifests with hypoglycemic episodes, lactic acidemia, hyperlipidemia, and neutropenia (GSDIb). Additionally, in the long term, these patients may present with kidney failure and hepatic malignancies [2]. When these patients require major surgery necessitating general anesthesia, they are at high risk of perioperative hypoglycemia, metabolic acidosis, rhabdomyolysis, myoglobinuria, acute renal failure, pancreatitis, and excessive bleeding [2,3].
The co-existence of this disease with an abdominal aortic aneurysm (AAA) is an extremely rare occasion that poses a serious therapeutic challenge, considering the perioperative and postoperative difficulties associated with an aortic repair. This case report aims to highlight the demanding peri-and postoperative management with increased rates of complications, and the absolute necessity of a multidisciplinary approach in order to achieve the best medical care for these patients.
This case report adheres to the ethical principles outlined in the Helsinki Declaration, including the principles of informed consent, confidentiality, and protection of patient privacy. The patient provided written informed consent for www.vsijournal.org anonymized information, including images, to be published in this article. Since the manuscript was a case report based on an observational study of a patient, Institutional Review Board approval was not required.
CASE
A 62-year-old female, previously diagnosed with GSDI, was presented to our outpatient care with the sonographic finding of an asymptomatic AAA measuring 5.6 cm in diameter. Hypertension, hepatomegaly, kidney dysfunction, and hyperuricemia were included as comorbidities. As an indication for surgical treatment was evident, a computed tomography angiography (CTA) was performed (Fig. 1). By evaluating the CTA images, we concluded that the characteristics (reverse tapered anatomy, infrarenal angulation >60°, diameter of the infrarenal neck <22 mm) were outside of the instruction for use for an endovascular aneurysm repair (EVAR); thus, from the morphological point of view, the open repair approach was preferred [4]. A fenestrated endovascular aneurysm repair (FEVAR) and chimney endovascular aneurysm repair (CHEVAR) approach was also considered, but neither option was selected since these complex endovascular techniques require a greater amount of contrast agent and thus increase the risk of acute renal failure.
On the other hand, the diagnosis of von Gierke disease made the required anesthesia for open surgical repair quite challenging due to the described risks [2,3]. Preoperatively, the patient underwent fibrate therapy for three days in order to achieve lower triglyceride levels and minimize the risk of postoperative pancreatitis. Additionally, dextrose solution was administered the day prior to the operation, and the aneurysm was successfully excluded by the placement of an aortoiliac Y-prothesis. The operation lasted two hours, with an overall aorta clamping time of 40 minutes. During the operation, minimal blood loss (400 mL) was observed. Additionally, a minimal dosage of catecholamines was administered. No hypoglycemic crisis or acidosis was noted perioperatively. The patient was successfully extubated, had palpable peripheral pulses, and stayed in the intensive care unit (ICU) for two days. During this time, the monitoring of glucose, lactate, and base excess through regular arterial blood analysis revealed normal-range glucose levels; this eliminated the possibility of a hypoglycemic or acidosis episode. A constant glucose perfusion was performed until the glucose levels were stable within the range of 90-120 mg/dL. Following that, Nutriflex, a nutritional solution consisting of lipids, carbohydrates, and amino acids totaling 765 kcal per liter, was administered through an IV at 40 mL/h. Lipofundin, containing medium-chain triglycerides and glycerol with a total caloric intake of 1,980 kcal/L, was also administered at 11 mL/h. Since the patient was 150 cm tall and weighed 80 kg, the body mass index was calculated at 35.6 kg/m 2 ; therefore, a total of 750 kcal for the first 12 hours was determined to be sufficient nutrition.
After two days in the ICU, the patient returned to the peripheral vascular station for four additional days. During the entire in-hospital postoperative period, the patient required transfusion of three units of erythrocyte concentrates; meanwhile, the triglyceride values decreased from 1,600 mg/dL to 1,052 mg/dL and therefore plasmapheresis was not necessary. Additionally, a temporary increase of 0.5 mg/dL in the creatinine levels was observed, reaching a maximum of 1.6 mg/dL; this was treated with adequate hydration, resulting in the reversion of creatinine levels to the normal range. On the day of discharge, the glomerular filtration rate (GFR) was 89 mL/min, the Hb level was >9 g/ dL and the patient was fully mobilized, free of symptoms, and in an overall good condition.
The 6-month follow-up was uneventful. A magnetic resonance tomography angiography of the abdomen showed exclusion of the aneurysm and stenosis-free flow to the groins (Fig. 2).
DISCUSSION
GSDI is a rare disorder with an incidence of one in 100,000 [1]. Meanwhile, AAA in females is an uncommon disease, with an estimated prevalence of 0.43% in females at the sixth decade of life [5].
Two of the most prominent articles were case series with 80 and 6 patients, respectively, where the patients suffered from GSDIa or GSDIb and required major surgeries [2,3]. Gurrieri et al. [3] in a series of six patients reported two intraoperative hypoglycemias that were treated with dextrose solutions and three perioperative metabolic acidosis that resolved after 24 hours. No postoperative pancreatitis or any other adverse event was reported. On the other hand, Boers et al. [2] in their study of 80 patients, reported four postoperative deaths (one suicide, one malignancy, one transplant rejection, and one death due to acute pancreatitis and sepsis two months after the surgery). They also reported eight acute and six chronic kidney failures; however, only three patients required dialysis. After reviewing the literature, it becomes apparent that surgery is associated with intraoperative hypoglycemic episodes and metabolic acidosis, as well as postoperative renal failure. Since this is the first documented case with combined GSDI and AAA, the vascular status of these patients has not yet been documented. As their triglyceride levels remain extremely high for their entire lives, it is quite logical to expect increased vascular tissue frailty with damaged arterial walls and an increased risk of thromboembolization. However, during the operation, the arterial wall of the abdominal aorta was similar to that of any other patient suffering from AAA, disproving this hypothesis.
Although our first choice of treatment was EVAR, the reverse tapering and the narrow and angulated infrarenal neck excluded the possibility of a minimally invasive treatment with conventional EVAR. Additionally, taking into consideration the risk of potential renal failure for a patient with GSDI, the nephrotoxicity of the contrast agent used in the more complicated endovascular techniques (FEVAR and CHEVAR) was another deterring factor of the endovascular approach.
On the other hand, the open approach hides the risk of intraoperative hypoglycemic episodes as well as acute postoperative pancreatitis. Even though we administered fibrates to counter hyperlipidemia, triglyceride values were constantly >1,200 mg/dL, further increasing the risk of acute pancreatitis. After a multidisciplinary meeting between the vascular and anesthesiology departments of our hospital, accompanied by a specialist for metabolic disorders, we decided to proceed with an open repair. A temporary creatinine spike, which reached 1.6 mg/dL and was resolved with hydration after 48 hours, was the only observed event. During the stay at the hospital, the metabolic status was stable and within normal ranges.
Based on our experience with postoperative management, we suggest regular monitoring of blood glucose levels and the provision of more frequent meals in order to minimize the risk of hypoglycemic episodes. In cases where feeding is not feasible, we suggest using appropriate parenteral nutrition. We also suggest regular monitoring of creatinine levels and adequate hydration. The use of fibrates or other cholesterol-lowering medications cannot be suggested since they did not appear to affect triglyceride values.
In conclusion, GSDI and life-threatening AAA is an extremely rare combination of diseases that impose a serious therapeutic challenge. The comorbidities and intraoperative and postoperative risks that come with GSDI are a prominent factor in therapeutic decision-making but should not be given more importance than any other factor. The anatomical characteristics of the aneurysm should dictate the surgical technique and the decision to perform an open or EVAR.
A multidisciplinary approach is required in order to establish a peri-, intra-, and postoperative plan with all the above-mentioned measures so that the risks of an adverse event can be minimized.
FUNDING
None. | 2023-06-20T06:17:11.201Z | 2023-06-19T00:00:00.000 | {
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269085280 | pes2o/s2orc | v3-fos-license | Retrospective case series of high-density silicone oil (Oxane HD) in severe proliferative vitreorretinal retinal detachment patients
Background Describe complications and clinical outcomes of heavy silicone oil (HSO) Oxane HD® use as an alternative to overcome the challenges of performing vitrectomy to treat tractional and rhegmatogenous retinal detachments with proliferative vitreoretinopathy (PVR). Methods A retrospective, observational study was performed on patients from one center from August 2014 to Aug 2023. It was included patients who underwent surgery using HSO Oxane HD® to treat rhegmatogenous retinal detachment with PVR or mixed tractional and rhegmatogenous diabetic retinal detachment. Severely ill patients who could not attend to follow up were excluded. The primary outcome was successful retinal attachment at first postoperative month. A descriptive analysis was performed. Results Among the 31 patients, 29 (93.5%) underwent surgeries due to rhegmatogenous retinal detachment and two (6.5%) for diabetic retinal detachment. The primary anatomic success was achieved in 27 (87.1%) patients. At the final visit, 17 (56.6%) had vision better than 20/400 (range, 20/30 to light perception). The vision was stable or improved in 22 (76.8%) patients at the end of follow-up. Nineteen (61.3%) patients required hypotensive eye drops after HSO use and twelve (38.7%) still required hypotensive eye drops at the final follow-up; three (9.7%) patients required additional glaucoma surgeries. Conclusions HSO is safe and useful for complex retinal detachments cases specially with inferior tears and PVR. Ocular hypertension is frequent and usually clinically controlled with hypotensive eyedrops. Close postoperatively follow-up is advised due to the ocular complications, particularly elevated intraocular pressure and emulsification.
Vitrectomy, the most common surgical technique performed to treat rhegmatogenous retinal detachment, can be combined with phacoemulsification and/or scleral buckling in selective cases to optimize surgical results.Surgical skill and the choice of proper vitreous substitutes are crucial for the final visual outcomes.The most common vitreous substitutes used in vitreoretinal surgeries are air, expandable gases, and silicone oil (SO).SO, which is hydrophobic and lighter than water, tends to float in the vitreous cavity and provides a good tamponade effect in the superior retina.However, it leaves an inferior residual meniscus that can accumulate fluid, proinflammatory proteins, and cellular elements such as retinal pigment epithelial tissue [1,2].This facilitates proliferative vitreoretinopathy (PVR) formation and recurrent retinal detachments which are the main reasons for surgical failure and blindness [3][4][5].
Heavy SO (HSO), which has been described since the late 1990s, is characterized by a density that exceeds 1, the ability to tamponade the inferior retina in horizontal gaze, and prevention of retinal reproliferation and redetachment [6].It is advocated for retinal detachments with large inferior breaks or inferior PVR in patients unable of proper head positioning, such as spinal osteoarthritis, or cognitive impairment [1].
The current study is a nine year retrospective case series of complicated retinal detachments using HSO and their clinical outcomes.
Methods
We retrospectively reviewed 31 patients from August 2014 to Aug 2023 (9 years) from one center in Assis, Sao Paulo, Brazil.The primary outcome was primary retinal attachment at first postoperative month.The inclusion criteria were rhegmatogenous retinal detachment with proliferative vitreoretinal disease or mixed tractional and rhegmatogenous diabetic retinal detachment treated with HSO (Oxane HD®, Bausch & Lomb, Toulouse, France).The patients had poor clinical prognoses and were informed of probable global atrophy due to anterior PVR and ciliary body traction resulting from advanced PVR.Severely ill patients who could not attend regular follow-up examinations were excluded.
One surgeon (MM) performed all four-port pars plana vitrectomies with Constellation® Vision System (Alcon, Fort Worth, Texas, USA) and a chandelier illumination.The surgery was combined with phacoemulsification and/or scleral buckling at the discretion of the surgeon.In all cases which perfluorocarbon liquids (PFCL) were used, they were aspirated during fluid-air exchange and then the Oxane HD® HSO was injected (example in Fig. 1).The HSO was removed at the surgeon's discretion based on the retinal status, intraocular pressure (IOP), and visual prognosis after several weeks in another surgical procedure.
The HSO removal was performed on bimanual technique and under direct visualization.A 18-gauge needle was connected to the extrusion line through one sclerotomy and another 18-gauge needle was connected to the vitreous cutter aspiration line.This technique is important for fast and safe HSO removal because extrusion line frequently partially or completely clogs during HSO aspiration, and the other needle keeps a continuous aspiration inside the SO bubble during the whole procedure.This prevents the release of a remaining HSO bubble back to the posterior pole [7].
Patients were evaluated on the postoperative day 1, week 1, months 1 and 3, and every trimester thereafter.Additional visits were scheduled as needed according to surgical complications.Best corrected visual acuity (BCVA), anterior biomicroscopy, IOP, and fundus photographs were performed at all appointments and further ancillary examinations in specific cases.
The preoperative and postoperative BCVA were analyzed after conversion to the logarithm of minimum angle of resolution (logMAR) BCVA.The results were detailed in a descriptive analysis.The local ethics committee approved the study, which followed the tenets of the Declaration of Helsinki.
No previous retinal surgery had been performed in fifteen (48.4%) patients; ten (32.2%) had undergone one retinal surgery, five (16.1%) had two previously retinal surgeries, and one patient (3.2%) had three previously retinal surgery before the HSO injection (Table 1).
Twenty-seven (87.1%) patients had primary anatomic success at the one-month postoperative clinical examination.Patients were scheduled for HSO removal depending on surgeon and patients' agreement regarding retina status, visual prognosis and patient's health and social availability: eleven patients had their HSO removed before 3 months, four patients between 4 and 6 months and nine patients more than 6 months after first surgery, seven patients did not remove HSO.The outcomes of these patients were: twelve kept their retina attached after HSO removal; eight had retinal redetachment after HSO removal and were retreated with standard SO or HSO; four patients had early retinal redetachment and were retreated with standard or HSO (primary anatomic failure), seven patients had not been submitted to further surgery (five patients refused HSO removal due to bad visual prognosis and were kept under close surveillance and two patients had short follow up period − 2 and 5 months (Fig. 2).
The mean baseline BCVA was 1.93 ± 1.17 logMAR and ranged from 20/30 to light perception.Ten (37%) patients had a BCVA better than 20/400 at baseline.At the final visit, the mean BCVA was 1.65 ± 1.06 logMAR.Fifteen After HSO surgery, 19 (61.3%) patients needed hypotensive drops for IOP control.The IOP rise was clinically well controlled and was not the reason for HSO removal in any patient.At the final follow-up, a total of 12 (38.7%)patients still required hypotensive eye drops and three (9.7%)patients had required additional glaucoma procedures: one patient needed selective laser trabeculoplasty, and two patients needed cyclophotocoagulation.These three glaucoma procedures were performed several months after HSO removal or HSO/SO exchange and all these patients had previously silicone oil emulsification.Two (6.4%) patients had been previously submitted to glaucoma drainage device implant surgery in the superior quadrant before the tractional retinal detachment.Other postoperative complications: one (3.2%)patient developed severe posterior capsule opacification; six (19,3%) patients developed clinically visible SO emulsification; two (6.4%) patients developed epiretinal membrane, two (6.4%) patients developed corneal decompensation, one (3.2%)patient had chronic hypotony and globe atrophy; two (6.4%) patients developed optic nerve atrophy (Table 2).
From our point of view, it is the proper vitreous tamponade for patients unable of face down positioning with high risk for PVR in inferior retinal detachments.Some examples are patients with cognitive impairment such as autism or dementia, obesity, spinal osteoarthritis, previously superior glaucoma drainage valve implant and inferior retinal detachment in patients with uveitis.It is important to mention that these complex cases require experienced surgeon able to identify and peel off all membranes and residual traction, especially anterior PVR, or associate scleral buckling or retinotomies/ retinectomies to relief longitudinal and tangential traction.
One of the concerns regarding HSO is about their removal.Because of its density, it progressively concentrates in the posterior pole during its removal and should be done under direct visualization and continuously aspirated inside the bubble during the whole procedure.In our experience, Oxane HD has higher viscosity and is more difficult to remove than Densiron.Intraoperative care is important to ensure there is no residual PFCL and avoid direct PFCL/HSO exchange to avoid sticky SO formation.In this series, there was not any retinal tear or early hypothony during HSO removal.
Clinical postoperative outcomes
The most common complication after HSO surgeries is development of cataract [9].For this reason, all phakic patients underwent combined phacoemulsification during the first procedure using HSO.We also believe this step facilitates access to vitreous base shaving which is essential for removing all the anterior vitreous.Another complication is the development of epiretinal membrane proliferation.Some authors believe it may be prevented if endotamponade is early removed [10].Moreover, some authors report intraretinal and subretinal fibrosis from 10 to 29.2% in complex retinal detachment cases treated with HSO.These conditions are sight-threatening and mentioned to occur in patients with previously PVR [11][12].In our cohort, two (6.9%) patients developed epiretinal membrane and intra/subretinal fibrosis were not detected.
Another described complication of HSO is inflammation and oil emulsification in the anterior chamber, especially in scenarios where the blood-retinal barrier is compromised or when SO is used over the long term [10,[13][14].Recent metanalysis did not identified increased risk for inflammation or oil emulsification with HSO comparing with standard SO [15].There are several theories about the mechanisms for development of intraocular inflammation: direct toxicity and immunogenicity (delayed type IV hypersensitivity), toxicity due to impurities or instability of the agent, oil emulsification, and mechanical injury due to gravity.[14,[16][17].In our series, there was two patients which had retinal detachment with bad PVR and developed early emulsification before 3 months.These cases were treated with HSO removal or exchange for standard SO.Sixteen patients had Oxane HD for more than six months because social/ health issues or unwilling to attend to further surgery due to bad visual recovery.Four of them developed later emulsification.
IOP elevation is another common postoperative complication.Previous studies have reported no difference in IOP elevation when comparing standard 5,000 cSt SO and HSO surgeries [18].In the current series, IOP rise was frequent while the patients had the HSO and were well controlled with hypotensive eyedrops.Two patients needed glaucoma procedures after receiving dexamethasone implants for macular edema several months after SO/HSO removal and one patient needed cyclophotocoagulation for uncontrolled neovascular glaucoma four years after Oxane removal and replacement for standard SO.At the last follow up, over than a third of the patients (38.7%) were kept with hypotensive eye drops.
Furthermore, aphakic patients require closer followups due to the risk of corneal decompensation.There were two aphakic patients in our study: one developed bullous keratopathy and the other developed globe atrophy.There was another patient that required corneal graft transplantation which had previously undergone The last but not the least, unexplained visual loss has been described associated in patients with SO/HSO more often than those with expandable gases.It is controversial if the incidence of unexplained visual loss after HSO removal has the same or different incidence with standard SO [19,20].Some investigators have theorized a sudden change in potassium ion concentrations, direct phototoxicity from the operating microscope, or the refractive effect of a shrinking SO bubble as possible pathogenesis.Particularly in HSO removal, the bubble shrinkage is concentrated near the posterior pole opposite to the position of the standard SO near the lens, which apparently may not be the cause.In our study, there was not any patient with unexplained visual loss, but it is important to highlight this pathology could be misdiagnosed because the cohort included patients with bad visual prognosis and recurrent retinal detachments.
Recent studies by Moussa et al. reported that the incidence rates of cataract, macular epiretinal membranes, IOP elevation, inflammatory reactions, and emulsification of HSO and standard SO were similar.On the other hand, some of these of these complications are more common in severe cases when there is need of retention of the SO.Moreover, it was noted less retinectomy rates in favor of HSO when compared to standard SO [14,20].
In several cases, the HSO remained in the eye for longer than 6 months without substantial complications.This is longer than reported in other studies (usually 3-6 months) because most patients had complex retinal detachments or persistent postoperative hypotony.Some of these patients had preserved ambulatory vision (≥ 20/800) and were afraid of consequent surgeries [2,10].In our series, there were 11 patients unwilling or incapable to be submitted to SO or HSO removal surgery and their tamponade for over a year, especially due to bad visual prognosis such as age macular degeneration, optic nerve atrophy, or bullous keratopathy.Only one of these eleven patients required further surgery: a corneal transplant after multiple glaucoma surgeries.
Regarding anatomic success and visual outcomes, HSO has been reported to be neither inferior nor superior compared with standard SO.It is important to highlight that HSO cannot prevent PVR formation, although it shifts the PVR formation to the superior retina.Future studies about drugs to inhibit PVR formation are needed as alternative/adjuvant therapies for these severe cases [3,5,21,22].In the current series, poor VA outcome at the final follow-up visit was associated with PVR formation and multiple surgeries, a finding that agreed with previously reported data [9].
This study has some limitations.It is retrospective and selected patients from 2014 onward from one single center.This series included some patients with bad visual prognosis that explains the re-detachments rate and the reason some patients were left with HSO for over than 6 months period.All patients at this study were treated with Oxane HD HSO and the results should not be addressed for other HSO such as Densiron.Furthermore, careful analysis of retinal layers on OCT should be evaluated in future prospective studies.
We concluded that HSO is safe and useful for complex retinal detachment patients with inferior tears and PVR.Follow up of these patients is advised postoperatively due to the ocular complications, particularly elevated intraocular pressure and emulsification.
Table 1
Characteristics of patients and pre and postoperative's eye status Fig.2Flow chart of the anatomical outcome after HSO use in complex retinal detachments cases (50%) patients had a BCVA better than 20/400 (range, 20/30 to light perception) at final visit.
Table 2
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55416953 | pes2o/s2orc | v3-fos-license | Can fish introductions alter nutrient cycles in previously fishless high-latitude lakes?
The additional input and enhanced cycling of nutrients derived from introduced fish can be a significant factor altering nutrient dynamics in oligotrophic lakes. To test this, we used a bioenergetic model to estimate the fish-derived nutrient load in Lake Kuutsjärvi, a historically fishless boreal lake of northern Fennoscandia. The lake was selected because of the absence of other anthropogenic stressors, a known stocking history and the possibility of quantitatively estimating the size-structure and biomass of the fish population through a mass removal. Subsequently, we used a mass balance model to compare fish-derived nutrients with other nutrient load pathways. For comparison over longer timescales, we used lake sediment records of diatoms, chlorophyll and carotenoid pigments, C:N ratios and stable isotopes to infer whether fish introduction produced detectable changes in the lake trophic state, primary productivity and terrestrial nutrient input. Based on the nutrient mass balance model, we found that phosphorus and nitrogen derived from fish were 0.46% and 2.2%, respectively, of the total load to the lake, suggesting that fish introduction could not markedly increase the nutrient load. Accordingly, the palaeolimnological record indicated little increase in primary production but instead a shift from pelagic to benthic production after fish introduction.
INTRODUCTION
Nutrient cycling between water, sediment and biota is the movement and exchange of organic and inorganic compounds between the environment and the living matter. Over long term (centennial-millenial) timescales, nutrient cycles in high-latitude lakes are influenced by temperature, atmospheric deposition and hydrography of the catchment area (Kernan et al., 2010). On a shorter term, other anthropogenic factors could be more relevant, ranging from forestry management (Ahtiainen and Huttunen, 1999) to fish introductions (Moss et al., 2003).
Unlike other clades, where introductions are often unintentional, fish introductions are commonly linked to deliberate sport fisheries manipulations (García-Berthou et al., 2005). Introduced fish can directly modify habitats (Dibble and Kovalenko, 2009;Weber and Brown, 2009), and alter the food webs of the host ecosystem through trophic cascades (Carpenter et al., 1985;Pace et al., 1999;Strock et al., 2013). Trophic cascades can modify the abundance and trophic relation of several elements of the food web, which can influence the way these elements exploit nutrients and ultimately affect the trophic status of water bodies. Experimental and survey studies have shown that fish introductions may change lake nutrient regeneration markedly (Vanni, 1996;Schindler et al., 2001) or more subtly (Schindler and Eby, 1997;Pace et al., 2004;Cole et al., 2006;Carpenter et al., 2011) but each highlighted the important role of fish in transporting nutrients between benthic and pelagic zone or between terrestrial and aquatic environments. Fish predation can move and transform nutrients derived from benthic or terrestrial prey to a bioavailable excretion in the pelagic areas of lakes, resulting in a net regeneration of nutrients. However, isolating the effects of fish introductions on the trophic state of lakes from other concurrent pressures remains challenging (Gąsiorowski and Sienkiewicz, 2013).
Fish are generally highly linked with several levels of the food web (Duffy et al., 2007) due to their complex trophic interactions (Dunne et al., 2002). This complexity of trophic linkages increases ecosystem resilience, but also the difficulties to track specific changes in lakes where fish are already present (Folke et al., 2004). Therefore, fish introductions should have the greatest impact in previously fishless lakes (Schindler et al., 2001).
Most small lakes in Fennoscandia were fishless for a long period after the last deglaciation, but fish were intentionally introduced to many of them in recent centuries (Tammi et al., 2003). While some studies exist on the food web changes induced by introduced fish in Fennoscandia (Bøhn and Amundsen, 2001;Hayden et al., 2013), none have focused on the consequences on nutrient cycling. Palaeolimnological analyses (i.e. the study of subfossil and physicochemical characteristics of lake sediment cores) can be used to reconstruct past trophic and production changes through subfossil proxies of primary producers (Battarbee, 2000) as well as changes in coupling with the terrestrial ecosystems through stable isotope analysis (McLauchlan et al., 2013). Therefore, palaeolimnology can be used to infer changes in nutrient cycling through changes in primary production, if the dates of fish introduction are known or can be estimated. However, few studies have yet used a palaeolimnological approach to examine this question (Leavitt et al., 1994).
Can fish introductions alter nutrient cycles in previously fishless high-latitude
We hypothesized that fish introductions have been significant in altering the nutrient balance of small high-latitude lakes. To test this hypothesis, we selected a small lake in northern Fennoscandia, with a known fish stocking history, isolated from other local anthropogenic stressors, and sufficiently shallow to enable the removal of the whole fish population. We used complete fish removal as a unique opportunity to quantitatively estimate the fish biomass, an essential parameter in nutrient dynamics models. We employed a bioenergetic model to evaluate the magnitude of nutrient cycling effects derived from introduced brown trout (Salmo trutta L.). Fish-derived nutrient loads were then compared with other nutrient sources in a comprehensive lake nutrient budget. The model was also used to test whether fish act as a net source or sink of nutrients. Taking advantage of the welldocumented disturbance history of the lake, we sought to validate our findings from the bioenergetic model and investigate how lake production responded to fish introduction and removal, over longer timescales. Therefore, we analysed diatom, chlorophyll and carotenoid pigments, and stable isotopes of carbon (δ 13 C) and nitrogen (δ 15 N) in sediment cores as proxies to test for changes in lake primary production and the input of terrestrial matter. In particular, we expected that diatom and algal communities would respond to changes in nutrient availability due to trout excretion, increasing the abundance of nutrient-sensitive species (e.g. Asterionella formosa). Furthermore, we expected that stable isotopes in the sediment would record more terrestrial matter (i.e., a depletion in δ 13 C ratios) if trout predation on terrestrial prey had created a new alternative pathway of nutrient transport.
Study site
Lake Kuutsjärvi (67°44'49.13"N, 29°36'35.47"E) is a small (circa 0.7 ha) north-boreal headwater lake, located at an altitude of 341 m above mean sea level, in northeastern Finnish Lapland (Fig. 1). Water temperatures, measured at 2 m of depth during 2009-2012, vary from a minimum of 1.5°C in January to a maximum of 11.8°C in July. The ice-free period typically lasts from late May to early October. The lake is mesotrophic (TP 13-26 µg L -1 , TN 67-152 µg L -1 ) and shallow with clear water (max depth 8.5 m, Secchi depth to the bottom). The catchment area is small (<1.5 km 2 ), with steep slopes surrounding the lake covered by north-boreal coniferous forest dominated by Scots pine (Pinus sylvestris), but the upper part of the catchment area is treeless tundra. The lake was originally fishless due to migration barriers (waterfalls, rocks and steep rapids) in its small outlet brook. However, in 1980 it was stocked with adult brown trout (hereafter trout) from nearby populations (E. Pulliainen, personal communication) to create local recreational fisheries. The trout quickly established a self-sustaining population that reproduces in the lake. A small outlet, blocked with a metal grill, allowed the water to flow out of the lake but enclosed the introduced fish. In 1981, the Värriö Natural Reserve was officially established, comprising an area of 125 km 2 roughly centred around Lake Kuutsjärvi and the nearby research station. Värriö Natural Reserve is a strict natural reserve and no human activities, other than research, are allowed in the area.
Fish sampling and bioenergetic model
Trout were removed from Lake Kuutsjärvi between 2010 and 2012. Fish were mainly captured using multimesh gillnets (mesh sizes of 10-60 mm, knot to knot), rodand-line and a baited longline, during sampling events at the beginning of June, July and August. Over 90% of the total individuals were caught already in August 2011 and fishing continued in 2012 when the last few individuals were captured. Given the high fishing effort concentrated in a small lake, fish removal was discontinued when catch per unit of effort declined to 0 for more than one week and the population was considered removed. Specimens were immediately sacrificed by head concussion and frozen at the Värriö Research Station, located in the immediate vicinity of the lake (Fig. 1). Subsequently, samples were thawed and the total length of fish was measured with a precision of 1 mm and weighed with a precision of 0.1 g. Stomach contents analysis (SCA) was performed using a volumetric point method (Windell, 1971), in which stomach fullness was visually estimated using a scale from 0 (empty) to 10 (full) and the relative proportion of each prey item to total stomach fullness was estimated. Each prey item was identified as accurately as possible to genus, family or sub-order level. Subsequently, the total volumes of prey items were pooled into five larger categories, which were terrestrial arthropods, aquatic arthropods, rodents, amphibians and indigestible content (detritus), to be used in the bioenergetic model.
The Monastyrsky formula was used to back-calculate size-at-age from scale rings and to reconstruct growth increments for years when direct measures at age were not available (Bagenal and Tesch, 1971). Weights were estimated based on a length/weight exponential equation (1): where W is weight (g), L is total length (cm) and parameters a and b were fitted on the distribution of lengths and weights of the whole population. To confirm age readings obtained from scales, sagittal otoliths of the sampled trout were also collected, stained with alizarin red S and examined under a stereomicroscope. If age readings differed, otolith readings were considered to be more reliable (Thaulow et al., 2017). Visual inspection of gonads was carried out to assess maturity of individuals and sex ratio of the population. A bioenergetic model was used to estimate nutrient regeneration rates of trout. The model estimated consumption rates and excretion rates for each cohort in the population (accounting for mortality), based on speciesspecific metabolic levels and measured water temperatures. To compute nutrient consumption and excretion rates, the model used diet proportions derived from SCA, energy/nutrient content for each prey item and average measured growth rates.
Consumption rates were calculated using a temperature-dependent bioenergetic model for cool-water species (2) (Thornton and Lessem, 1978;Hanson et al., 1997): Where C is consumption, T is temperature, CA is the intercept of the mass dependence function for a 1 g fish at the optimum water temperature and CB is the coefficient of the mass dependence. CQ is the lower water temperature at which the temperature dependence is a small fraction (CK1) or the maximum rate. CT0 is the water temperature corresponding to 0.98 of the maximum consumption rate. CTM is the temperature (≥CT0) at which dependence is still 0.98 of the maximum and CTL is the temperature is some reduced fraction (CK4) of the maximum rate.
Excretion rates were calculated as a function of consumption, with the expression (3) (Kitchell et al., 1977): where UA is a constant proportion of assimilated energy, times consumption (C) minus egestion (F, a constant proportion of consumption). The bioenergetic model took into account all the different fish cohorts and their respective biomasses, as the different age-classes have different metabolic levels and thus different regeneration rates. Population parameters were reconstructed for 2009, before sampling began, and were used to model the population in undisturbed conditions. Species-specific parameters were derived from values reported in Dieterman et al. (2004) for stream dwelling populations of trout. Temperatures were continuously recorded at different depths with HOBO water temperature data loggers throughout the study period (2009)(2010)(2011)(2012). In the bioenergetic model, daily tempera- tures, recorded at 12:00 at a depth of 2 m throughout year 2009, were used to estimate the feeding activity of trout. This is the depth of the littoral zone, a preferred depth for trout activity (since trout are often littoral and surface-oriented predators) and at this depth temperatures are less susceptible to small-scale daily variations.
As different prey has variable energy and nutrient contents, they are processed differently by the fish metabolism. Therefore, stomach content data were used to define prey proportions in the model (Tab. 1) in order to derive specific assimilation and excretion rates. Energy contents of specific prey items and trout were derived from direct calorimetric measures by Cummins and Wuycheck (1971). Detritus (i.e., plant matter) was considered to be non-energetic. Values of N content in trout muscle and their prey were directly measured in samples from the lake food web with an elemental analyser (TruSpec Micro, LECO Corporation, St. Joseph, Michigan, U.S.A.). Phosphorus (P) concentration in prey and trout was derived from Penczak et al. (1985), Nakashima and Leggett (1980), Elser et al. (2000) and Dierenfeld et al. (2002). An assimilation coefficient of 0.72 for the uptake of both nutrients (N and P) was used (Nakashima and Leggett, 1980). Consumption of all terrestrial prey items was set to 0 during ice-cover, as they would be inaccessible. The remaining proportion of the diet was assumed to shift towards a purely aquatic diet (Tab. 1). The model also accounted for annual population dynamics, with an average 10% weight loss at spawning, due to the release of gametes (Jonsson and Jonsson, 1999) and 10% natural mortality for all mature cohorts (Jonsson and Jonsson, 2011).
Nutrient load in trout excretion was estimated through a function of the bioenergetic model that takes into account specific nutrient content of both consumed prey and trout (Kraft, 1992). N and P not assimilated by trout (egested in faeces) would not be immediately available to phytoplankton. In contrast, assimilated nutrients subsequently evacuated (excreted in urine) were assumed to be directly available to primary producers and to contribute to internal load (Brabrand et al., 1990;Lall, 1991).
To assess the extent of trout being either a sink or a generator of nutrients we compared the amount of nutrients segregated into fish bodies (sink) versus the amount of nutrients regenerated via excretion (generation). The wet mass composition of nutrients in fish bodies was calculated based on the population biomass of 2009, as used in the bioenergetic model, and accounting for mortality rates. Both the annual biomass gain and the total biomass were compared with nutrient regeneration rates.
Nutrient mass balance model
Internal and external nutrient loadings were calculated on an annual basis and year 2009, before the sampling began, was taken as a model year for calculations. We assumed that the main factor affecting the internal load of the lake would be fish-derived nutrients. This was supported by the negligible re-suspension of the sediment due to short fetch, the presence of a thick bryophyte mat covering the lake bottom and high depth-to-surface ratio of the lake. The good oxygenation throughout the water column at most times of the year (Milardi, unpublished data) suggests a minimal nutrient re-dissolution. In addition, the well-established sediment chronology suggests a stable and undisturbed sedimentation environment.
We assumed that the external load would derive from the catchment area and the atmospheric load, as there are no other significant external sources of nutrients in the area. Catchment and lake areas were calculated using a polygonal approximation in Arc-GIS. Catchment nutrient load of P and N was then estimated on a surface-unit basis, taking into account that most of the catchment area consists of unmanaged forest. Values of 110 kg km -2 year -1 for N and 4.2 kg km -2 year -1 for P were used (Kortelainen et al., 2006). P and N fallout was estimated from atmospheric deposition measures taken by the Finnish Environmental Centre. Measures were taken on rainwater/snow collected monthly between 2004 and 2012 in Sodankylä (about 130 km west from Lake Kuutsjärvi). Annual atmospheric average loads Tab. 1. Prey items and their energy and nutrient content in the diet of trout. Nutrient content is expressed as a proportion of wet weight. Brown trout itself has an energy content of 6247 j g -1 , N and P content of 2.6% and 0.5% of wet weight, respectively (f, a). The composition of prey items is expressed as the sum of ingested prey volume across the whole fish population.
Prey item
Energy content (j g -1 ) N content (%) P content (%) Trout diet (%) were estimated to be 3.67±1.44 kg km -2 and 216.89±38.97 kg km -2 of P and N, respectively. P and N atmospheric deposition was assumed to originate from precipitation and dry fallout directly on the lake surface. Internal and external nutrient loadings were then compared to assess the difference in loading with and without fish regeneration.
Palaeolimnological analyses and nutrient model validation
A HTH-Kajak type gravity corer (Renberg and Hansson, 2008) was used to derive a 19.5 cm long sediment sequence from the deepest part of Lake Kuutsjärvi in spring 2009. The core was sub-sampled for loss-on-ignition (LOI), radiometric dating, diatoms, and chlorophyll and carotenoid pigment analysis at intervals of 2.5 mm, representing a temporal resolution of ca. 1-10 years. A second core was obtained in spring 2011 and sub-sampled for LOI and stable isotopes of C and N at intervals of 5 mm. Both cores were analysed for LOI and sedimentation rate in order to correlate the chronology obtained through radiometric dating. LOI was performed measuring the weight of sediment samples after heating in ceramic cups at 105°C overnight and successively igniting at 550°C for 4 hs (Heiri et al., 2001).
Sediment samples were analysed for 210 Pb, 226 Ra and 137 Cs by gamma spectrometry in the Environmental Radioactivity Laboratory of Liverpool University. The radiometric dating chronologies were calculated using the Constant Rate of Supply (CRS) dating model (Appleby and Oldfield, 1978). Based on the known low sediment accumulation rates of northern oligotrophic lakes (Korhola and Weckström, 2004) the basal sample for diatom analysis (depth of 19.5 cm) was estimated to cover at least the last five hundred years.
Diatoms were prepared using H 2 O 2 digestion and HCltreatment and cleaned diatoms were mounted in Naphrax (Battarbee, 1986). A minimum of 300 diatom valves from each sample were identified and counted along random transects at 1000x magnification. Diatom identification was based mainly on Krammer and Lange-Bertalot (1986, 1988, 1991a, 1991b. For more details concerning the procedure and taxonomic literature used, see Weckström et al. (1997). Counts of 300 specimens per sample were converted into percentages, and these were plotted as a stratigraphical frequency diagram using the program C2 version 1.72 (Juggins, 2007). In order to identify the periods of time of the most significant shifts in diatom assemblages, the constrained optimal sum of squares partitioning was used to zone the diatom core data (Birks and Gordon, 1985). The number of statistically significant zones was calculated using the broken-stick model and the associated approach described by Bennett (1996). Optimal partitioning was implemented using the program ZONE 1.2 (Lotter and Juggins, 1991).
To quantify past abundance of algal groups, pigments were quantitatively extracted from freeze-dried sediments in acetone: methanol: water (80:15:5), filtered (0.2 μm PTFE), dried under nitrogen gas, re-dissolved into acetone, ion-pairing reagent and methanol (70:25:5) and injected into an Agilent 1200 series high performance liquid chromatography (HPLC) system (Leavitt and Hodgson, 2001). Separation conditions were a modification of Chen et al. (2001) using solvent A (80:20 methanol: 0.5 M ammonium acetate), solvent B (9:1 acetonitrile: water) and solvent C (ethyl acetate) with a Thermo Scientific ODS Hypersil column (205 x 4.6 mm; 5 µm particle size) for the stationary phase (Thermo Scientific, Bremen, Germany). Pigments were identified based on spectra and retention times, and quantified by calibration with commercial standards (DHI, Denmark). Pigment concentrations are reported as molar weights per unit weight of organic material (estimated by LOI). Sub-samples for isotopic analysis were ground to a fine powder using a mechanical mortar. To compute the C:N ratio, the masses of C and N were determined using an elemental analyser (TruSpec Micro, LECO Corporation, St. Joseph, Michigan, U.S.A.). Samples for C isotopes analysis were weighed and loaded into silver cups, then fumigated for 24 hours under 37% HCl vapours to dissolve inorganic C. A minimum of 0.3 mg of sediment was measured for each C sample. Samples for N isotope analysis were weighed and loaded into tin cups, then directly analysed. A minimum of 0.4 mg of sediment was measured for each N sample. Sub-samples were analysed using a Finnigan DeltaPlusAdvantage mass spectrometer (Thermo Scientific, Bremen, Germany) connected on-line to an elemental analyser NC 2500 (CE Instruments, Milan, Italy) via a ConFlow III interface (Thermo Scientific, Bremen, Germany). The resulting isotopic ratios were expressed in terms of relative concentrations referred to a chitin standard, equivalent to Pee-Dee belemnite.
The diatom, pigments, sediment stable isotope and C:N ratios data were analysed using a non-parametric multivariate analysis of variance (NPMANOVA, Anderson 2001) to test the null hypothesis that the periods before and after fish introduction would have differences in values, species composition and/or abundance of the different variables examined. The analysis was performed using the vegan package (Oksanen et al., 2016) under statistical software R version 3.2.0 (R Core Team, 2015). which, before fish removal in 2009, were 0-12 years old. The regression parameters for the length/weight equation were estimated as a=0.0045 and b=3.2443 (R 2 =0.82). Ageing using combined scales and otoliths indicated that three year-classes (2000,2001,2002) constituted 79.25% of the population (whole range: 1997-2009) and no fish hatched in 2006 or 2007. Visual inspection of gonads indicated that trout were mature after 4 years of age, at a size of 29.4 cm and 272 g of weight, and that the sex ratio was equal. The SCA results underlined that aquatic arthropods were the most important food item for trout (56% of the total prey volume, Tab. 1).
Nutrient mass balance model
Based on the bioenergetic model for year 2009, annual nutrient load of fish derived nutrients was low, 0.72 kg and 0.13 kg for N and P respectively, relative to the external loads (Tab. 2). Annual external loads of N and P from the catchment area were estimated to be the biggest component, 151.8 kg and 5.8 kg respectively, whereas atmospheric loads of N and P (1.52 kg and 0.03 kg respectively) were more than one order of magnitude lower than that from the catchment area (Tab. 2). Therefore, internal P load due to fish presence (2.2% of the total P load) represented a more important load component for P than the external load due to atmospheric deposition. Runoff from the catchment area dominated the N input (Tab. 2) and internally regenerated N from fish was a negligible component (0.46% of the total N load).
The initial trout biomass used to model the population was 47.81 kg in 2009. According to the model, after one year the total biomass of the population should have been 47.55 kg, due to the effects of mortality. Without mortality, the biomass should have been 52.83 kg, which was practically equivalent to the total biomass of 52.55 kg removed between 2010 and 2012.
Even if 2.6% of the fish wet mass is composed of N and 0.5% is composed of P (Tab. 1), the total amount of N and P sequestered in fish bodies during 2009 was 0, due to the loss of biomass at the population level. Without accounting for mortality, only 0.13 kg of N and 0.02 kg of P would have been sequestered. Therefore, fish biomass could, in ideal conditions with no mortality, sequester only 18% and 15.2% of the N and P it regenerates. Overall, the entire biomass of trout removed contained only 1.37 kg of N and 0.26 kg of P; this is the total amount of nutrients sequestered by the whole population over a timespan of several years.
Palaeolimnological analyses and nutrient model validation
Seventy diatom taxa were enumerated from the subsamples. The diatom stratigraphy (Fig. 2) was divided by the constrained optimal sum of squares partitioning to zone into two statistically significant diatom assemblage zones (DAZ II and DAZ III) and one additional non-statistically significant diatom zone (DAZ I). DAZ I represents the 'reference' period dating back at least five hundred years (Korhola and Weckström 2004) and was dominated by the planktonic Aulacoseira subarctica and Asterionella formosa (~20%). DAZ II covers the period prior to AD 1980, when trout were stocked in the lake. In this period, Aulacoseira subarctica still dominated the diatom community, whereas the relative abundance of Asterionella formosa was significantly lower. At the end of this zone Aulacoseira subarctica started to slowly decrease, whereas Fragilaria pinnata (Staurosirella pinnata), a benthic diatom, increased. DAZ III represents the time after fish stocking (1980-present). This zone is characterized by a gradual decrease in the proportion of Aulacoseira subarctica from ~60 to ~30% and a gradual increase of Staurosirella pinnata from ~0 to ~25%. The NPMANOVA also indicated a statistically significant change in diatom abundances after fish introduction (P=0.001, pseudoF=7.0542, df=16).
The pigment assemblages indicated that siliceous algae were abundant in the lake and that cyanobacteria, chlorophytes and cryptophytes were also present (Fig. 3). The presence of the carotenoid okenone from anoxic photosynthetic bacteria (Fig. 3f) indicates both light penetration, consistent with the clear water conditions recorded (light penetration to the lake bottom at 8.5 m), and oxygen depletion in the lake hypolimnion (McGowan et al., 2008). The NPMANOVA indicated that introduction of trout in 1980 had a significant impact on the composition or abundance of primary producers in the lake (NPMANOVA P=0.0243, pseudoF=4.3574, df=15), driven mostly by the increase in the cryptophyte pigment alloxanthin (Fig. 3d), after fish stocking.
The C:N ratio (mean 12.2) was typical of an aquatic system with some terrestrial linkages and showed moderate changes. Some shifts in stable isotope ratios occurred after fish introduction, including a shift towards lower δ 13 C and δ 15 N values (Fig. 4 b,c) compatible with an increase in benthic primary production but not with an increase in terrestrial linkage. However, shifts in C:N ratio and δ 13 C and δ 15 N values were not significant (NPMANOVA P=0.395, pseu-doF=0.9594, df=15).
Tab. 2.
Internal and external loads of N and P in Lake Kuutsjärvi on annual basis. Atmospheric deposition is expressed as mean and standard deviation.
DISCUSSION
The results of the nutrient load model, which did not predict a significant increase in bioavailable nutrients due to fish presence, were in accordance with palaeolimnological analysis, which indicated only few apparent changes in lake primary productivity corresponding with fish introduction.
Is fish presence a significant element altering nutrient cycles in small boreal lakes?
Our bioenergetic model suggests that nutrients derived from fish excretion were not an important component of the total load, even if fish-derived P load was higher than the atmospheric-derived P load. Catchment area load dominated the nutrient input even if nutrient loading from catchment runoff is probably overestimated in the model, because part of the catchment basin is located above the treeline and no catchment area load estimates were available for such landscapes (Kortelainen et al., 2006). As a result of this overestimation, fish excretion could be a more important nutrient source than estimated in our study. However, fish-derived nutrient load seems to be unlikely to have a relevant effect on nutrient dynamics of lake ecosystems, contrarily to previous evaluations (Hansson et al., 1987).
Terrestrial prey, in particular, contributed significantly to the trout diet in Lake Kuutsjärvi (31.5% of the diet during the open water period, Tab. 1). This is an important factor, as it creates an additional flow of terrestrial nutrients into the system (Mehner et al., 2005;Cole et al., 2006;Milardi et al., 2016a). Trout simultaneously recycle aquatic nutrients and introduce nutrients derived from the terrestrial system, by preying on animals which would not otherwise enter the lake food web (e.g., swimming rodents, terrestrial flying insects). Thus, predation on terrestrial sources and subsequent excretion of terrestrial nutrients by introduced trout apparently increases terrestrial nutrient transport from the catchment to lakes. When fish die and decompose, these terrestrial nutrients are released in the aquatic system. This mechanism should further magnify the nutrient load to the lakes derived from fish presence, although the uptake of these sequestered nutrients is not always linear (Vanni et al., 2013). Similarly, fish egestion does not immediately affect nutrient dynamics of the lakes, as the portion of fish diet that is egested is, for the largest part, not readily available to primary producers without prior microbial breakdown (Brabrand et al., 1990;Vanni, 1996). However, eventually, egestion might stimulate the growth of bacterial decomposing organisms, and consequently enhance the growth of mixotrophic phytoplankton such as cryptophytes (Boros et al., 2014). As there are no estimations on the timing and extent of such processes in Lake Kuutsjärvi, we focused on the most direct and immediate effects of fish predation derived from excretion, i.e. the cause of a fertilising effect on autotrophic production. However, egestion is a factor that likely plays a role in the overall nutrient load, as large numbers of aquatic and terrestrial organisms are killed by trout and then processed by decomposers.
The size-structure of the population is important when modelling the magnitude of nutrient cycling. Small-sized individuals feed on smaller prey at higher rates than largesized specimens (Tarvainen et al., 2005) but cycle nutrients at lower rates because of their higher allocations to somatic growth. However, the removal of trout showed that the population of Lake Kuutsjärvi in 2009 was rather peculiar and mainly composed of relatively large and slow-growing old individuals, with only few small and younger specimens. Larger trout individuals feed less on zooplankton and the results from SCA suggest that direct top-down control of fish on zooplankton, if any, was very limited. This could be of importance in our study lake because lower trophic levels typically have a higher biomass and therefore higher nutrient regeneration rates. While previous studies have noted that zooplankton might be more significant regenerators of P than fish (Sereda et al., 2008), our study could not account for such regeneration rates.
The amount of nutrients regenerated by the trout population in a single year, even if low, still exceeds the nutrients sequestered in fish bodies. Therefore, fish act as generators, rather than sinks, of nutrients. According to the model, there should be a positive net gain in bioavailable nutrients in the system due to fish presence, even if a very small one.
Can the impact of fish introduction on the aquatic ecosystem be tracked in the sediment record?
Fish introductions often lead to changes in food web structure and, due to their feeding ecology or habitat alteration, ultimately to trophic cascade effects (Carpenter et al., 1985). While young trout might partially feed on zooplankton, older trout feed mainly on the aquatic stages of terrestrial insects and on aquatic macro-invertebrates decreasing their abundance (Epanchin et al., 2010) which could relieve invertebrate predation pressure from small plankton grazers (Wilhelm and Schindler, 1999). This can result in more abundant grazer populations and increased grazing pressure on algal production (Milardi et al., 2016b).
High concentrations of diatom frustules and siliceous algal pigments suggest that diatoms are an important component of overall lake primary production. Based on diatom analysis, no inferred increase in pelagic nutrient concentration has occurred in Lake Kuutsjärvi during the last centuries, as the proportion of A. formosa was higher in the reference sample (from ca. 500 years ago) than today in the most recent sediments. This species is known to favour elevated nutrient concentrations, especially nitrogen (Lund, 1950;Krivtsov et al., 2000;Saros et al., 2005). The most evident change in the diatom stratigraphy occurs after the introduction of fish in 1980 (Fig. 2), with a decrease of the planktonic A. subarctica and the increase of the benthic S. pinnata. The increase of S. pinnata indicates a shift from planktonic to benthic primary production and also suggests a change from a relatively stable environment to an unstable, more turbulent/chaotic environment. Staurosirella (Fragilaria) species are often associated with high environmental instability and are known to tolerate broad environmental gradients (Haworth, 1976;Smol, 1988;Denys, 1990;Korhola, 1995). The timing of the gradual increase of S. pinnata during the last 34 years, following fish introduction, suggests that the main reason for the change in the diatom community was the introduction of fish. Diatom influx rates of Lake Kuutsjärvi are almost tenfold compared to e.g. Lake Nautajärvi, southern Finland (Weckström, unpublished data). Lake Kuutsjärvi has a very high sedimentary silica concentration compared to other boreal lakes (Tallberg et al., 2014), which might support a longer diatom bloom time and negate the effects of silica limitation. This is also supported by the occurrence of diatoms such as A. formosa and A. subarctica which bloom in the spring and favour waters with elevated silica concentration (Krivtsov et al., 2000;Horn et al., 2011).
Even if there was a significant change in pigments after fish introduction, pigment analysis did not reveal a clear change in lake trophic state, as Chlorophyll-a and other pigment indicators (cyanobacteria, chlorophytes and purple sulphur bacteria) did not show significant trends that can be related to fish presence. The change was mainly related to diatom pigments, which showed an increase, but this may have been due to an increase in the abundance of benthic species, such as S. pinnata, and therefore a further confirmation of the switch to benthic production, which enhances pigment preservation in the sediment (Cuddington and Leavitt, 1999). The increase in alloxanthin could indicate an increase in bacterial production after fish introduction, which stimulated the growth of potentially mixotrophic cryptophytes ('the microbial loop'). Other pigments (fucoxanthin, Chl a, pheophytin a) also increased over a decade after the stocking of trout but this may be caused by degradational changes in the upper sediments because fucoxanthin and Chl a are quite labile (McGowan, 2013); however the subsequent decline in Chl a in the uppermost sample suggests that the patterns are not exclusively degradation-driven. It is also possible that fish stocking increased heterotrophic nutrient processing pathways by increasing the availability of organic matter through faeces and urine. Cryptophytes are capable of mixotrophy and so may have been able to di-rectly utilise these energy sources. Organic sources from fish wastes can serve as an energy source for heterotrophic bacteria which are grazed by smaller zooplankton and phagotrophic phytoplankton (Jansson et al., 2000). The presence of okenone indicating anoxia implies active decomposition of organic material in the deeper waters of the lake. However, okenone concentrations were low and did not rise significantly after fish introduction and there was a lag in the increase of alloxanthin, suggesting a rather subtle or indirect effect on lake microbial processing. It is possible that there was a cascading effect on algal communities through fish-induced changes in the food web structure (Strock et al., 2013). Consumption of aquatic arthropods in benthic and pelagic areas by fish might either enhance or depress algal production depending on whether prey items are primary (e.g., crustacean zooplankton, chironomidae) or secondary consumers (e.g., Odonata). However, this predation will fluctuate in time with variations in population structure, as different sized individuals focus on different prey. It is accepted, however, that trophic cascades exert more influence on lower trophic levels in oligotrophic lakes (McQueen et al., 1989), and so the rather high nutrient concentrations in the study lake may counter such effects. Chlorophylla concentrations suggest that algae have slightly increased overall; this is still likely affected by the increase in benthic primary producers.
Trout introduction effects were not significant in the C:N ratio series, which remained stable over a long period of time and did not show the shift towards higher C:N ratios, as predicted by the results of the nutrient mass balance model. The C:N ratio of Lake Kuutsjärvi sediments was typical of headwater lakes that have some linkage with the terrestrial system (Kortelainen et al., 2013). In Lake Kuutsjärvi, decreasing δ 15 N and δ 13 C ratios, although not significant, could further indicate a production shift from pelagic to benthic habitats (as shown by diatom analysis) but could also be the result of an increase in primary production of diatoms (Hundey et al., 2014). Taking into account the pigment trends and the accumulation rates of diatoms (Weckström, unpublished data), which do not show any significant trend, it is unlikely that a production increase has actually occurred. Previous studies noted that δ 15 N ratios should increase 3-4‰ in lakes where fish populations have cascading effects on phytoplankton production, as increased phytoplankton biomass usually produces higher values of δ 15 N due to a preferential utilization of 15 N (Gąsiorowski and Sienkiewicz, 2013). Furthermore, increased phytoplankton biomass often decreases δ 13 C ratios in sediments, due to the preferential utilization of 12 C (France, 1995). On the contrary, increases in phytobentos biomass have been associated with increases in δ 13 C as benthic algae have higher δ 13 C ratios than planktonic ones (France, 1995; N o n -c o m m e r c i a l u s e o n l y Wang et al., 2013). Together, the stable C:N ratios and the slight decrease in δ 15 N and δ 13 C ratios could suggest that benthic production after fish introduction did not increase significantly and that trouts could have slightly increased the linkage with the terrestrial system, which is 13 C depleted.
In conclusion, most palaeolimnological proxies used in this study coherently suggested that there was a shift from planktic to benthic primary production. However, the palaeolimnological proxies confirmed the prediction of the nutrient mass balance model that bioavailable nutrients in the lake did not significantly increase after fish introduction. The water retention time (under 2 months) could be short enough to rapidly cycle lake water flushing some of the nutrients regenerated by trout out of the system before they are uptaken by planktic and benthic micro-organisms (Kozerski et al., 1999, Köhler et al., 2005. Macro-organisms, on the other hand, would be reduced by trout predation, therefore potentially reducing the animal-derived overall nutrient regeneration rates (Vanni, 2002). Moreover, as the lake receives groundwater input through bottom springs, there might have been switches between closed and open basin hydrology. Also climate changes could influence the effects of nutrient loading, if these shorten the open water period, and affect the water mixing dynamics (Douglas and Smol, 2000). However, average summer air temperatures, precipitation and the ice cover period have all remained fairly stable in the past 25 years (Milardi et al., 2016b), suggesting that these might not explain the changes seen in the lake. Finally, the model estimates are valid for a limited 'snapshot' of time, as the complete history of the trout population dynamics is unknown. Although wide fluctuations in the trout population structure are unlikely over a short time span (ca. 5 years, Platts and Nelson, 1988), they could have been significant over the 34 years since introduction, thus influencing the overall effect.
CONCLUSIONS
Our study suggests that Lake Kuutsjärvi has not significantly increased its production but might have increased slightly its dependency upon terrestrial C. However, there is strong evidence for a shift from planktonic to benthic algal production after fish introduction, suggesting that introduced fish enhanced nutrient transport to the bottom of the lake, most likely through the production and sinking of wastes. Subtle increases in mixotrophic algae could suggest changes in microbial/ bacterial processing. These, coupled with C:N ratios and δ 13 C trends, could be an indication of a subtle increase in the terrestrial energy pathway to the lake.
Further studies would be needed to explore the relation between fish introduction and nutrient balance on a wider range of high-latitude lakes, using different models and different species to test their validity. However, future studies will face a major challenge in selecting study systems where a sufficient level of historical information (e.g., date of fish introduction) is available. | 2018-12-12T15:16:54.229Z | 2016-04-15T00:00:00.000 | {
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257540670 | pes2o/s2orc | v3-fos-license | Application of Amyloid-Based Hybrid Membranes in Drug Delivery
The properties of amyloid fibrils, e.g., unique structural characteristics and superior biocompatibility, make them a promising vehicle for drug delivery. Here, carboxymethyl cellulose (CMC) and whey protein isolate amyloid fibril (WPI-AF) were used to synthesize amyloid-based hybrid membranes as vehicles for the delivery of cationic and hydrophobic drugs (e.g., methylene blue (MB) and riboflavin (RF)). The CMC/WPI-AF membranes were synthesized via chemical crosslinking coupled with phase inversion. The zeta potential and scanning electron microscopy results revealed a negative charge and a pleated surface microstructure with a high content of WPI-AF. FTIR analysis showed that the CMC and WPI-AF were cross-linked via glutaraldehyde and the interacting forces between membrane and MB or RF was found to be electrostatic interaction and hydrogen bonding, respectively. Next, the in vitro drug release from membranes was monitored using UV-vis spectrophotometry. Additionally, two empirical models were used to analyze the drug release data and relevant rate constant and parameters were determined accordingly. Moreover, our results indicated that in vitro drug release rates depended on the drug–matrix interactions and transport mechanism, which could be controlled by altering the WPI-AF content in membrane. This research provides an excellent example of utilizing two-dimensional amyloid-based materials for drug delivery.
Amyloid fibrils are formed by dense packing of hydrogen bonds that contributes to the backbone-backbone interactions of the intermolecular polypeptide chains [20][21][22]. The rigidity of amyloid fibrils' cross β-sheet core structure, which is formed through the noncovalent interactions, is considered to be responsible for the excellent mechanical properties of
Thioflavin T (ThT) Binding Assay
The ThT dye powder was first weighed and then dissolved in ethanol to prepare the ThT stock solution. The actual concentration of the ThT stock solution was determined to be 189 µM, using a Cary 50 UV-Vis Spectrophotometer (Varian, Palo Alto, CA, USA), and also spectrophotometrically, using the extinction coefficient of 26,600 M cm −1 at 416 nm. Next, the ThT working solution was prepared by diluting the ThT stock solution to a final concentration of 10 µM using PB buffer. Afterward, 20 µL aliquots of the sample solutions were mixed with 480 µL of the ThT working solution, and 350 µL of the mixture solutions were then pipetted into a 1 cm light path quartz cuvette. The ThT fluorescence spectra of the samples were obtained using a Cary Eclipse Fluorescence Spectrophotometer (Varian, USA) with the excitation wavelength set at 440 nm and the emission wavelength set at a range of 450 to 550 nm. Finally, the data of ThT fluorescence intensity at 485 nm were plotted against incubation time and fitted by the Boltzmann sigmoidal equation shown below [40,41]: where F is the ThT fluorescence intensity (at 485 nm); F i and F f are the ThT fluorescence intensities at 485 nm at the initial and final times; t 1/2 is the time required to reach half of the elongation phase; and τ denotes the elongation time constant.
Transmission Electron Microscopy (TEM)
10 µL of the sample solutions were placed on a 200 mesh carbon-stabilized/formvarcoated grid for 20 s, and the excess sample solutions on the grid were thoroughly removed using filter paper. Next, the samples remaining on the grid were negatively stained using 10 µL of 1% (w/v) uranyl acetate solution for 90 s and left to air-dry for 30 min. Afterward, the sample grids were examined and photographed using a Hitachi H-7650 Transmission Electron Microscope with a Gantan model 782 CCD Camera (Japan) at an accelerating voltage of 75 kV.
Zeta-Potential Measurements
The zeta-potential measurements were performed using a Malvern Zetasizer (Nano ZS, Malvern Instruments, UK) with a folded capillary zeta cell (DTS1070, Malvern Instruments, UK). The measurement parameters for each sample were set as follows: 60 s equilibrium duration, 10 s measurement duration, 10 measurements, and water as the dispersant/solvent at room temperature. The mean ± standard deviation of the zeta-potential value of samples was evaluated using Malvern Zetasizer software.
Fourier Transform Infrared (FT-IR) Spectroscopy
The functional groups of the CMC/WPI-AF membranes with different CMC:WPI-AF mass ratios and loading drugs (e.g., MB and RF) were investigated using a Perkin Elmer Spectrum 1000 FT-IR Spectrometer. Prior to the measurements, all the membranes were dehydrated via air-drying at 100 • C for a day and then grind-mixed following a 1:100 weight ratio with potassium bromide (KBr) powder. Next, the sample powder was compressed into tablets. Afterward, the FT-IR spectra of the sample tablets were obtained by means of an FT-IR spectrometer using a wavenumber range of 450-4000 cm −1 .
Scanning Electron Microscopy (SEM)
The surface microstructures of the CMC/WPI-AF membranes with different CMC:WPI-AF mass ratios were examined using a scanning electron microscope (FEI Inspect S; FEI, USA). In a typical experiment, the CMC/WPI-AF membranes were dehydrated via airdrying at 100 • C for a day. Next, the surface of each membrane was coated with platinum using a metal coating device (VD MSP-1S) under vacuum conditions. Finally, the surface microstructure of each membrane coated with platinum was examined and imaged by means of SEM.
Absorption of the Drug in the CMC/WPI-AF Membrane
MB, as a model of the cationic drug, and RF, as a model of the hydrophobic drug, were loaded into the CMC/WPI-AF membranes. First, stock solutions of 0.1 M MB and 0.1 M RF were prepared by dissolving appropriate amounts of MB and RF in PBS buffer and 0.5 N NaOH solution, respectively. Next, immersion solutions of 0.02 M MB and 0.01 M RF were produced by diluting the stock solutions with PBS buffer. To allow each drug to be loaded into the CMC/WPI-AF membranes, the membranes were cut into 4 cm 2 (length × width = 2 cm × 2 cm) pieces and then immersed in the MB and RF immersion solutions until absorption equilibrium was achieved, thereby obtaining both MB-loaded CMC/WPI-AF membranes and RF-loaded CMC/WPI-AF membranes. The concentrations of the drugs (MB or RF) loaded into the CMC/WPI-AF membranes were determined using a UV-Vis spectrophotometer (Thermo Fisher, Waltham, MA, USA) and the pre-calibration curves (see Figure S2). Finally, to assess the ability of the CMC/WPI-AF membranes with different CMC:WPI-AF mass ratios with regard to MB and RF loading, the two principal parameters, namely, the encapsulation efficiency (EE%) and the loading capacity (LC%), were determined. The evaluation of the two parameters was performed as expressed in Equations (2) and (3), respectively: EE (%) = weight of drug loaded in the membrane (g) weight of drug in the solution (g) ×100% (2) LC (%) = weight of drug loaded in the membrane (g) total weight of membrane (g) ×100% (3)
In Vitro Passive Drug Delivery Studies
The in vitro drug release of the MB and RF from the CMC/WPI-AF membranes was performed in the form of passive drug delivery. The procedure for the passive drug delivery was as described in a previous study [42], albeit with minor modifications. First, the MB-loaded CMC/WPI-AF membranes and RF-loaded CMC/WPI-AF membranes were immersed in PBS buffer solution (pH 7.4) at room temperature. Next, the MB and RF release experiments were conducted in dark vessels for 3 h and 0.5 h, respectively. During the passive drug release kinetics, a UV-vis spectrophotometer (Thermo Fisher, USA) was utilized to monitor the absorbance of the MB and the RF at a wavelength of~665 nm and 445 nm at different time points. The percentage of drugs released was as expressed in Equation (4): where [Drug] released was the drug released concentration (mM) and [Drug] total was the total drug concentration in the CMC/WPI-AF membrane. The relevant kinetic parameters of the passive drug delivery were further estimated using both the first-order model (Equation (5)) and the Korsmeyer-Peppas model (Equation (6)), based on the gathered experimental data: where M ∞ was the mass of the drug released at an infinite time; M t was the mass of the drug released at time t; and k f , k p , and n were the representative of the first-order rate constant, a parameter corresponding to the structural and geometric characteristics of the dosage form, and a diffusional exponent corresponding to the release mechanism, respectively.
Statistical Analysis
The data obtained from n independent measurements were calculated as the mean ± standard deviation. A statistically significant difference was determined by referring to the p ≤ 0.05 obtained using Student's t-test on n independent measurements.
Formation and Characterization of the WPI-AF
WPI is composed of a variety of proteins, including β-lactoglobulin (β-Lg), α-lactalbumin (α-La), and bovine serum albumin (BSA), which account for 51%, 19%, and 6% of its composition, respectively [43]. To form WPI-AF via the amyloidogenesis process, the acidic (pH 2.0) WPI solution was incubated at 80 • C with 600 rpm of magnetic stirring for 24 h. Next, as the characteristics of the fluorescence emission following the addition of the ThT molecules were specifically binding on the anti-β sheet structure of the amyloid fibril [44], the fibrillation kinetic of the WPI-AF formation was monitored by means of a ThT binding assay. Figure 1A presents the ThT fluorescence spectra of the WPI-AF at 0 h and 24 h of incubation. As shown in the ThT fluorescence spectra, no significant characteristic peak was observed at 0 h of incubation time, which indicates that the WPI had not yet been induced to form the amyloid fibril. However, as the incubation time increased, there appeared an apparent characteristic peak at~485 nm, indicating the formation of WPI-AF [44,45]. Furthermore, the ThT fluorescence intensity of the WPI-AF was detected on the basis of the incubation time. The results concerning the WPI fibrillation kinetics were consistent with the Boltzmann sigmoidal curve (see Figure 1B), which suggests that the fibrillogenesis process of the WPI was nucleation-dependent [41,44]. The nucleation-dependent amyloid fibrillogenesis process involves three phases: nucleation, elongation, and stationary [40,44]. Within the initial~2 h, the WPI monomers aggregated to form oligomer nuclei during the nucleation phase. Next, the oligomer nuclei aggregated into protofibrils during the process of elongation from~2 h to 12 h, before eventually entering the stationary phase to form mature fibrils after~12 h. Moreover, the time required to reach half of the elongation phase (t 1/2 ) and the elongation time constant (τ) were determined by analyzing the kinetics of the WPI-AF formation via the Boltzmann sigmoidal curve (see Figure 1B). The morphology of the WPI-AF was shown to comprise fibrillar aggregates with a high aspect ratio in the TEM micrograph (see Figure 2), which was similar to the morphology of previously reported WPI-AF [43,46]. The ThT binding assay and TEM results revealed that the WPI successfully formed amyloid fibril from the native protein. The ThT binding assay and TEM results revealed that the WPI successfully formed amyloid fibril from the native protein.
Synthetic Mechanism of the CMC/WPI-AF Membranes
A schematic diagram of the synthesis strategy of the CMC/WPI-AF membranes is shown in Figure 3A. First, the WPI-AF and CMC were chemically crosslinked to form composites with interconnected networks. Within these composites, the amino and hydroxyl groups on the WPI-AF and CMC molecular chains could react with the aldehyde group of the glutaraldehyde to produce chemical crosslinking [47]. The detailed crosslinking reaction is presented in Figure 3B. In this regard, the three possible chemical crosslinking reactions are as follows. First, the substituent (amino group) of the WPI-AF could react with the aldehyde group of the glutaraldehyde to form imine-type bonds. Second, the single hydroxyl group of the CMC could react with the aldehyde group of the glutaraldehyde to form semiacetal. Third, the two hydroxyl groups of the CMC could react with the aldehyde group of the glutaraldehyde to form acetal-type rings [47]. Next, the CMC/WPI-AF membranes were synthesized by means of a phase inversion-based immersion precipitation method. With this method, a non-solvent induces the phase inversion of the polymer solution, resulting in the precipitation of the polymer species to form a membrane [48]. The chemically crosslinked CMC/WPI-AF blend solution was then cast on a glass platform and immersed in a precipitation bath containing ethanol. During this immersion process, the solvent in the CMC/WPI-AF blend solution and the non-solvent in the precipitation bath were exchanged through mass transfer, resulting in the CMC/WPI-AF membranes being synthesized via phase inversion. Figure 3C shows the CMC/WPI-AF membranes to be yellow in color, indicating that the WPI-AF are crosslinked with the CMC via the glutaraldehyde crosslinker. Furthermore, the physical appearance of the CMC/WPI-AF membrane maintained its integrity in the PBS buffer. Conversely, the membrane prepared using CMC alone disintegrated in the PBS buffer, thereby indicating that CMC cannot crosslink on its own.
Synthetic Mechanism of the CMC/WPI-AF Membranes
A schematic diagram of the synthesis strategy of the CMC/WPI-AF membranes is shown in Figure 3A. First, the WPI-AF and CMC were chemically crosslinked to form composites with interconnected networks. Within these composites, the amino and hydroxyl groups on the WPI-AF and CMC molecular chains could react with the aldehyde group of the glutaraldehyde to produce chemical crosslinking [47]. The detailed crosslinking reaction is presented in Figure 3B. In this regard, the three possible chemical crosslinking reactions are as follows. First, the substituent (amino group) of the WPI-AF could react with the aldehyde group of the glutaraldehyde to form imine-type bonds. Second, the single hydroxyl group of the CMC could react with the aldehyde group of the glutaraldehyde to form semiacetal. Third, the two hydroxyl groups of the CMC could react with the aldehyde group of the glutaraldehyde to form acetal-type rings [47]. Next, the CMC/WPI-AF membranes were synthesized by means of a phase inversion-based immersion precipitation method. With this method, a non-solvent induces the phase inversion of the polymer solution, resulting in the precipitation of the polymer species to form a membrane [48]. The chemically crosslinked CMC/WPI-AF blend solution was then cast on a glass platform and immersed in a precipitation bath containing ethanol. During this immersion process, the solvent in the CMC/WPI-AF blend solution and the non-solvent in the precipitation bath were exchanged through mass transfer, resulting in the CMC/WPI-AF membranes being synthesized via phase inversion. Figure 3C shows the CMC/WPI-AF membranes to be yellow in color, indicating that the WPI-AF are crosslinked with the CMC via the glutaraldehyde crosslinker. Furthermore, the physical appearance of the CMC/WPI-AF membrane maintained its integrity in the PBS buffer. Conversely, the membrane prepared using CMC alone disintegrated in the PBS buffer, thereby indicating that CMC cannot crosslink on its own.
Zeta Potential Properties of the CMC/WPI-AF Membranes
As the pH value of the solvent affects the charged functional groups on the surface of the WPI-AF and CMC, the zeta potentials of the WPI-AF, CMC, and CMC/WPI-AF membranes were analyzed (see Figure 4). As the isoelectric point (pI) of the WPI was~5.2, the zeta potential of the WPI-AF in an acidic condition (pH 2.0) was positive (27.27 ± 0.91 mV). However, when adjusted to a pH value higher than the pI, the zeta potential of the WPI-AF changed from positive to negative. This change in the zeta potential was attributed to the increase in the amount of negatively charged carboxyl groups (-COO − ) within the protein and the neutralization of the positively charged amino groups (-NH 3 + ) [49]. As depicted in Figure 4, the zeta potential value of the CMC was negative in both the acidic and neutral environments, and it can be observed that the amount of negative charges in the CMC increased as the pH increased, which is similar to previous findings [49,50]. These zeta potential results confirm that the CMC and WPI-AF were not only cross-linked by chemical bonds but also exhibited electrostatic interactions. Notably, the CMC/WPI-AF membranes with different CMC:WPI-AF mass ratios had zeta potentials more negative than −30 mV in a neutral environment, indicating high negative charge and stability [51].
Zeta Potential Properties of the CMC/WPI-AF Membranes
As the pH value of the solvent affects the charged functional groups on the surface of the WPI-AF and CMC, the zeta potentials of the WPI-AF, CMC, and CMC/WPI-AF membranes were analyzed (see Figure 4). As the isoelectric point (pI) of the WPI was ~5.2, the zeta potential of the WPI-AF in an acidic condition (pH 2.0) was positive (27.27 ± 0.91 mV). However, when adjusted to a pH value higher than the pI, the zeta potential of the WPI-AF changed from positive to negative. This change in the zeta potential was attributed to the increase in the amount of negatively charged carboxyl groups (-COO -) within the protein and the neutralization of the positively charged amino groups (-NH3 + ) [49]. As depicted in Figure 4, the zeta potential value of the CMC was negative in both the acidic and neutral environments, and it can be observed that the amount of negative charges in the CMC increased as the pH increased, which is similar to previous findings [49,50]. These zeta potential results confirm that the CMC and WPI-AF were not only cross-linked by chemical bonds but also exhibited electrostatic interactions. Notably, the CMC/WPI-AF membranes with different CMC:WPI-AF mass ratios had zeta potentials more negative than −30 mV in a neutral environment, indicating high negative charge and stability [51].
Surface Microstructure Characterization of the CMC/WPI-AF Membranes
SEM has been widely used to characterize the surface microstructures of membranes. In this study, the samples of CMC/WPI-AF membranes were first air-dried at room temperature, and then the surface microstructures of the membranes were examined via SEM. As shown in the SEM images, there were differences in the surface microstructures of the CMC/WPI-AF membranes synthesized with different CMC:WPI-AF mass ratios ( Figure 5A,B). More specifically, the surface microstructure of the membrane synthesized with a CMC:WPI-AF mass ratio of 1:1 were found to be dense and smooth at high magnifications ( Figure 5C). When compared with the membrane with a CMC:WPI-AF mass ratio of 1:1, the surface microstructure of the membrane synthesized with a CMC:WPI-AF mass ratio of 1:2 showed pleated bulges at high magnifications ( Figure 5D). These results suggest that CMC/WPI-AF membranes with a high content of WPI-AF exhibited surface structure of more corrugated protrusions, which may contribute to increasing their surface area [52]. We speculate that a large amount of WPI-AF was
Surface Microstructure Characterization of the CMC/WPI-AF Membranes
SEM has been widely used to characterize the surface microstructures of membranes. In this study, the samples of CMC/WPI-AF membranes were first air-dried at room temperature, and then the surface microstructures of the membranes were examined via SEM. As shown in the SEM images, there were differences in the surface microstructures of the CMC/WPI-AF membranes synthesized with different CMC:WPI-AF mass ratios ( Figure 5A,B). More specifically, the surface microstructure of the membrane synthesized with a CMC:WPI-AF mass ratio of 1:1 were found to be dense and smooth at high magnifications ( Figure 5C). When compared with the membrane with a CMC:WPI-AF mass ratio of 1:1, the surface microstructure of the membrane synthesized with a CMC:WPI-AF mass ratio of 1:2 showed pleated bulges at high magnifications ( Figure 5D). These results suggest that CMC/WPI-AF membranes with a high content of WPI-AF exhibited surface structure of more corrugated protrusions, which may contribute to increasing their surface area [52]. We speculate that a large amount of WPI-AF was deposited and stacked on the membrane surface after the phase inversion, which resulted in apparent pleat bulges on the membrane surface. deposited and stacked on the membrane surface after the phase inversion, which resulted in apparent pleat bulges on the membrane surface.
FT-IR Analysis of the CMC/WPI-AF Membranes
To verify whether the crosslinking of the CMC and WPI-AF following the addition of glutaraldehyde to the solutions containing the CMC/WPI-AF samples was successful, an FT-IR spectroscopy analysis was conducted on the samples with CMC:WPI-AF mass ratios of 1:1 and 1:2. Figure 6A presents the FT-IR spectra of the native WPI, WPI-AF, CMC, and CMC/WPI-AF membranes. The secondary structure of the protein is known to be associated with a specific region of amine I (1625-1750 cm −1 ) and amine II (1475-1575 cm −1 ), which are related in the C=O stretching and N-H stretching of the peptide linkage in the protein [46,53]. The FT-IR spectra of the native WPI showed an amide I band at 1654 cm −1 , which was downshifted to 1643 cm −1 following incubation at 80 •C and pH 2.0, thereby indicating the formation of WPI-AF concomitant with an increase in the β-sheet structure [54]. Figure 6A illustrates the characteristic peaks of the CMC at 1111 cm −1 and 1160 cm −1 , which were attributed to the stretching vibration of the C-OH groups. Furthermore, the strong peaks at 1616 cm −1 and 1420 cm −1 were attributed to the carboxylic group's asymmetric and symmetric stretching vibration [55].
In the FT-IR spectra of the CMC/WPI-AF membranes, all the characteristic peaks of the WPI-AF and CMC were observed. Due to the crosslinking of the WPI-AF and CMC via glutaraldehyde, a shift in the characteristic peaks and the formation of new peaks occurred. First, the amide II band that emerged at 1538 cm −1 in the FT-IR spectrum of the WPI-AF shifted to that observed at 1543 cm −1 in the CMC/WPI-AF membranes, indicating that the amide groups of WPI-AF can react with the carbonyl groups of glutaraldehyde to form imine group [56]. Second, the new peak seen at 1711 cm −1 in the FT-IR spectrum of
FT-IR Analysis of the CMC/WPI-AF Membranes
To verify whether the crosslinking of the CMC and WPI-AF following the addition of glutaraldehyde to the solutions containing the CMC/WPI-AF samples was successful, an FT-IR spectroscopy analysis was conducted on the samples with CMC:WPI-AF mass ratios of 1:1 and 1:2. Figure 6A presents the FT-IR spectra of the native WPI, WPI-AF, CMC, and CMC/WPI-AF membranes. The secondary structure of the protein is known to be associated with a specific region of amine I (1625-1750 cm −1 ) and amine II (1475-1575 cm −1 ), which are related in the C=O stretching and N-H stretching of the peptide linkage in the protein [46,53]. The FT-IR spectra of the native WPI showed an amide I band at 1654 cm −1 , which was downshifted to 1643 cm −1 following incubation at 80 •C and pH 2.0, thereby indicating the formation of WPI-AF concomitant with an increase in the β-sheet structure [54]. Figure 6A illustrates the characteristic peaks of the CMC at 1111 cm −1 and 1160 cm −1 , which were attributed to the stretching vibration of the C-OH groups. Furthermore, the strong peaks at 1616 cm −1 and 1420 cm −1 were attributed to the carboxylic group's asymmetric and symmetric stretching vibration [55].
In the FT-IR spectra of the CMC/WPI-AF membranes, all the characteristic peaks of the WPI-AF and CMC were observed. Due to the crosslinking of the WPI-AF and CMC via glutaraldehyde, a shift in the characteristic peaks and the formation of new peaks occurred. First, the amide II band that emerged at 1538 cm −1 in the FT-IR spectrum of the WPI-AF shifted to that observed at 1543 cm −1 in the CMC/WPI-AF membranes, indicating that the amide groups of WPI-AF can react with the carbonyl groups of glutaraldehyde to form imine group [56]. Second, the new peak seen at 1711 cm −1 in the FT-IR spectrum of the CMC/WPI-AF membranes was mainly associated with C=O stretching in the pendant aldehyde [47]. Third, the region of the peaks seen at~1700-1585 cm −1 in the CMC/WPI-AF membranes was broader than that observed in relation to the WPI-AF and CMC, meaning that the peaks corresponding to the C=O stretching (in the amine I of the WPI-AF and the carboxylic group of the CMC) and C=N stretching (in the formed Schiff bases) overlapped [57]. These findings support the proposed crosslinking mechanism between CMC and WPI-AF, as shown in Figure 3B. [58,59]. Moreover, the downshifting of the peak at 1402 cm −1 associated with the positive charge of nitrogen on the MB indicated an electrostatic interaction between the MB and membrane [58]. On the other hand, after the RF had absorbed into the CMC/WPI-AF membranes, the characteristic peak of the CMC/WPI-AF membrane shifted from 1627 cm −1 to 1640 cm −1 . Moreover, the peaks seen at 3100-3600 cm −1 corresponding to the O-H and N-H stretching of the RF vanished. These peak shifts and disappearances indicated that the carboxyl groups of the CMC/WPI-AF membranes interacted with the N-H and O-H groups of the RF via hydrogen bonding [42,60]. Furthermore, the peak at 1700 cm −1 corresponding to the carbonyl stretching of the isoalloxazine ring of the RF disappeared after the RF had been absorbed into the CMC/WPI-AF membranes, implying that the RF was successfully absorbed and encapsulated in the membranes [61]. Table 1 shows that the EE% and LC% of the CMC/WPI-AF membranes were increased with an increasing amount of WPI-AF in the membranes. The possible reason for this is that the WPI-AF possessed the characteristics of a negative charge (see Figure 4) and a hydrophobic pocket mediated by aromatic residues [62,63]. Moreover, previous Following the drug absorption into the CMC/WPI-AF membranes, the interactions between the drugs (MB or RF) and the CMC/WPI-AF membranes were further revealed in the FT-IR spectrum (see Figure 6B). Following the absorption of the MB into the CMC/WPI-AF membranes, the peaks at 1604 cm −1 and 1487 cm −1 corresponding to the C-N and C-C bonds of the aromatic rings of the MB shifted and vanished, respectively, suggesting an interaction between the MB and membrane involving π-π interaction [58,59]. Moreover, the downshifting of the peak at 1402 cm −1 associated with the positive charge of nitrogen on the MB indicated an electrostatic interaction between the MB and membrane [58]. On the other hand, after the RF had absorbed into the CMC/WPI-AF membranes, the characteristic peak of the CMC/WPI-AF membrane shifted from 1627 cm −1 to 1640 cm −1 . Moreover, the peaks seen at 3100-3600 cm −1 corresponding to the O-H and N-H stretching of the RF vanished. These peak shifts and disappearances indicated that the carboxyl groups of the CMC/WPI-AF membranes interacted with the N-H and O-H groups of the RF via hydrogen bonding [42,60]. Furthermore, the peak at 1700 cm −1 corresponding to the carbonyl stretching of the isoalloxazine ring of the RF disappeared after the RF had been absorbed into the CMC/WPI-AF membranes, implying that the RF was successfully absorbed and encapsulated in the membranes [61]. Table 1 shows that the EE% and LC% of the CMC/WPI-AF membranes were increased with an increasing amount of WPI-AF in the membranes. The possible reason for this is that the WPI-AF possessed the characteristics of a negative charge (see Figure 4) and a hydrophobic pocket mediated by aromatic residues [62,63]. Moreover, previous studies have shown that amyloid fibril can provide multiple binding sites [42,64], thereby effectively improving the EE% and LC% of CMC/WPI-AF membranes in terms of the loading of cationic and hydrophobic drugs. Furthermore, with regard to the microstructures (see Figure 5), as the amount of WPI-AF increased, the surface microstructures of the membranes exhibited more corrugated protrusions, which provided a larger surface area for the absorption drugs on the membrane surface [52]. Yet, it can be observed that the CMC/WPI-AF membranes had higher EE% and LC% in relation to the loading of cationic drugs than hydrophobic drugs. The possible reason for this, as derived from our zeta-potential and FT-IR results, may be that the CMC in the membranes can also interact with MB via electrostatic interaction. Therefore, the presence of WPI-AF and CMC in the membranes can have a synergic effect on the absorption of cationic drugs. In addition, previous literature results have shown that drugs are more adsorbable when interacting with substrate surfaces through electrostatic interactions than hydrogen bonding [65].
In Vitro Passive Drug Release of MB and RF from the CMC/WPI-AF Membranes
To explore the in vitro passive release of the drugs from the CMC/WPI-AF membranes, the drug release procedure was conducted in PBS at pH 7.4. Given that the release behavior of a drug in a matrix-type drug delivery system is associated with the drug-matrix interaction and the diffusion of the drug within the matrix material [66,67], our drug release condition included different types of drugs (i.e., MB and RF) and different compositions of membranes (i.e., CMC:WPI-AF mass ratios of 1:1 and 1:2). The release profiles of the MB and RF from the membrane samples with CMC:WPI-AF mass ratios of 1:1 and 1:2 were obtained by plotting the cumulative drug release percentages as a time function, as shown in Figure 7A and 7B, respectively. Then, a nonlinear regression analysis was performed to fit the MB and RF release kinetics data via the utilized empirical models, namely the first-order and Korsmeyer-Peppas models (as shown in Equations (5) and (6)), to determine the corresponding kinetic parameters and coefficients of determination (R 2 ). Notably, the first-order model, which was used to describe the burst-release phenomenon, and the Korsmeyer-Peppas model, which was used to distinguish the different release mechanisms, were selected to predict the MB and RF release rate constants and decipher the diffusion behavior of the MB and RF [68,69].
According to the drug release results (as shown in Figure 7), the percentage of MB released from the CMC/WPI-AF membranes leveled off within 180 min, while the percentage of RF released from the CMC/WPI-AF membranes leveled off within 30 min. In addition, it should be noted that the percentages of MB and RF released from the membranes were reduced by increasing the WPI-AF content in the CMC/WPI-AF membranes. Concerning the different drug release behaviors upon drug release from each group, we further quantitatively analyzed the drug release results using the first-order model. As shown in Table 2, the data concerning the MB and RF released from the CMC/WPI-AF membranes adequately fit the first-order model, in which the R 2 values for all the release curves were determined to be greater than 0.95. The fitting results of the first-order model revealed the following. First, the mass of the drug released at infinite time from the membrane was CMC:WPI-AF = 1:1 > CMC:WPI-AF = 1:2. Second, the magnitude of the first-order rate constant (k f ) of the RF release was much larger than that of the MB release. Third, the magnitude of the first-order rate constant for the release of RF from the membrane with CMC:WPI-AF at 1:1 was found to be larger than that from the membrane with CMC:WPI-AF at 1:2, whereas the values of k f (or magnitude of the first-order rate constant) for the release of MB from both membranes (CMC:WPI-AF = 1:1 and CMC:WPI-AF = 1:2) were found to be very close.
To examine the mechanism of MB and RF release from the CMC/WPI-AF membranes, the Korsmeyer-Peppas model was used to fit the drug release kinetics data. The n value determined using the Korsmeyer-Peppas model has previously been employed to determine the mechanisms by which drugs were released from various geometric matrices (e.g., slab, cylinder, and sphere) [69,70]. More specifically, for the drug release system of polymeric films, the diffusional exponent n was found to be equal to 0.5, be between 0.5 and 1.0, or to be equal to 1.0, indicating the diffusion mechanism to be Fickian diffusion, anomalous transport, or case-II transport, respectively. We show in Table 3 that, in this study, the n value for the drug release from each group was within the range of 0.5-1.0, suggesting that the mechanism was anomalous transport [69]. Our results indicated that the value of n for the drug release from the membrane with CMC:WPI-AF at 1:1 was found to be larger than that from the membrane with CMC:WPI-AF at 1:2.
Finally, the drug release results can be interpreted as indicating the following. First, as the CMC and WPI-AF were negatively charged at pH 7.4, they could electrostatically interact with the MB, leading to a large amount of drug absorption into the membrane. Conversely, when the MB interacted with the membranes through electrostatic interactions, it resulted in a diffusion barrier/resistance to drug release, leading to a lower drug release rate [65]. Second, increasing the WPI-AF content in the membranes rendered the membrane structure compact and increased the interaction between WPI-AF and drug, resulting in a significantly lower amount of drug release. Third, the MB and RF release mechanisms corresponded to non-Fickian transport controlled by diffusion and chain relaxation. In addition, the mechanism of drug release from the membranes was changed from being swelling controlled to being diffusion controlled by increasing the WPI-AF content in the membranes [42], suggesting different drug release behaviors to be presented by membranes with different CMC:WPI-AF mass ratios. These findings confirmed that CMC/WPI-AF membranes exhibit the potential to modulate the drug release rates and drug release behaviors of cationic (MB) and hydrophobic (RF) drugs through altering the CMC:WPI-AF mass ratio or WPI-AF content.
release rate [65]. Second, increasing the WPI-AF content in the membranes rendered the membrane structure compact and increased the interaction between WPI-AF and drug, resulting in a significantly lower amount of drug release. Third, the MB and RF release mechanisms corresponded to non-Fickian transport controlled by diffusion and chain relaxation. In addition, the mechanism of drug release from the membranes was changed from being swelling controlled to being diffusion controlled by increasing the WPI-AF content in the membranes [42], suggesting different drug release behaviors to be presented by membranes with different CMC:WPI-AF mass ratios. These findings confirmed that CMC/WPI-AF membranes exhibit the potential to modulate the drug release rates and drug release behaviors of cationic (MB) and hydrophobic (RF) drugs through altering the CMC:WPI-AF mass ratio or WPI-AF content.
Discussion
Previous investigations have indicated that the polymeric vehicles (e.g., polyvinyl alcohol cryogel [71], polyethersulfone/polyacrylic acid composite hydrogel membrane [72], polyethylene oxide/pentaerythritol triacrylate/multi-walled carbon nanotubes composites [73], copolymer (PVA-co-PE) nanofiber membrane [74], and gelatin-polyacrylamide hydrogel [75]) have been commonly utilized in TDD systems. However, there are some potential problems/concerns that may arise when using the polymeric vehicles for drug delivery systems. These potential problems/concerns include cytotoxicity and lack of biocompatibility, generation of hazardous byproducts upon degradation, and usage of toxic additives or solvents during fabrication [76].
Amyloid fibrils exhibit remarkable properties, such as structural stability, mechanical stiffness, and abundance of functional groups [30]. Evidence has suggested that amyloid fibrils derived from food/milk proteins (e.g., whey, soybean, and egg white) show negligible cytotoxic effects toward human cells (Caco-2 and Hec-1a) in vitro [77], highlighting the biocompatible nature of food/milk protein-derived amyloid fibrils (e.g., WPI-AF). In addition, it was previously demonstrated that AFs derived from milk proteins (e.g., βlactoglobulin) are able to enhance skin bio-adhesivity and anti-inflammatory activity of drugs used for topical treatments in vivo [78]. As a result, amyloid-based materials are suitable for a variety of biomedical applications, including drug delivery, biomineralization, and cell scaffolds [79]. Moreover, the above-mentioned potential problems and/or drawbacks associated with polymeric vehicles may be overcome when using amyloid-based materials (e.g., WPI-AF) as drug delivery carriers. Table S2 summarizes amyloid-based materials with different forms (e.g., fibrillar aggregates, membranes, and hydrogels) used in drug delivery systems. Application of amyloid-based materials have been explored in intravenous and gastrointestinal drug delivery systems, but very few reports have examined their usage in transdermal drug delivery (TDD) systems. To that end, we have prepared | 2023-03-16T15:06:55.575Z | 2023-03-01T00:00:00.000 | {
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219705841 | pes2o/s2orc | v3-fos-license | Effect of intraoperative nerve monitoring on postoperative vocal cord palsy rates after thyroidectomy: European multicentre registry‐based study
Background Intraoperative nerve monitoring (IONM) of the recurrent laryngeal nerve (RLN) predicts the risk of vocal cord palsy (VCP). IONM can be used to adapt the surgical strategy in order to prevent bilateral VCP and associated morbidity. Controversial results have been reported in the literature for the effect of IONM on rates of VCP, and large multicentre studies are required for elucidation. Methods Patients undergoing first‐time thyroidectomy for benign thyroid disease between May 2015 and January 2019, documented prospectively in the European registry EUROCRINE®, were included in a cohort study. The influence of IONM and other factors on the development of postoperative VCP was analysed using multivariable regression analysis. Results Of 4598 operations from 82 hospitals, 3542 (77·0 per cent) were performed in female patients. IONM was used in 4182 (91·0 per cent) of 4598 operations, independent of hospital volume. Postoperative VCP was diagnosed in 50 (1·1 per cent) of the 4598 patients. The use of IONM was associated with a lower risk of postoperative VCP in multivariable analysis (odds ratio (OR) 0·34, 95 per cent c.i. 0·16 to 0·73). Damage to the RLN noted during surgery (OR 24·77, 12·91 to 48·07) and thyroiditis (OR 2·03, 1·10 to 3·76) were associated with an increased risk of VCP. Higher hospital volume correlated with a lower rate of VCP (OR 0·05, 0·01 to 0·13). Conclusion Use of IONM was associated with a low rate of postoperative VCP.
Introduction
According to the literature, vocal cord palsy (VCP) rates range from 3 to 5 per cent in the early postoperative period following thyroidectomy, and permanent VCP is diagnosed in 1-2 per cent of patients [1][2][3] . Hospital-specific variation may in part be related to the routine or targeted use of postoperative laryngoscopy, due to symptoms or intraoperative findings. The most common mechanism of injury of the recurrent laryngeal nerve (RLN) during thyroidectomy is traction at the suspensory ligament of the thyroid gland (Berry's ligament), which is in close anatomical relation to the nerve. Transection or thermal damage during surgery leads to more serious RLN lesions 4,5 . Depending on the mechanism of injury, global (type 1) or segmental (type 2), loss of RLN signal may occur during surgery, with VCP being transient or permanent. Early VCP was observed in 95 per cent of patients with segmental loss of signal, whereas VCP occurred in 70 per cent of patients who experienced the less severe global loss of signal 4,6 .
Apart from causing voice impairment, VCP is associated with an increased risk of aspiration pneumonia 7,8 . Bilateral VCP may lead to a mechanically based respiratory insufficiency, which potentially requires tracheostomy [7][8][9] . In past decades, intraoperative nerve monitoring (IONM) has been shown to contribute to intraoperative identification of the RLN 10,11 . Identification of the nerve is essential to preserve its functional integrity during surgery 12,13 . The advantage of IONM to reduce the risk of transient 14 or permanent 3 VCP has been substantiated in various comprehensive studies. However, meta-analyses 12,15,16 have concluded that use of IONM did not contribute to reduced rates of VCP. Apart from the debate on the influence of IONM, thyroiditis (Graves' disease or Hashimoto's thyroiditis) 17,18 and malignancy 1,19 have a proven effect on postoperative rates of VCP. In addition to factors influencing the complexity of the individual surgical procedure, the experience and training of the operating surgeon play an important role, which is why hospital volume is a relevant factor when considering rates of postoperative VCP 1,3,19 .
The aim of this large-scale multicentre study was to analyse the impact of IONM, and other factors, on the rate of postoperative VPC in a homogeneous patient cohort undergoing first-time thyroidectomy for benign thyroid disease.
Methods
Patients undergoing first-time surgery with thyroidectomy (bilateral thyroid lobe resection, 'total thyroidectomy') for underlying benign thyroid disease (nodular goitre, follicular adenoma, Graves' disease, Hashimoto's thyroiditis, Riedel's thyroiditis and other (unspecified) benign thyroid tumours) in hospitals using the EUROCRINE ® database, between May 2015 and January 2019, were included in the study. As laryngoscopic assessment is essential to diagnose VCP 7 , patients who did not have postoperative laryngoscopy were excluded from the analysis. The number of RLN palsies was counted per patient.
EUROCRINE ® registry
Since May 2015, the European database EUROCRINE ® has been available for registration of endocrine surgical procedures. The aim of EUROCRINE ® is increase the quality of surgical treatment of endocrine disease, and to disseminate clinical standards and reduce the differences among hospitals that perform surgery on patients with endocrine disease 20 .
Data variables
Data on vocal cord function and clinical parameters were collected prospectively. Clinical parameters included sex, age, use of IONM, thyroiditis, and damage to the RLN during surgery (noted and reported by the operating surgeon). Hospital volume (defined by the total number of operations in this study performed per hospital), and surgeon qualification (consultant versus assistant surgeon) were registered and analysed. The primary endpoint of the study was postoperative VCP, and the secondary endpoint was the frequency of permanent VCP.
Statistical analysis
Data were documented and described using Excel ® (Microsoft, Redmond, Washington, USA). Statistics were performed using R v. 3 To investigate the influence of different parameters on postoperative VCP, univariable analyses were performed, using the glm function in the stats R package to perform logistic regressions analysis. Variables with a P < 0⋅250 were included in a multivariable model. Resulting odds ratios (ORs) and their 95 per cent confidence intervals are reported. For comparison, continuous variables were standardized using the R scale function. The analysis was explorative; no adjustment for multiple testing was performed. The significance level was set at 5 per cent.
For an analysis of risk factors for permanent VCP, palsies persisting for more than 6 months after surgery were considered as permanent. Two theoretical models were analysed depending on whether patients with postoperative VCP lacked long-term follow-up or not: patients with postoperative VCP and lacking long-term control with laryngoscopy were assumed to have transient VCP in model 1, and to have permanent VCP in model 2. Owing to the small sample size of only six permanent palsies in total, the estimators of model 1 are not stable.
Results
A total of 8751 first-time thyroidectomies performed for benign disease were registered in EUROCRINE ® during the study period. After excluding 4153 operations due to lack of postoperative laryngoscopy, 4598 operations were included in the present analysis ( Fig. 1) from 82 different hospitals participating in EUROCRINE ® . Some 94⋅6 per cent of analysed patients had both preoperative and postoperative laryngoscopy (Fig. 1). Postoperative laryngoscopy was performed a mean(s.d.) of 6(22) days after surgery. Postoperative VCP n = 37 (0·8%) No long-term control n = 28 Thyroidectomy without IONM n = 416 Thyroidectomy without laryngoscopy control n = 4153 The percentage values shown are based on the total number of included operations (n = 4598). IONM, intraoperative nerve monitoring; VCP, vocal cord palsy.
There were 3542 female patients (77⋅0 per cent) and the the median (range) age of patients was 54 (3-92) years. The number of procedures entered into the register per hospital ranged from 1 to 683. The mean(s.d) duration of surgery was 112(45) min. Approximately three-quarters of the operations were carried out by a consultant surgeon ( Table 1).
Subgroup with postoperative vocal cord palsy
The overall rate of VCP was 50 of 4598 (1⋅1 per cent). Hospitals with a lower patient volume were more likely to have a higher rate of postoperative VCP (Fig. 2). Except for one patient, VCP was unilateral. Among the 50 patients in the group with postoperative VCP, it was more common to have postoperative laryngoscopy only, rather than preoperative and postoperative laryngoscopy as assessment of vocal cord function ( Table 1). The distribution of age and sex was similar in the patient groups with and without VCP. Median hospital volume was significantly lower in the group diagnosed with postoperative VCP ( Table 1).
The rate of thyroiditis on final histological examination was higher in the group with postoperative VCP: 25 of 50 (50 per cent) versus 1275 of 4548 (28⋅0 per cent) without VCP. The rate of IONM was lower in patients who had postoperative VCP than in those with a normal finding on postoperative laryngoscopy (74⋅0 versus 91⋅1 per cent respectively) ( Table 1).
Of the 50 patients diagnosed with postoperative VCP, 37 did not undergo long-term laryngoscopy control. Of the remaining 13 patients who underwent long-term control, six had permanent pareses; seven pareses were transient (Fig. 1).
Intraoperative nerve monitoring
IONM was used in 4182 (91⋅0 per cent) of the 4598 operations. There were no differences in hospital volume or patient age in the groups with and without use of IONM ( Table 2 and Fig. 3). In patients with IONM, RLN damage was noted more often than in patients without IONM (3⋅9 versus 1⋅9 per cent respectively) ( Table 2). A higher rate of postoperative VCP was registered in the group without the use of IONM: 3⋅1 per cent versus 0⋅9 per cent in the group that had IONM ( Table 2). Values in parentheses are percentages. Reference groups are shown in parentheses. Values in parentheses are percentages. Reference groups are shown in parentheses. Thirty-seven postoperative palsies without follow-up are assumed *transient and †permanent. n.a., Not assessable.
Risk factors for early postoperative vocal cord palsy
Univariable analysis revealed a significant impact of the use of IONM, hospital volume, damage to the RLN noted (as reported by the operating surgeon) and thyroiditis on postoperative VCP ( Table 3). Multivariable regression analysis confirmed the impact on VCP for these factors ( Table 3). The strongest effect on the risk of VCP in multivariable analysis was documented for damage to the RLN noted during surgery and hospital volume, followed by use of IONM and thyroiditis. When RLN damage was noted during surgery, the risk of postoperative VCP was 25-fold higher than in patients without observed or suspected damage. When IONM was not used, the risk of postoperative VCP was threefold higher than when it was used. The presence of thyroiditis was associated with a twofold greater risk of postoperative VCP than in patients without thyroiditis. A reduction in hospital volume by one standard deviation (211 patients) resulted in a 22-fold higher risk of postoperative VCP. Surgeon qualification, patient sex and patient age did not influence the rate of VCP.
Risk factors for permanent vocal cord palsy
Among the 50 patients with postoperative VCP, 37 (almost three-quarters) did not have long-term follow-up by laryngoscopy. Risk factors for permanent VCP in model 1 (assumed transient VCP) and model 2 (assumed permanent VCP) were RLN damage noted during surgery and hospital volume. Thus, independent of potential long-term laryngoscopic follow-up, these factors were relevant for the risk of permanent VCP. The risk of permanent VCP was 5-34-fold higher when suspected RLN damage was documented during the operation. A reduction in hospital volume by one standard deviation (211 patients) resulted in a 7⋅7-fold increase in the risk of permanent VCP, if the VCP in the 37 patients without laryngoscopic follow-up was considered transient. If the VCP in the 37 patients without follow-up was considered permanent, the risk would have increased by 100-fold ( Table 4). In model 2 (patients without follow-up assumed to have permanent VCP), thyroiditis and the use of IONM were also associated with a decreased risk of permanent VCP (Table 4). Age, sex and type of surgeon did not influence the risk of permanent VCP.
Discussion
This analysis found that postoperative laryngoscopy to exclude VCP after thyroidectomy was not standard in the countries and clinics participating in EUROCRINE ® . Of 8751 patients who had a thyroidectomy, only 4598 were investigated with postoperative laryngoscopy. This lack of standard use of postoperative laryngoscopy is in agreement with the American Thyroid Association guidelines 21 , which recommend laryngoscopy neither before (unless there are risk factors or suspicion or proven thyroid malignancy) nor after (unless voice abnormality is documented) surgery. In contrast, German Association of Endocrine Surgeons (CAEK) guidelines 22,23 recommend preoperative and postoperative laryngoscopy, independent of preoperative suspected or proven malignancy. In the present study, patients without postoperative laryngoscopy were excluded from analysis, as VCP could not be assessed objectively.
In addition to the CAEK guidelines, Dionigi and co-workers 24 recommend that postoperative laryngoscopy be performed on postoperative day 2, owing to its high sensitivity for VCP, based on patient compliance. In the present cohort, a high proportion of patients with documented postoperative VCP had only postoperative laryngoscopy, and not systematic preoperative and postoperative assessment. The underlying reason for this may be that hoarseness noted after surgery led to the suspicion of VCP, which resulted in verification of the suspected diagnosis by laryngoscopy rather than a routine investigation, as recommended by CAEK.
The use of targeted postoperative laryngoscopy, due to loss of signal during IONM or for other clinical reasons, leads to overestimation of the rate of VCP in these patients 3 . Furthermore, approximately three-quarters of patients diagnosed with postoperative VCP did not have long-term follow-up for potential permanent paresis. Therefore, the present study does not allow for a precise analysis of risk factors of permanent VCP. IONM was used in over 90 per cent of operations, a high proportion compared with that in other studies in the field 2,3,19 . Considering that thyroid redo surgery and operations for malignant disease were excluded from the present analysis, this finding emphasizes that in recent years IONM has commonly been done in clinics using the EUROCRINE ® database for routine procedures. Moreover, IONM was used independently of hospital volume of the participating centres. In the present study, use of IONM had a significant impact on the rate of postoperative VCP (0⋅9 per cent versus 3⋅1 per cent without use of IONM). Among other reasons, IONM facilitated detection of intraoperative RLN damage (3⋅9 per cent versus 1⋅9 per cent without use of IONM). However, information on RLN damage may be biased by the information given by the loss of signal during IONM, as the detection of RLN damage without the use of IONM is restricted to severe RLN injury, such as by transection or thermal damage.
An RCT by Barczynski and colleagues 14 showed that use of IONM led to a reduction in the rate of transient VCP. However, the study did not show an effect on permanent VCP 14 . In contrast, a retrospective observational multicentre study by Bergenfelz et al. 3 of Swedish departments showed that the use of IONM led to a reduced rate of permanent VCP. As an advantage, the multivariable analysis in this study 3 crucially included information on whether postoperative laryngoscopy was used routinely or targeted due to patient symptoms and signs. In the present study, the effect of IONM on the postoperative VCP rate was analysed, and found to be significant. In addition, two theoretical models for permanent VCP were analysed, and gave a strong indication that IONM also influenced postoperative rates of VCP. However, the fact that 37 patients with documented postoperative VCP did not have long-term follow-up did not allow precise estimation of the rate of permanent VCP, and hence the precise impact of IONM.
A weakness of this study is that it did not assess whether continuous or intermittent IONM was used. Intermittent IONM can be used to identify the RLN, follow its anatomical course, and assess RLN function before and after resection of the thyroid lobe. This technique enables the surgeon to change strategy in patients with suspected RLN palsy, and therefore prevents bilateral VCP (according to the International Neural Monitoring Study Group (INMSG) 2018 recommendation 7,9 ). In contrast, continuous IONM additionally permits real-time evaluation of RLN function and enables the surgeon to react to electromyographic signal changes (so-called combined events) [25][26][27] with a change of surgical strategy, depending on information on the effect on RLN function indicated by continuous IONM. Owing to the documentation structure of EUROCRINE ® , the exact surgical management prompted by intermittent or continuous IONM information cannot be retrieved. Data on which operations with loss of signal were terminated as a lobectomy rather than the intended thyroidectomy were not included during the analysed time period. However, as the use of IONM was associated with a reduced VCP rate, this implies that the INMSG recommendations 7,9 for intermittent and continuous IONM were practised successfully in hospitals that used the EUROCRINE ® database for quality control.
However, postoperative laryngoscopy cannot be replaced by IONM to evaluate the rate of VCP, as the literature is inconclusive regarding the value of IONM 15,16 .
Apart from IONM, factors influencing postoperative VCP rates are thyroiditis (Graves' disease or Hashimoto's thyroiditis) 17,18 and malignancy 1,19 . Especially in thyroid malignancy, deliberate resection of the RLN for oncological reasons is inevitably associated with permanent VCP. Thyroid redo surgery may be associated with increased rates of RLN palsy of up to 20 per cent 1,2 . The a priori exclusion of thyroid reoperation and surgery for malignant disease excluded these factors from the present analysis. As already described by other authors 17,18 , the present study has confirmed that thyroiditis increases the rate of postoperative VCP. In contrast, for permanent VCP, thyroiditis is debatable as a risk factor based on the theoretical models calculated for the present cohort.
In the present analysis, hospital volume (defined by the total number of operations in the study that were entered in the registry per hospital) had a significant impact on postoperative VCP, with lower volume being associated with an increased risk of VCP. However, the fact that hospitals joined EUROCRINE ® at different time points might have biased the results, as hospitals with a higher volume (and greater experience) might be represented with an incorrectly low patient number in the registry. However, hospital volume significantly influenced postoperative VCP, as well as permanent VCP development. Surgeon qualification (consultant versus assistant) did not significantly influence VCP rates.
Postoperative laryngoscopy is not used as a standard to evaluate vocal cord function during first-time thyroidectomy for benign disease in a number of European countries and hospitals. The use of IONM is common during thyroidectomy in this subgroup of patients, and is associated with a reduced risk of early postoperative and permanent VCP. The impact of hospital volume on the risk of VCP after thyroidectomy is significant. | 2020-06-16T20:06:50.454Z | 2020-06-16T00:00:00.000 | {
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202222928 | pes2o/s2orc | v3-fos-license | Frictional Properties of Rotary Glyd-ring under Water Lubrication
The ocean, which covers about 70% of the earth surface, contains abundant natural resources, such as organisms, minerals, energy resources, and etc. Development and utilization of ocean resources is an important way to solve the problems of population, resources and environment that human beings are confronting with. Besides, the underwater operation equipment plays an important role in the development and utilization of the ocean. The rotary dynamic seal, which exerts great effects on the static and dynamic performances, reliability, life span and etc. is a key technology of the underwater rotating equipment. This research focuses on the frictional properties of rotary dynamic Glyd-ring under water lubrication. A physical model is proposed to study the effects of rotational speed and working pressure on the maximum Von Mises stress numerically. A rotary seal test bench is developed and experiments have been carried out to investigate the friction, torque of Glyd-ring under different rotational speed, different sealing pressures on one or both seal sides. The research results provide a basis for the design of dynamic seals for deep sea equipment.
Introduction
In order to improve the sealing performance, domestic and foreign scholars have carried out a series of research on dynamic sealing performance. For example, Robert Bosch GmbH of Germany [1][2][3] used the finite element analysis software ABAQUS to establish a model to verify the accuracy of the lubrication theory of the ring sealing surface. Aachen University of Technology uses the test bench for four different sealing rings [4] (Stern seal, Glyd-ring, PU compound seal ring, Combined sealing ring) in different working conditions (different pressure (25bar~100bar), different oil temperature (30°C~70°C), different motor frequency (2.8Hz~12.5Hz), different speed (0~10m/s); and the dynamic sealing performance under the test has been studied. It is concluded that the combined sealing and PU compound seals composed of PTFE and O-ring seals have the advantages of good lubricity, and are more suitable for dynamic sealing; University of Alabama Huntsville [5] used the analysis software ABAQUS to establish a simulation model of the O-ring seal, and analyzed the change of the seal ring load of the O-ring at different compression ratios. Tests show that as the compression ratio increases, the load rate of the O-ring gradually decreases; Tsinghua University [6][7] uses the analysis software to establish the axisymmetric model of the nitrile rubber O-ring, the static seal of the nitrile rubber Oring and the micro-motion sealing performance was simulated and experimentally studied. In addition, universities such as North University of China [8] , Beijing University [9] of Aeronautics and Astronautics, and Jiangsu University [10] have also conducted research on the dynamic sealing performance of seal rings.
Grid model
The physical model of the rotary dynamic seal is established. The specific specification of the Glyd ring is RC11-Q-8×12.9×2.2, and the overall structure has axis symmetry. It is assumed that the piston rod and the sleeve will not be deformed and is defined as an analytical rigid body. The sealing structure is expanded to convert the rotational motion into a linear motion. For the convenience of calculation, the expanded 1/10 model is built and the mesh is divided. Figure 1 shows a simplified mesh model of a rotating dynamic seal. During the conversion process, the speed conversion formula is: Where: ν is the tangential velocity; n is the motor speed; d is the inner diameter of the Glyd-ring.
Material model
The O-ring of the Glyd Ring is a nitrile rubber material, which is an incompressible superelastic body [11] . It is described by a two-parameter Mooney-Rivlin model [12] . The Mooney constant is C 10 =1.87, C 01 =0.47. The material of the square ring is PTFE, the material of the sleeve is aluminum bronze QAL9-4, the material of the piston rod is stainless steel 316L, the friction coefficient of O-ring is 0.3, the friction coefficient of square ring is 0.05, the specific parameters are as follows in Table 1.
Boundary Condition
In the process of simulation, there are more boundary conditions, considering the actual situation and the feasibility of the simulation, within the error tolerance, a series of assumptions are made. The material stiffness of the piston rod and the sleeve is much higher than the stiffness of the Glyd ring, so the deformation can be neglected and defined as the analytical rigid body. Ignore the temperature change and wear caused by the friction during the process of simulation analysis. The Glyd ring is considered to be caused by a certain displacement of the boundary of the constrained piston rod.
Analysis
Step According to the model, the sleeve is fixed during the whole analysis process, and the sleeve is completely fixed when the boundary condition is set. The analysis of the rotary dynamic sealing characteristics of the Glyd ring is divided into three analysis steps: Applying a certain amount of pre-compression to the Glyd ring, that is, setting the displacement of the piston rod in the Y-axis direction; Applying a certain working pressure to both sides of the Glyd Ring; Apply a certain speed to the piston rod (Rotary axis).
Test Plan and Data Handle
First, the air in the two water chambers is taken out, and the water is filled into the two water chambers; the AC servo motor is started. After the motor speed is stabilized, the sliding table on the rodless cylinder starts to operate, and each sensor works normally, the dynamic sealing characteristic test bench data The display in the acquisition system is normal, and the test runs for several cycles. The mounting position of the sleeve assembly Glyd ring is A, B, C, as shown in Figure 2. Apply different pressure to one side of the Glyd ring.Firstly, the unilateral compression of the Glyd ring is tested. The Glyd ring is not set at the B point, the cavity 1 and the cavity 2 are connected, and the rotation speed is set to a certain value, in the step of 2 MPa, in the circle. The pressure of water in the two chambers was recorded. When the pressure was 0 to 10 MPa, the tensile sensor data is recorded as F 1 , and the data F 1 obtained at this time. It is the friction force of the two Grad rings, so the friction force of the Glyd ring on one side under pressure is F a1 =F 1/2 .
The values of P a and F a1 were obtained through experiments, and the graph of a was drawn to analyze the relationship. The contact pressure distribution of the sealing ring of the Glyd ring is obtained. The curve of contact pressure with water pressure is obtained, and a graph is drawn to analyze the relationship. The P b and Fb values were obtained through experiments, and a graph was drawn. The conclusion was obtained by comparing a.
3.1.3.
Apply different pressure to the sides of the Glyd ring. Install the Glyd ring at all of A, B and C. The two chambers are not connected. Adjust the pressure of the water in the chamber to a certain value, set the rotation speed to a certain value, and record the pressure P c of the chamber 2 as 0 to 10MPa, the pressure sensor shows the number F 3 . When the pressure of the two chambers is the same as the pressure of the chamber 1 at the same time, the pressure sensor shows F 4 . Compared with the case a, the gray circle can be obtained. Friction force Fc=F 3 -F 1 -F 4 /2. The P c and F c values were obtained through experiments, and a graph was drawn. Comparing the case a, and the conclusion was drawn.
Experiments
The rotary seal test bench is shown in Figure 3. The overall physical map of the test bench is shown in Figure 4. The AC servo motor 1 drives the shaft 8 to rotate continuously through the couplings 3, 7. The motor speed is controlled by the driver, the pressure regulating device is responsible for adjusting the pressure of two water chambers, and the pressure sensor is responsible for recording the pressure of the water chambers, the torque-speed sensor 6 is responsible for recording the rotational speed and the friction torque of the sealing ring. Finally, through the data acquisition system of test bench, the data of rotating speed, pressure and friction torque will be stored and processed.
Effect of Speed on Contact Pressure
The moving speed of the piston rod is set to 0.2m/s, and the working pressures of 0MPa, 2MPa, 4MPa, 6MPa, 8MPa and 10MPa are applied on one side of the Glyd ring, and the analytical working pressure is obtained for the Glyd ring at different compression ratios. The experimental results of the influence curve of Mises stress show that the larger the compression ratio after applying the pressure load, the smaller the maximum Mises stress of the Glyd ring. When the compression ratio ω is 17%, the Mises stress changes more smoothly with the increase of the pressure, so the pre-compression rate ω analyzed in the experiment of performing the rotary dynamic sealing is selected to be 17%. When the pre-compression amount is 17%, the working pressure of 2 MPa is applied on both sides of the Glyd ring respectively, and the distribution of contact pressure of the sealing surface of the Glyd ring is obtained by simulation at 500 rpm, 1000 rpm, 1500 rpm, 2000 rpm, and the test results are shown in Figure 5. It can be concluded that the Maximum contact pressure of the sealing surface of the glyph ring is basically stable at about 3.6 MPa, which is less affected by the rotational speed.
The effect of Working Pressure on Contact Pressure
According to the results of the modeling, get the Bode simulation in the ABAQUS/CAE. The simulation of the modeling as follows. It can be seen from the above figure that under the three kinds of compression conditions, the maximum contact pressure of the sealing surface of the Glyd ring is greater than the current water pressure. The maximum contact pressure gradually increases with the increase of water pressure, and the maximum contact pressure of the sealing ring on both sides is less than the maximum contact pressure when one side is pressed.
The effect of Constant Pressure Shift on Friction Torque
When the pressure of the water on both sides of the Glyd ring is 0 MPa, the friction torque curve of the Glyd ring at different rotational speeds is obtained.
It is concluded that when the two sides of the Glyd ring are not under pressure, the frictional moment of the Glyd ring is less affected by the rotational speed, fluctuating around 0.042 N•m, and the variation is small, and the contact with the simulation is obtained. The pressure is consistent with the lesser effect of the speed.
The Effect of Constant Speed Variable Pressure on Friction Torque
When the rotation speed n=200 rpm, the pressure method corresponding to the simulation was tested, and the curve of the Glyd ring under three kinds of pressures was obtained, as shown in Figure 9.
It can be seen from Figure 10 that the friction torque of the Glyd ring increases with the increase of the working pressure when the rotation speed is constant, and the friction torque when the two sides are pressed is smaller than the friction torque when the pressure is pressed on one side; The friction torque at the same pressure on both sides is smaller than the friction torque at different pressures on both sides. The test results are in agreement with the simulation results.
Conclusion
In this paper, the Glyd Ring is taken as the research object, and the test bench for rotating dynamic sealing characteristics is developed. The simulation and experimental research on the dynamic sealing characteristics under water lubrication conditions are carried out, and the following conclusions are obtained: The maximum Mises stress and the maximum contact pressure on both sides of the Glyd-ring are smaller than those on the single side; Under constant working pressure, the rotational speed exerts little effects on the frictional torque; Under constant rotational speed, the frictional torque of the Glyd-ring gradually increases with the increase of the working pressure, the maximum Mises stress and friction torque of the Glyd-ring under both sides with pressures are smaller than one side with pressures. | 2019-09-11T02:02:50.448Z | 2019-08-01T00:00:00.000 | {
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155535936 | pes2o/s2orc | v3-fos-license | Microstructure of magnetite - polyvinylidene fluoride (Fe3O4/PVDF) nanocomposite prepared by spin coating method
Recently, nanomaterials in the world have a very promising potential in the world of technology and industry. One of them is the development of nanocomposite materials. Composite is a combination of two or more different materials, one as a matrix component and the other as a filler component. This study aims to synthesize and characterize Fe3O4/PVDF nanocomposite thin film from natural iron sand using the spin coating method. The thin films nanocomposite magnetite - polyvinylidene fluoride (Fe3O4 - PVDF) was prepared using a solgel method and then developed employing a spin coating device with a mirror substrate. The Fe3O4/PVDF thin film nanocomposite was characterized using XRD and SEM to determine its microstructure. In this paper, the microstructure of Fe3O4/PVDF nanocomposite will be discussed in depth and elaborate the parameters that affect the Fe3O4/PVDF microstructure.
Introduction
The development of nanostructured materials today has very promising potential, ranging from nanoparticle, nanowire [1], nanotubes, to nanocomposite materials [2]. Composites are a combination of two or more different materials, one as a matrix component and the other as its filler component. While the nanocomposite itself is a solid structure with nanometer-sized dimensions wich are arranged repeatedly with the distance between different constituent shapes.
Researchers have done a lot of research on polymer materials to develop nanocomposite materials, which, by using nano-sized fillers, will be dispersed into the polymer matrix system. The mixing of nanoparticles into the constituent matrix is part of the development of the nanotechnology world. After adding a number of nanoparticles into the matrix material, the resulting nanocomposite exhibits superior properties compared to the properties of the prior material [3].
The advantages of this nanocomposite depend on the structure, properties and composition of the material itself. Polymer-based nanocomposites have advantages over other conventional composite materials. This advantage depends on the material properties, structure and composition of the constituent of the nanocomposite material.
A thin films nanocomposite Magnetite -Polyvinylidene fluoride at this time is growing very rapidly because it can be used in various fields, especially in the field of sensor. Magnetite in nano size contained in iron sand has advantages compared to other compounds, because it is superior in response to external magnetic field. So it has a big hope to apply latest various fields like electronics and industry as well as in the manufacture of thin layers of nanocomposite. A thin film of magnetic nanoparticles inside a polymer matrix is possible to be applied in areas such as electronics, magnetic, optical and mechanical [4,5].
Polyvinylidene Fluoride or PVDF is a pure thermoplastic fluoropolymer that has a low melting point making it easier to melt [6]. PVDF an actual normally worn applications demanding purity, strength, resistance for solvents, acids, and heat. compared with other fluoropolymers. This PVDF is a remarkable semi-crystalline polymer. So, the use of PVDF as a matrix in making nanocomposite is one of the key parameters for various applications. One application of the PVDF-based thin film polymer is that it can be used as a capacitive biosensor to measure the glucose content of a material [7], as a chemical reaction sensor [8], as piezoelectric materials for liquid viscosity sensor [9]. The microstructure of this nanocomposite depends on several parameters, one of which is the spread of nanoparticles into the polymer matrix.
A thin films is a layer of a thickness of size in a mirkometer order to a nanometer made of organic, inorganic, or metallic material having properties such as a conductor, an insulator or a semiconductor, develop to the incorporation based the atomic equity. There are several methods that can be used to grow thin films such as DC magnetton sputtering method [10,11], sputtering [12], electrophoresis [13], slip casting [13], as well as pyrolysis spray [14]. However, this technique has the detriment that the cost is very expensive, requires sophisticated instruments and wide area formed on a small layer.
Spin coating method is the most simple and easy method. Spin coating is a method of growing a thin films through a process of rotation or spin. Where the advantages of this method is that the resulting thin film has a pretty good quality and manufacturing costs are relatively cheap compared with other methods [15]. As for the preparation of a thin film of the best method used is the method of sol-gel because this method has several advantages, one of which is to produce a thin films with high levels of homogeneity [16].
The spin coating process is divided into several stages, namely the penetration stage of the solution on the substrate, the acceleration stage (Spin up), the level of smoothing (Spin off) and the last drying stage. The first step of the spin coating process is the penetration stage of the solution on the substrate. Then in the next stage is the acceleration stage, where the solution will be attracted to the edge of the substrate and spread evenly [17], this is because at this stage there is a centrifugal force that affects. The thin film will then spread evenly on the spin off and then in the last stage the solvent will be absorbed into the atmosphere so that a thin film of thickness is formed.
Experimental
Research here an experiment research. The sample used latest this research is magnetite (Fe3O4) prepared from iron sand and polymer used is polyvinylidene fluoride. On this study the device worn are spin coating, HEM-E3D, glasses of chemistry, magnetic stirrer equipped with hot plate, permanent magnet, and digital scales. While other materials needed to make the nanocomposite magnetitepolyvinylidene fluoride (Fe3O4 -PVDF) are aquabidest, ethanol, nitric acid, ethylene glycol and oxalic acid.
Before Iron sand is prepared by sol-gel method, iron sand must be purified first. In this research, the process of purification of iron sand is by means of iron sand purified by means of drawn using permanent magnet 30 times, to separate it from impurities (residue). Then the iron sand was washed with aquabidest, and dried. Once dry, the iron sand is pulled employing a permanent magnet. Then pure iron sand act made into nanoparticles using the HEM-E3D tool as long as 30 hour, because on milling 30 hours shows the loss away the hematite phase, such that alone one phase remains, the magnetite phase. Then in the washing to remove dirt or mixture on the iron sand and then dried again [18].
The precursor used to make the magnetite sol gel is Precursor (Fe(NO3)3.9H2O), preceded by reacting 17.4 g of magnetite in 4.5 g oxalic acid (C2H2O4) and 42 mL nitric acid (HNO3) at 110 0 C. Then add ethylene glycol to the solution. Stir for 2 hours at 80 0 C using a magnetic stirrer [19]. the magnetite sol-gel is then mixed into Tetrahydrofuran (THF) and then fed into the ultrasonic cleaner for 2 hours (Solution 1). Next make polyvinylidene fluoride polymer precursor (PVDF) by dissolving PVDF into Tetrahydrofuran (THF) by comparison (3:70). The mixture is continuously stirred using a magnetic stirrer until homogenously at 75 0 C (Solution 2).
Then the nanocomposite is grown on a glass substrate using a spin coating tool. The growth of a thin film of nanocomposite magnetite -polyvinylidene fluoride (Fe3O4 -PVDF) was performed at a rotational speed of 3000 rpm for 60 seconds. Furthermore, a thin film that has been formed in annealing using the furnace for 30 minutes at 60 0 C [20]. The thin film of Fe3O4-PVDF nanocomposite are employing X-Ray Diffraction (XRD) to investigate its microstructure and use Scanning Electron Microscopy (SEM).
Results and discussion
The results of characterization using XRD showed that a thin films of nanocomposite magnetite -Polyvinylidene fluoride Fe3O4 -PVDF) was developed on the glass substrate, characterized by the presence of magnetite peaks in X-Ray diffraction patterns with variations of Fe3O4 and PVDF compositions. Based at Figure 1 can be seen the influence of the Fe3O4 -PVDF composition on nanocomposite thin film crystal structure. Increasing the amount of Fe3O4 composition in a thin films of nanocomposite will result in the peak intensity of Fe3O4 also becoming higher or more prominent. this is because more of the Fe3O4 content in the solution will affect the number of atoms that make up the crystalline plane of the Fe3O4 thin film.
In the growth of thin film of nanocomposite, there are several things that will affect the results of characterization so that not all results will be in accordance with the theory. One that affects the homogeneity of the thin film made, the level of flatness of the thin layer formed, and the difference in gel mass dripped on the substrate [21].
Based on data obtained from the XRD results it can be known the magnitude of the crystal diameter (D) thin layer of nanocomposite. the diameter of the crystal (D) can be calculated using the Scherrer equation.
where λ (lamda) is the wavelength of 1.54 Å, B act FWHM like the preferred peak, θB act the angle of Bragg and K is the actual constant whose price is fewer than one. By using the value of FWHM it can be determined the size of crystallite from a thin films. The magnitude of the crystal size of the Fe3O4 -PVDF nanokomposite thin films can be seen in the Figure 2. The average crystallite size of the Fe3O4-PVDF nanocomposite increases with the addition of polyvinylidene fluoride polymer matrix in the nanocomposite, and vice versa the crystallite size decreases with the increasing amount of Fe3O4 composition in the nanocomposite sol-gel. This result corresponds to the results shown in XRD which show that in addition to Fe3O4 the peak crystallinity composition becomes more prominent and narrow. This crystallization peak shows the distribution of crystallite size [22]. So that more concentrations of Fe3O4 in the nanocomposite result in less crystallinity.
Based on the Figure 3 above can be seen that the morphology of thin films of nanokomposite well distributed throughout the PVDF matrix although there is still formed aglomerisasi on a thin films of nanokomposite. With the increasing concentration of Fe3O4 in the nanocomposite, the agglomerates are also getting bigger. In addition, from this SEM data can also be obtained the size of the grain from the thin films of nanocomposite for variation of F3O4: PVDF composition shown in Figure 4.
Variation of Fe3O4 Composition: PVDF in the Figure 4 shows generally that with the increase of Fe3O4 composition the grain size of nanocomposite will decrease further. enlarged grain size was obtained on the composition ratio (Fe3O4: PVDF) of 10:20. This grain size depends on the composition of the composite material material. The thickness of the Fe3O4 -PVDF nanokomposite thin films using SEM for 5 variations of Fe3O4 -PVDF composition at magnification 500 times can be seen in the Figure 5. The effect of thickness of the thin layer of nanocomposite on Fe3O4: PVDF composition to be more clearly can also be seen in Figure 6. Based on Figure 6, it can be seen that with the addition of the composition of Fe3O4 in the process of making nanocomposite thin films it results in a decrease in the thickness away the thin films of nanocomposite. This is in accordance with Vegonopal's research, et al, 2014 [23] which says that the more composition of Fe3O4 we add in a thin film of nanocomposite, the thinner the thickness of the layer. And vice versa, with the addition of a PVDF polymer, the thickness of the thin films will be even greater, we can see the composition ratio Fe3O4: PVDF (10:20). But in the composition away Fe3O4: PVDF as much as 10:30, the thickness of the thin films decreases. The thickness of these different thin layers is influenced by the degree of homogeneity of the nanocomposite thin films made, the level of flatness of the thin film formed, and the difference in gel mass dripped on the substrate [21]. So the results obtained are less significant, due to differences in Fe3O4 and PVDF compositions used in making nanocomposite thin films. So it can be seen in all test results that the microstructure of the magnetite nanocomposite -PVDF depends on the composition of Fe3O4 -PVDF used. Where PVDF as a matrix in making this nanocomposite thin film is one of the important parameters that will influence the spread of magnetite nanoparticles into the polymer matrix.
Conclusion
The results showed that a thin films of Fe3O4 -PVDF nanocomposite thin film on a glass substrate with 5 variation of Fe3O4 -PVDF composition was performed. One of the parameters that affect the microstructure of the nanocomposite is the spread of magnetite nanoparticles into the polymer matrix. The spread of these magnetite nanoparticles is influenced by the composition of Fe3O4 -PVDF compositions used. In addition the Fe3O4 composition results in increasingly prominent and narrower | 2019-05-17T14:23:52.394Z | 2019-04-01T00:00:00.000 | {
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235297948 | pes2o/s2orc | v3-fos-license | Congenital Obstructive Müllerian Anomaly: The Pitfalls of a Magnetic Resonance Imaging-Based Diagnosis and the Importance of Intraoperative Biopsy
A retrospective cohort study of the concordance between the magnetic resonance imaging (MRI) diagnosis and final diagnosis in patients with Müllerian duct anomalies (MDAs) was conducted, and diagnostic clues were suggested. A total of 463 cases of young women who underwent pelvic MRIs from January 1995 to February 2019 at Seoul Asan Medical Center were reviewed. Interventions consisted of clinical examinations, abdominal or transvaginal/rectal ultrasound, MRI, and operative procedures, including hysteroscopy and laparoscopy. The concordance of the diagnosis between the results obtained with MRI and those obtained with surgeries was evaluated. It was found that a total of 225 cases (48.6%) showed genital tract anomalies on MRI. Among them, 105 cases (46.7%) underwent reconstructive surgery. Nineteen cases (8.4%) revealed discrepancies between the final diagnosis after surgery and the initial MRI findings and eleven cases (57.9%) had cervical anomalies. Incorrect findings associated with the MRIs were particularly evident in biopsied cases of cervical dysgenesis. A combination of physical examination, ultrasound, and MRI is suitable for preoperative work-up in the diagnoses of congenital obstructive anomalies. However, it is recommended that a pathologic confirmation of tissue at the caudal leading edge be made in obstructive genital anomalies, in cases of presumptive vaginal or cervical dysgenesis.
Introduction
Congenital Müllerian duct anomalies (MDAs) are relatively common, and diverse types of this condition have been reported [1]. MDAs are observed in 2-3% of fertile women, 3% of infertile women, and 5-10% of patients with repeated miscarriages [2], and various classifications have been proposed [3,4]. MDAs can also be categorized as obstructive and nonobstructive anomalies. Obstructive reproductive tract anomalies include an imperforate hymen, distal vaginal atresia or agenesis, transverse vaginal septum, cervical dysgenesis or agenesis, and an obstructive hemivagina with an ipsilateral renal anomaly (OHVIRA) [5], although an imperforate hymen is derived from the urogenital sinus and does not have a Müllerian duct origin.
Among various obstructive reproductive tract anomalies, differential diagnoses between cervical dysgenesis or agenesis and vaginal agenesis are especially important because the impact of these conditions on women's sexual and reproductive potential can be significantly different. However, differentiation between the two is often difficult, even for experienced gynecologists and radiologists. The incidence of vaginal agenesis ranges from 1/4000 to 1/5000 [6,7]. For cervical agenesis or dysgenesis, the exact incidence is unknown, but it is considered to be approximately between 1/80,000 and 1/100,000 [6]. Cervical dysgenesis or agenesis is difficult to diagnose, and this can also contribute to its perceived rarity, with only approximately 200 cases reported to date. Moreover, 50% of affected patients have congenital vaginal agenesis [8].
In terms of treatment and prognosis of these two obstructive MDAs, treatment for vaginal agenesis is reconstructive vaginoplasty, after which, patients can plan a pregnancy [9]. However, the classical treatment of congenital cervical agenesis or dysgenesis has been total hysterectomy due to the increased risk of infection, sepsis, and even death after canalization surgery [10][11][12][13][14]. Nonetheless, many cases of successful reconstructive surgery, even with vaginal [15] or minimally invasive laparoscopy [16,17], and some instances of successful pregnancies and deliveries after surgery in patients with cervical agenesis or dysgenesis, have also been reported [18]. Therefore, conservative surgical management, such as uterovaginal or uterovestibular anastomosis, has been considered as a treatment option by several authors [18,19]. However, consensus on the optimal treatment for these rare obstructive MDAs remains controversial.
An accurate diagnosis of the type of MDA is important to determine the appropriate treatment option, timing or type of surgery, and counseling about reproductive and sexual outcomes. Symptoms, physical examination, imaging modalities, including ultrasonography (US), magnetic resonance imaging (MRI), hysterosalpingography, and diagnostic operative procedures including hysteroscopy and laparoscopy, all help in the differential diagnosis of MDAs. In terms of imaging studies, US may be the first screening tool, and the introduction of three-dimensional US has increased the accuracy comparable to that of pelvic MRI; however, it is not very reliable in the diagnosis of cervical atresia or dysgenesis [20]. The gold standard imaging modality for MDAs is known to be pelvic MRI. MRI is a well-established, excellent diagnostic tool in the evaluation of the female pelvis and has also been a preferred imaging modality for pediatric patients when transvaginal or transrectal US is not available. Compared with US or computed tomography (CT), MRI provides greater tissue detail and focuses more on the contrast of soft tissue; therefore, MRI has been considered to be superior to US or CT for examining the cervicovaginal anatomy or lesions [21,22].
Interpretation of an MRI finding of a cervical anomaly is based on the contour and signal intensity of the genital tract in multiplanar images [21]. However, some discrepancies between MRI findings and surgical diagnoses have been documented, especially in cervicovaginal anomalies [23][24][25].
This study was, therefore, performed to evaluate the accuracy of MRI-based diagnoses for MDAs and to reinforce the importance of intraoperative biopsies for the exact diagnosis and selection of the proper surgical methods for obstructive MDAs. In this study, we performed the diagnosis and classification of MDA as per the guidelines of the American Society for Reproductive Medicine (ASRM), which remains the standard till date [26]. Based on our experience, we were able to reduce re-obstructions by applying appropriate reconstructive surgery according to an accurate diagnosis based on intraoperative biopsy results relating to whether the obstructive MDA was vaginal or cervical dysgenesis or agenesis.
Materials and Methods
A total of 463 cases of young women, aged 9-25 years, who underwent pelvic MRIs for various reasons from January 1995 to February 2019 at Seoul Asan Medical Center, were retrospectively reviewed. The indications for MRI were the clarification of imaging findings of suspected MDAs on US or CT in cases of clinical symptoms, such as amenorrhea, pelvic pain, dysmenorrhea, and dyspareunia. Patients were excluded from the study if they were not diagnosed with genital tract anomalies as determined by MRI. Among 463 patients, a total of 225 patients diagnosed with genital tract anomalies, as indicated by the final analysis using MRI, were included in the study. The final diagnosis of the MDAs, the postoperative diagnosis after hysteroscopic or pelviscopic reconstructive surgery, and the preoperative MRI findings in patients who underwent reconstructive surgery were compared. Patients with classical Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, with or without accompanying anomalies, were excluded. In this study, we specifically focused on the preoperative description of MR images that showed vaginal or cervical dysgenesis or agenesis. We compared the final surgical diagnosis of the obstructive MDAs with the preoperative MRI diagnosis to determine the accuracy of MRI in the diagnosis of obstructive MDAs.
MRI Assessment
All patients underwent MRI prior to surgery using 1.5T (Achieva; Siemens Healthcare, Erlangen, Germany) or 3.0T (Skyra; Siemens Healthcare or Ingenia, Philips Healthcare, Best, Netherlands) systems with a phased-array body coil. Sequence selection included axial, coronal, and sagittal T2-weighted images, T1-weighted images, diffusion-weighted images of the pelvis, and T2-weighted turbo spin echo images covering the abdomen and pelvis. Intravenous gadolinium administrations inducing T1-weighted, contrast images were also obtained. All MR images were preoperatively assessed by experienced radiologists at our hospital who were blinded to the surgical findings and final diagnosis. The features of the genital tract, including the congenital anomaly of the uterus, cervix, vagina, and ovaries, were also interpreted. The contour, shape, and uterine wall thickness, as well as the signal intensity of the hematometra or hematocolpos were described. The preoperative MRI diagnosis was compared with the final diagnosis after surgery.
Surgical Assessment
All patients were examined at the outpatient clinic before surgical assessment. The timing and method of surgical procedures were determined on the basis of the presumptive diagnosis, combined with the clinical diagnosis and imaging study results. All surgeries were performed by a single gynecologist (B.M.K.) with experience of more than 30 years in adolescent gynecology, in our adolescent gynecology clinic, designated as a training center for the surgery of congenital MDAs by the International Federation of Pediatric and Adolescent Gynecology (FIGIJ) in 2018. Surgeries performed on each case of the obstructive reproductive tract anomalies were as follows. Cruciate and ovoid incisions to the thin imperforate hymen were made to open the vaginal orifice. Continuous locking sutures with 2-0 Vicryl (Ethicon Inc., Somerville, NJ, USA) were used for hemostasis of the edge of the circumferentially incised hymen. The thin transverse vaginal septum could be directly resected followed by end-to-end anastomosis of the lower and upper vagina [27]. If the normal vaginal tissue with the thick septum was not enough, the septum was divided into a distal section in an "X" fashion and a proximal section in a "+" fashion [5]. Created leaflets were interdigitated to bridge a larger distance [5].
In cases of vaginal agenesis, the vagina was first gradually dilated with the Frank method, which was successful in creating a functional vagina in most cases, as reported by other researchers [28,29]. However, when the Frank method fails, surgical interventions, including the McIndoe technique [30] or Davidov surgery [31], were performed. In undistinguishable cases of vaginal or cervical dysgenesis, we chose the surgical approach. First, we obtained access to the lesion with the hematoma under US guidance; as soon as we noticed the old blood spilling from the lesion on aspiration, we performed an intraoperative tissue biopsy at the caudal leading edge of the hematoma mass for confirmation of the presence of vaginal or cervical tissue in six cases of obstructive MDAs. After confirming whether the tissue was from the cervix or vagina, we proceeded with the reconstructive surgery accordingly. In cases of cervical agenesis or dysgenesis, the atretic portion of the cervix was opened until the uterine cavity was encountered. The surgical technique required a complete dissection of the rectouterine and vesicouterine space to expose the vagina, allowing circumferential anastomosis of the vagina to the lower uterine segment.
In terms of the extent of tissue dissection, vaginal dissection with the Davidov method, a vaginoplasty for vaginal agenesis, was performed in the usual manner. However, in cases of cervical dysgenesis or agenesis, the vaginal lateral dissection was extended deeper, in a more lateral direction compared to that in vaginal agenesis, to decrease re-obstruction and avoid re-operation.
This study was approved by the Asan Medical Center Institutional Review Board (approval No. 2020-1035). Informed consents were waived by our Institutional Review Board because of the retrospective chart-review study design.
Statistical Analysis
All data are presented as frequencies and percentages. A chi-square test was performed to compare the proportions of the categorical variables between the two groups. A p-value < 0.05 was considered to be statistically significant. Statistical analyses were performed using R, a language and software environment for statistical computation (R Foundation for Statistical Computing, Vienna, Austria) [32].
Results
The mean age of the initial cohort (n = 463) was 18.05 ± 4.59 years. Among the 463 cases, 225 (48.6%) were interpreted as having genital tract anomalies on MRI. The mean age of the included patients (n = 225) was 17.30 ± 4.21 years. In terms of diagnosis, the MRKH syndrome was the most common anomaly (75 cases, 33.3%). Of the 75 cases, 42 (33.3%) had uterine didelphys, including OHVIRA, and 27 (12.0%) exhibited anomalies of the hymen and lower one-third of the vagina, including an imperforate hymen, distal vaginal atresia or agenesis, and a transverse vaginal septum (Table 1). Among the 225 cases, 105 (46.7%) underwent reconstructive surgery according to their diagnosis. In cases of MRKH, the Frank method was performed in 52 cases (69.3%) instead of reconstructive surgery. Table 2 shows each case of disagreement between the preoperative MRI findings and the final surgical diagnosis. We defined final surgical diagnoses on the basis of pathologic confirmation by intraoperative tissue biopsies at the caudal leading edge of the hematoma lesions and findings of diagnostic hysteroscopy and pelviscopy. Nineteen of the 225 cases (8.4%) showed a discrepancy between the MR interpretation and the final diagnosis. Among the considered patients, the outcomes of the MRI-based diagnosis did not match the final diagnosis in patients with cervical anomalies in cases 1, 8, 9, 11, and 13-19. The discordance rate for cervical or vaginal dysgenesis was significantly higher (57.9% (11 out of a total of 19 cases)) than that of other types of MDAs (4.08% (8 of 196 cases), p = 8.36 × 10 −9 ) (Table 2, Figure 1). For example, in Patient 15, preoperative MR images showed a clear cervical contour and were assumed to represent vaginal stenosis with a presumed hematocolpos (hematoma in vagina); therefore, a vaginoplasty for vaginal agenesis was planned ( Figure 2). However, an intraoperative tissue biopsy at the caudal edge of the hematoma lesion, producing presumptive vaginal tissue, revealed the presence of cervical gland and stroma material. Therefore, the final diagnosis of the case was changed to cervical dysgenesis ac- For example, in Patient 15, preoperative MR images showed a clear cervical contour and were assumed to represent vaginal stenosis with a presumed hematocolpos (hematoma in vagina); therefore, a vaginoplasty for vaginal agenesis was planned ( Figure 2). However, an intraoperative tissue biopsy at the caudal edge of the hematoma lesion, producing presumptive vaginal tissue, revealed the presence of cervical gland and stroma material. Therefore, the final diagnosis of the case was changed to cervical dysgenesis accompanied with vaginal agenesis. In addition, diagnostic hysteroscopy showed no resistance when the telescope entered the uterus-like lesion, in which this surgical finding also supported the final diagnosis, cervical dysgenesis accompanied with vaginal agenesis.
Figure 1.
A graph of the discordance between the magnetic resonance imaging-based diagnosis and the final diagnosis in patients with genital tract anomalies (* p < 0.001, chi-square test).
For example, in Patient 15, preoperative MR images showed a clear cervical contour and were assumed to represent vaginal stenosis with a presumed hematocolpos (hematoma in vagina); therefore, a vaginoplasty for vaginal agenesis was planned ( Figure 2). However, an intraoperative tissue biopsy at the caudal edge of the hematoma lesion, producing presumptive vaginal tissue, revealed the presence of cervical gland and stroma material. Therefore, the final diagnosis of the case was changed to cervical dysgenesis accompanied with vaginal agenesis. In addition, diagnostic hysteroscopy showed no resistance when the telescope entered the uterus-like lesion, in which this surgical finding also supported the final diagnosis, cervical dysgenesis accompanied with vaginal agenesis. (Table 2). However, the intraoperative biopsy, which was performed at the caudal leading edge (arrow), revealed the presence of cervical gland and stroma material. (Table 2). However, the intraoperative biopsy, which was performed at the caudal leading edge (arrow), revealed the presence of cervical gland and stroma material.
Discussion
Our study showed that the concordance rate between the clinical presumptive diagnoses and MRI interpretation was 81.9% (86 out of 105) for patients who underwent reconstructive surgery, and the discordance rate was 18.1% (19 out of 105). Eleven cases were misdiagnosed as cervical atresia in 29 patients with cervicovaginal atresia.
Clinical examination, US, MRI, hysteroscopy, and laparoscopy can help diagnose MDAs. There have been many studies evaluating the accuracy of MRI and US in determining the anomalies of the female genital tract. Earlier studies described MRI as a highly accurate tool for the evaluation of MDAs. Pellerito [33] reported an accuracy of 100% for MRI and 92% for US for the evaluation of MDAs in 12 cases. Pompili et al. [34] found a sensitivity and specificity of 100% using MRI for diagnosing MRKH in 56 patients. Preoperative MRI also produces excellent manifestations and accurate diagnoses in terms of the classification of oblique vaginal septum syndrome [35]. MRI was superior to US in diagnosing uterovaginal malformations in a subgroup of women with MRKH (n = 7) [36]. However, in another report, Soares et al. [37] described a sensitivity of 44.4% and a specificity of 100% for diagnosing uterine malformations with US. Lermann et al. [38] reported that a combination of clinical examination and US is as accurate as MRI alone. A high correlation between the diagnosis by MRI and surgical findings was demonstrated in previous studies. MRI results concurred with the diagnosis of 24 out of 24 cases of surgically proved anomalies and demonstrated a sensitivity and specificity of 100% in the diagnosis of a septate uterus [33]. Santos et al. [23] found that MRI was consistent with surgical findings in 88.5% of cases.
However, there has been a study revealing that the accuracy of MRI differs between uterine and vaginal anomalies [24]. An excellent agreement between MRI and clinical diagnoses in uterine anomalies has been noted; however, 2 out of 11 incorrect MRI diagnoses of vaginal anomalies were found in two menstruating, adolescent patients with cerebral palsy. In both patients, the diagnosis of vaginal stenosis was inferred from the presence of a vaginal cavity distended with urine due to spasticity of the pelvic floor. There was uterine didelphys with an obstructed left hemivagina case on the pelvic MRI, but the surgical finding was an obstructed, non-communicating left rudimentary horn [23]. Assessment of the cervix and vagina on MRI is not usually requested because they can be examined clinically through direct visualization and misdiagnosed on MRI, as has been indicated in these reports.
Thus far, no study has reported on the correlation between the MRI and final diagnoses using an intraoperative tissue biopsy for the confirmation of cervical or vaginal tissues in cases of presumptive cervicovaginal dysgenesis. The emphasis of this study is that reconstructive surgery should be planned according to the pathologic confirmation of the tissue. Because there is a paucity of accuracy when diagnosing cervicovaginal anomalies in MRI, a precise diagnosis is required through the histological confirmation of the caudal leading edge during surgery. Based on the MR image, the biopsy should be performed at the leading edge of the hematometra or hematocolpos lesion, and according to the result of the pathology, the final diagnosis should be confirmed. Kimble et al. [39] also stated the limitations of MRI and described the histological specimen results in their two cases of partial cervical agenesis and complete vaginal atresia. The presence of the endocervix, but absence of the ectocervix, was shown in the histological specimen after hysterectomy [39].
Prior to discussion about surgical treatment options, we should be better informed regarding the differences in the tissue compositions of the cervix and vagina. Vaginal tissue is much more distensible, and therefore, a larger amount of hematoma can be retained. In contrast, the hard tissue nature of the cervix enables only a small amount of hematoma to be retained in the endocervical canal. This can lead to an earlier diagnosis of cervical or vaginal dysgenesis as there are earlier and more severe presentations of symptoms, such as cyclic abdominal pain, associated with even a small amount of hematoma, compared with a transverse vaginal septum or imperforate hymen, both of which manifest delayed symptoms. The tissue compositions of the uterine body and cervix are also different in that the uterine body is mostly composed of muscular layers, in contrast to the cervix, which is composed of collagen and elastin, and in which the muscle components are below 10%. In addition, some degree of hardness and resistance can be encountered in the narrow endocervical canal when the telescope enters into this canal during hysteroscopy. This may also explain why canalization techniques are less invasive [40]; however, they are linked to a high risk of restenosis of the cervix, up to 40-60% [6]. Hence, uterovaginal or uterovestibular anastomosis should take preference over canalization techniques for cervical agenesis or dysgenesis [15,18].
In terms of treatment options for vaginal agenesis, nonsurgical vaginal canalization is the first-line approach. Frank has described a conservative method of vaginal dilatation using Pyrex tubes [41]. Several surgical methods of vaginoplasty have also been developed. McIndoe used a skin graft with a mold to canalize the vagina, but it has been found that restenosis was a common complication [42,43]. In our experience, there have been no cases of postoperative re-obstruction in patients with vaginal agenesis who underwent lateral deep resection. The vaginal dissection was initially performed to enter the plane between the bladder anteriorly and the rectum posteriorly. The lateral wide, deep resection prevents re-obstruction after surgery (Figure 3).
Deffarges et al. [18] confirmed that cervical atresia was successfully treated by uterovaginal anastomosis even when associated with vaginal agenesis. An inverted U-shaped incision was made on the perineum or vaginal tissue for anastomosis with a pulled-down uterus [18]. Laparoscopically assisted uterovestibular anastomosis was also performed [44]. By preserving the uterus, this process allows the patient to become pregnant and have a successful pregnancy outcome. The uterovaginal anastomosis was performed in our patients with tissue-confirmed cervical agenesis or dysgenesis. Thus, the effective conservative treatment and proper surgical option can be achieved by accurate preoperative and intraoperative diagnoses. We also support conservative uterovestibular or uterovaginal anastomosis after confirmation of the presence of cervical agenesis with an intraoperative tissue biopsy, as there were no cases of infection, sepsis, or death in our institution [15]. To prevent re-stenosis after the surgery and preserve future fertility, we recommend more lateral deep dissection before anastomosis for cervical dysgenesis.
are linked to a high risk of restenosis of the cervix, up to 40-60% [6]. Hence, uterovaginal or uterovestibular anastomosis should take preference over canalization techniques for cervical agenesis or dysgenesis [15,18].
In terms of treatment options for vaginal agenesis, nonsurgical vaginal canalization is the first-line approach. Frank has described a conservative method of vaginal dilatation using Pyrex tubes [41]. Several surgical methods of vaginoplasty have also been developed. McIndoe used a skin graft with a mold to canalize the vagina, but it has been found that restenosis was a common complication [42,43]. In our experience, there have been no cases of postoperative re-obstruction in patients with vaginal agenesis who underwent lateral deep resection. The vaginal dissection was initially performed to enter the plane between the bladder anteriorly and the rectum posteriorly. The lateral wide, deep resection prevents re-obstruction after surgery (Figure 3). Deffarges et al. [18] confirmed that cervical atresia was successfully treated by uterovaginal anastomosis even when associated with vaginal agenesis. An inverted Ushaped incision was made on the perineum or vaginal tissue for anastomosis with a pulled-down uterus [18]. Laparoscopically assisted uterovestibular anastomosis was also performed [44]. By preserving the uterus, this process allows the patient to become pregnant and have a successful pregnancy outcome. The uterovaginal anastomosis was performed in our patients with tissue-confirmed cervical agenesis or dysgenesis. Thus, the effective conservative treatment and proper surgical option can be achieved by accurate preoperative and intraoperative diagnoses. We also support conservative uterovestibular or uterovaginal anastomosis after confirmation of the presence of cervical agenesis with an intraoperative tissue biopsy, as there were no cases of infection, sepsis, or death in our institution [15]. To prevent re-stenosis after the surgery and preserve future fertility, we recommend more lateral deep dissection before anastomosis for cervical dysgenesis.
This study has some strengths. It considers a relatively large number of cases of congenital uterovaginal anomalies through diagnosis to treatment. However, there are several factors that contribute to the discordance between MRI readings and surgical outcomes. This could be considered as a major limitation of the study. First, since cases of cervical dysgenesis are rare among MDAs, there are only few references for radiologists to carry out further analysis. Second, three-dimensional sequences are considered as the most effective approach for the diagnosis of MDAs; however, MRI is not a threedimensional imaging technique. Third, interpretational errors can be caused due to poor depictions of some structures on the MRI. These errors could be attributed to small size (e.g., atretic uterus), physiological status (e.g., collapsed vagina) because of a large amount This study has some strengths. It considers a relatively large number of cases of congenital uterovaginal anomalies through diagnosis to treatment. However, there are several factors that contribute to the discordance between MRI readings and surgical outcomes. This could be considered as a major limitation of the study. First, since cases of cervical dysgenesis are rare among MDAs, there are only few references for radiologists to carry out further analysis. Second, three-dimensional sequences are considered as the most effective approach for the diagnosis of MDAs; however, MRI is not a threedimensional imaging technique. Third, interpretational errors can be caused due to poor depictions of some structures on the MRI. These errors could be attributed to small size (e.g., atretic uterus), physiological status (e.g., collapsed vagina) because of a large amount of hematoma persistent for a long period of time, or deformations from previous surgeries (e.g., stenosis or adhesion formation). These factors could result in suboptimal signals, resolution, or visualizations on the MRI. Lastly, there could be an interobserver bias owing to the interpretation and experience of each radiologist. MRI is usually interpreted by two radiologists from the radiology department. One of the two radiologists takes the initial reading and then the senior radiologist, who specializes in pelvic radiography, reviews and provides a final diagnosis. The images from the radiology department are diagnosed in a pragmatic manner in most hospitals, and therefore attain excellent interobserver variability.
Conclusions
MRI is an excellent imaging modality for the diagnosis of MDAs. However, clinicians should be aware of this modality's limitations, especially in cases where cervical or vaginal dysgenesis is suspected. Intraoperative pathologic confirmation of the tissue through a biopsy of the caudal leading edge of the hematoma is often essential for the accurate diagnosis and proper surgical treatment of a congenital obstructive genital anomaly, especially in cases of cervical dysgenesis and vaginal dysgenesis. Informed Consent Statement: Patient consent was waived by our Institutional Review Board because of the retrospective chart review study design.
Data Availability Statement:
The excel data used to support the findings of this study were supplied by Sa Ra Lee under license, and requests for access to these data should be made to S.R.L. leesr@amc.seoul.kr. | 2021-06-03T06:17:22.907Z | 2021-05-29T00:00:00.000 | {
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24774418 | pes2o/s2orc | v3-fos-license | World Journal of Experimental Medicine Potential for a pluripotent adult stem cell treatment for acute radiation sickness
Accidental radiation exposure and the threat of deliberate radiation exposure have been in the news and are a public health concern. Experience with acute radiation sickness has been gathered from atomic blast survivors of Hiroshima and Nagasaki and from civilian nuclear accidents as well as experience gained during the development of radiation therapy for cancer. This paper reviews the medical treatment reports relevant to acute radiation sickness among the survivors of atomic weapons at Hiroshima and Nagasaki, among the victims of Chernobyl, and the two cases described so far from the Fukushima Dai-Ichi disaster. The data supporting the use of hematopoietic stem cell transplantation and the new efforts to expand stem cell populations ex vivo for infusion to treat bone marrow failure are reviewed. stem cells derived from bone marrow or blood have a broad ability to repair and replace radiation induced damaged blood and immune cell production and may promote blood vessel formation and tissue repair. Additionally, a constituent of bone marrow-derived, adult pluripotent stem cells, very small embryonic like stem cells, are highly resistant to ionizing radiation and appear capable of regenerating radiation damaged tissue including skin, gut and lung.
INTRODUCTION
Accidental radiation exposure and the threat of deliberate radiation exposure have been in the news and are a public health concern. This paper will describe the state of the art of stem cell treatment of acute radiation sickness. Acute radiation sickness is defined as "a combination of clinical syndromes occurring in stages during hours to weeks after exposure as injury to various tissues and organs is expressed" [1] . Experience with acute radiation sickness has been gathered from atomic blast survivors of Hiroshima and Nagasaki and from civilian nuclear accidents as well as experience gained during the development of radiation therapy for cancer. Based on these sources, an approximate dose threshold for each target organ (Table 1) and a time course of illness can be estimated ( Table 2).
Potential for a pluripotent adult stem cell treatment for acute radiation sickness EDITORIAL Note that bone marrow failure (infection, hemorrhage) is not the exclusive cause of death. High dose radiation can kill with cerebral edema and enteritis and pneumonitis, independent of infection. These syndromes are unlikely to be treatable with hematopoietic stem cell transplantation. Pluripotent (ability to differentiate into all three germ layers) stem cells with potential to regenerate multiple tissue types would add an important benefit for the treatment of acute radiation syndrome. Very small embryonic-like stem cells that can be obtained from adults in autologous cell-dose quantities offer such an advantage and are discussed in more detail latter in this paper.
The standard of care is described in the United States Armed Forces Radiobiology Research Institute's "Medical Treatment of Radiological Casualties" [2] . There are many complexities to caring for patients after radiation exposure. Patients who have been exposed to an explosion may have life-threatening injury not related to radiation exposure. Patients may be externally or internally contaminated with radioactive particles. Rapid and effective decontamination can prevent serious sequelae including bone marrow failure. Another complication is radiation induced emesis which can be dehydrating and limit the utility of orally administered countermeasures. Medical countermeasures for radiation exposure can be classified into 3 groups [3] : (1) Radioprotectants prevent radiation damage to cells (e.g., amifostine); (2) Radiation mitigators limit radiation damage (e.g., pentoxifylline); and (3) Radionuclide eliminators enhance excretion of radionuclides (e.g., Prussian Blue).
The aspects of acute radiation sickness for which hematopoietic stem cell transplantation is appropriate is amelioration of bone marrow suppression and immune suppression and tissue damage repair. This would fit the classification of "radiation mitigators" because it limits damage that has already occurred. For purposes of describing the value of hematopoietic stem cell transplantation in acute radiation sickness, patients who received between 2 Gy -10 Gy are recommended to be treated with white blood cell supporting cytokines, either G-CSF (filgrastim, peg-filgrastim) or GM-CSF (sargramostim). Cytokines are unlikely to be clinically useful in most cases where exposure exceeds 4 Gy. Patients for whom CSFs are unsuccessful are candidates for hematopoietic stem cell transplantation. The published data on the success of bone marrow transplantation following nontherapeutic radiation exposure include the experience of 13 Chernobyl victims described in the next section. In total reports from 58 people exposed to radiation in excess of 5 Gy, half of whom had an allogeneic transplant, revealed that only three of 29 patients transplanted were alive at one year post exposure. Deaths occurred due to the development of graft-vs-host disease and other complications unique to allogeneic transplant that could be avoided if autologous bone marrow or blood-derived stem cells were collected and stored before the exposure and used in place of allogeneic cells.
ACCIDENTAL (CIVILIAN) OR DELIBERATE (MILITARY, TERRORISM) RADIATION EXPOSURE
In addition to approximately 20 civilian and 60 military nuclear accidents, there have been 3 major nuclear accidents as of June 2011: Three Mile Island in the United States, Chernobyl in the former Soviet Union and Fukushima Dai-Ichi in Japan. In these accidents, it is very difficult to quantify the amount of radiation released, but some information is available on acute radiation sickness following the accidents. The Three Mile Island accident during which a portion of the nuclear fuel melted down in a TMI-2 reactor, but did not breach the containment walls, occurred on March 28, 1979. The widespread perception of great danger from this accident was based on expert's concern that the containment vessel might explode, widely distributing radioactive material. In fact, the containment vessel maintained integrity. Even though increased radiation levels were detected inside the plant and at least 50 workers were exposed, no acute radiation sickness from this accident has been reported [4,5] .
The Chernobyl accident on April 26, 1986 was much more serious with significant public health consequences. The difficulty in verifying documentation and medical records of the government of the Soviet Union makes an assessment of the extent of acute radiation sickness due to the Chernobyl accident impossible to reconstruct. Nevertheless, a comprehensive review of available information was published by the New York Academy of Sciences in November 2009 [6] . The lowest estimate of acute mortality from the Chernobyl disaster is 9000 victims [7] . Soviet physicians reported on 13 bone marrow transplantations for acute radiation sickness due to exposure at Chernobyl. Twelve of 13 patients had skin injuries resembling burns from 20%-100% body surface area in addition to decreasing white blood cell counts. Four of 8 patients with non-HLA identical donors received T-cell depleted bone marrow transplants. Only 2 of the transplant recipients survived to the 3 year follow up. The deaths reported were not attributed to prolonged neutropenia/infection or to thrombocytopenia/bleeding. Interestingly, two of the transplant recipients had evidence of transient engraftment with donor cells followed by recovery of autologous bone marrow [8] .
Most recently, on March 11, 2011, a magnitude 9 earthquake followed by a tsunami estimated at 14 meters high, destroyed part of TEPCOs Fukushima Dai-ichi nuclear power plant and resulted in several explosions. The International Atomic Energy Commission Briefing disclosed Fukushima prefecture received 1.5 microSv/h on March 31 over a natural background of 0.1 microSv/ h [9] . Two workers were reported to have received radiation burns to ankles when wading in contaminated water [10] . These are the only two cases of acute radiation sickness reported to date. In October 2011, a consensus document was published that includes additional individual case reports from sparsely documented historical civilian accidental exposures with the caveat that information from those reports was insufficient to guide future therapy [11] .
From a perspective on military use of nuclear weapons, the acute radiation sickness due to use of atomic weapons on Hiroshima and Nagasaki during World War Two has been reviewed [12] . survey of 1216 survivors of the blast in Hiroshima, sheltered in a building, revealed that 451 died on the first day and 201 died in the succeeding 2 mo, presumably from the hematopoietic component of acute radiation syndrome. Since transplantation had not been developed, there are no data on bone marrow transplant or stem cell treatment of acute radiation sickness after weapons discharge. It is the mortality figures from the Hiroshima and Nagasaki bombs that form the basis of military mathematical models to predict acute radiation sickness following nuclear weapons discharge.
The United States Health and Human Services' Office of Preparedness and Emergency Operations has made public the scenarios being used to prepare the United States. Two of these scenarios (#1 and #11) include nuclear weapons. These scenarios are being used to plan public health resource prioritizations and can be applied to estimate the number of patients who would potentially benefit from hematopoietic stem cell transplantation. National Planning Scenario #1 envisions a 1 KT nuclear detonation [13] . Col. Jarrett published an estimate of a 4 × 3 km oval that would receive 4 Gy from a 1 kT nuclear detonation [1] . Utilizing published population densities (New York City = 4500 people/km 2 and San Francisco = 5400 people/km 2 ), this area (9.4 km 2 ) would represent between 42 000 and 50 000 victims. National Planning Scenario #11 envisions a Radioactivity Dispersal Device ("Dirty Bomb") which would produce a "no entry" zone (> 1 Gy exposure) of 500 m in diameter (0.2 km 2 ). Utilizing the same published population densities as above, this would represent between 900 and 1080 victims [14] . These estimates demonstrate that even "small" events in a crowded environment may create enormous demands on the local medical system, and would probably exceed the capabilities of almost all facilities.
As discussed earlier, currently available treatment for radiation exposures of greater than 1 Gy are palliative. Hematopoietic stem cell transplantation to rescue patients for whom cytokine therapy failed has several limitations. The primary limitation is that the donor pool is limited by the need for at least partial HLA matching. As an example, the United States National Marrow Donor Program reports among 9 million donors, only 650 000 (7%) are African American, making bone marrow matching for African Americans difficult [15] . Similar problems probably exist for other under-represented ethnic groups. Once the hematopoietic transplant has engrafted, there is continuous need for immunosuppression. In addition to the risks of life-threatening infection during titration of immunosuppressant medication, some of these medications have dose limiting acute and chronic toxicity independent of graft-vs-host disease [16] .
Autologous hematopoietic stem cell treatment would solve the problems of immunosuppression and graft-vshost disease. If people at risk were to receive G-CSF mobilized cells collected prior to exposure than there would be sufficient cells available to prevent the profound cytopenia and immune suppression that follows exposure to 4 Gy or more of radiation. In addition, a small volume of bone marrow (100-200 mL) collected prior to exposure and then expanded ex vivo post exposure, may also be sufficient to reconstitute hematopoiesis and immune function. The major concern is whether hematopoietic stem cells capable of re-constituting the bone marrow could be expanded ex vivo from 100-200 mL, before bone marrow suppression became life-threatening. Clinical studies using marrow, mobilized blood and cord blood have demonstrated the feasibility of doing so [17][18][19] . Harvesting hematopoietic stem cells from damaged marrow is being done with complex protocols in cancer treatment. However, there are many reports of protocols failing to mobilize hematopoietic stem cells sufficient for reconstitutive use. For example, fludarabine exposure in adults with follicular lymphoma predicted a poor hematopoietic stem cell harvest evidenced by > 5 d apheresis requirement [20] . In a retrospective analysis of 204 patients, Ford et al [21] calculated that platinum based drugs and etoposide exposure were most highly correlated with poor hematopoietic stem cell mobilization as reflected by the absence of CD34+ cells on the first day that the white blood cell count was greater than 500. Stem cell mobilization was reported successful in only 12 of 20 (60%) patients with chronic lymphocytic leukemia [22] . These results suggest that chemotherapy treatment at a minimum impairs hematopoietic stem cell mobilization. In addition, a new study confirms expectations, that age between 65-69 years impairs hematopoietic stem cell mobilization relative to younger patients with the same disease [23] . In contrast, hematopoietic stem cell harvest in children is not limited by mobilization, but by scaling factors in extracorporeal volumes and anticoagulation necessary for the apheresis machine and vascular access for sufficient flow [24] . No reports of hematopoietic stem cell harvest from pregnant women could be found on PubMed search. Ford et al [21] did not find any correlation of poor hematopoietic stem cell mobilization with prior radiotherapy which gives these authors hope that victims of acute radiation sickness could have their hematopoietic stem cells successfully harvested. However, radiation damage may result in long term issues such as myelodysplasia and leukemia so use of cells previously stored and not exposed to radiation would appear optimal.
New mobilizing agents
New mobilizing agents are being developed to replace the colony stimulating factors. The hematopoietic stem Modified from [51] .
Burdelya et al [28] reported mouse radio-protective activity from a Salmonella enterica flagellin derivative given 1 h after radiation exposure and rhesus monkey protection when given 45 min prior to radiation exposure. Its putative mechanism of action is via toll like receptor 5 (TLR5) to nuclear factor-κB (NF-κB) signaling to multiple cytokines including G-CSF. This product is under active development by Cleveland Biolabs, Inc., (Buffalo, NY).
Parathyroid hormone (PTH) appears to mobilize stem cells to peripheral blood in mice with a distinct mechanism from G-CSF [26] . In a Phase 1 study in patients with at least one failed peripheral stem cell harvest attempt, the combination of PTH (teraparitide) for days 1-14 and G-CSF (filgrastim) for days 10-14 prior to apheresis resulted in 9/20 (45%) patients meeting pre-specified mobilization criteria. The authors note that this level of success was also seen in second mobilization attempts with a combination of filgrastim (G-CSF) and sargramostim (GM-CSF). A spontaneous observation study of patients with primary hyperparathyroidism showed CD45+/CD34+/c-kit+ and CD45+/CD34+/CXCR4+ bone marrow progenitor cells were increased relative to matched controls. Interestingly, the primary hyperparathyroidism patients had lower G-CSF dosing than controls, while stem cell factor and erythropoietin were not different between groups [29] . Another spontaneous observation study evaluated hemodialysis patients with varying levels of secondary hypo-and hyper-parathyroidism. In patients with high PTH, circulating hematopoietic stem cells were higher than controls or patients with normal PTH levels. In patients with low PTH, circulating stem cell numbers were lower than patients with normal or elevated PTH [30] .
Lastly, α-tocopherol succinate is being explored as a single dose inducer of endogenous G-CSF equivalent to a multiday course of G-CSF (filgrastim) [31] .
Induced pluripotent stem cells
Another source of cells to reconstitute radiation damaged bone marrow would be adult induced pluripotent stem cells (iPS). Recent laboratory work shows that fibroblasts can be induced to become pluripotent stem cells without using retroviruses, only using mRNA for key transforming factors [32] . Human iPS maintain a flat colony morphology in the laboratory when maintained with basic Fibroblast Growth Factor. Induction of iPS into hematopoietic stem cells covering all three lineages (myelopoietic, erythropoietic and thrombopoietic) has not yet been described. Current laboratory confirmation of pluripotency is the ability of iPS to form teratomas in vitro and in vivo. Though this is a large potential clinical problem, progress continues to be made and a recent report describes reprogramming skin fibroblasts from a patient with thalassemia (single gene mutation) [33] . Laboratory size colonies appeared in 3 wk following transfection with an engineered retrovirus and were induced to form "hemaglobinized" (HbF producing) colonies in another 2 wk using specially prepared growth media. No description of resulting myeloid or thrombopoietic cells were offered [33] . In addition, the "retro-differentiation" process currently requires approximately 100 adult cells to create 1 induced pluripotent stem cell (1% efficient) and the cells so produced exhibit early senescence [34] . Recently Zhao et al [35] have shown that iPS can be rejected due to abnormal gene expression in some iPS cells which can induce a T-cell-dependent immune response. These authors recommend that the immunogenicity of autologous cells should be carefully evaluated before these cells are considered for therapeutic purposes. So as of this writing, iPS are not on the near-horizon for clinical use in acute radiation sickness. Barriers that will need to be overcome to use iPS to treat acute radiation sickness include: production of all three bone marrow lineages from iPS; managing the risk of using oncogene sequences for induction or the identification of alternate induction techniques; improving the speed of hematopoietic stem cell production to a timeframe consistent with rescuing the patient from bone marrow failure; eliminating the risk of rejection; and scaling up production for mass casualties.
Mesenchymal stem cells
A commercial product of human mesenchymal stem cells (MSC) prepared from multiple bone marrow donors (Prochymal ® ) is being developed for acute Graftvs-Host Disease in bone marrow transplants. Since one lineage of MSC matures into bone marrow stromal cells, MSC have been considered as a supportive treatment for primary engrafting of bone marrow transplants. The initial publication in 2007 that human MSC home to radiation-damaged tissue in mice provided evidence of the potential for restorative therapy outside of the bone marrow [36] . Hu et al [37] demonstrated that MSC rescued fatally irradiated mice. Lange et al [38] extended the findings to show that MSCs effect rescue in fatally irradiated mice by anti-inflammatory and "hematopoietic stem cell niche modulating" effects such that endogenous hematopoiesis recovers. Thus, it is possible that MSCs may indeed have a beneficial effect in acute radiation sickness, however it is credible that this effect is due to paracrine, trophic and endogenous stem cell recruitment, rather than a regenerative capability. As the work of Heider et al [39] would suggest, bone marrow derived pluripotent stem cells give rise to MSCs and thus are supportive of the role of MSCs in hematopoiesis, but MSCs themselves are not regenerative.
In this setting, Osiris Therapeutics and Genzyme Corporation are collaborating to develop Prochymal ® for the treatment of acute radiation sickness. Current clinical trials in steroid refractory graft-vs-host disease, where patients have received significant radiation exposure to treat their underlying disease, will provide a human model on which to base animal studies of Acute radiation sickness [40] .
Myeloid progenitor cell product
Though not a stem cell product, CLT008, being developed by Cellerant Therapeutics (San Carlos, CA), is a multi-person sourced human cell population derived from donor bone marrow. This product is reported to have capability to mature into monocytes, neutrophils and red blood cells. Though it does not have the potential to mature into T and B lymphocytes, it is being considered as a temporary therapy until the host marrow recovers [41] .
Very small embryonic-like cells
Very small embryonic-like cells (VSELs) are pluripotent and present in many tissues and circulate in peripheral blood. The properties and therapeutic potentials of VSELs have been recently reviewed [42] . VSELs can differentiate into multiple cell types in vitro and in mice [43] . Kassmer et al [44] recently reported that bone marrow derived stem cells that were not hematopoietic were able to differentiate into type 2 pneumocytes in fatally irradiated mice. These small bone marrow cells were reported to be identical to VSELs (personal communication, DS Krause, 2010). VSELs have been documented to be present during routine bone marrow or hematopoietic stem cell harvesting [45] . Ratajczak and colleagues report that in addition to hematopoietic stem cells, VSELs are mobilized during burn injury [46] . This adds important information to the mobilization of VSELs during acute myocardial infarct and stroke in humans [47,48] . So it is likely that some spontaneous mobilization of VSELs will be happening during acute radiation sickness. Murine VSELs are highly radiation-resistant relative to a general population of hematopoietic stem cells, tolerating 1 Gy of γ radiation and retaining ex vivo pluripotent differentiating activity ( Figure 1) [43] . Also important is that the ex vivo expansion of VSELs requires only 5-10 d in culture [43] . Barriers that will need to be overcome to use VSELs to treat acute radiation sickness include: confirming radio-resistant characteristics of VSELs in humans; confirming that VSEL expansion using growth media doesn't activate oncogenes; and scaling up production for mass casualties.
CONCLUSION
Bone Marrow reconstitution as a partial treatment for acute radiation sickness has developed significantly since bone marrow transplantation was utilized for Chernobyl disaster victims in 1986. Use of autologous bone marrow or mobilized and harvested hematopoietic stem cells should eliminate the risk of graft-vs-host disease. The potential of autologous sourced stem cells is being evaluated now. Autologous cell sources include induced hematopoietic stem cells, induced pluripotent stem cells from adult differentiated tissue, MSC from bone marrow, myeloid progenitor cells from bone marrow, and VSEL stem cells from peripheral blood. Autologous human VSELs are emerging as fully functional stem cells that not only have wide-ranging regenerative competence, but have the critically important attribute of radiation resistance. The ultimate goal will be utilizing autologous, expanded stem cell infusions that would reconstitute many of the tissues damaged by radiation exposure. | 2022-06-22T21:10:54.963Z | 0001-01-01T00:00:00.000 | {
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118686825 | pes2o/s2orc | v3-fos-license | A Regularity Result for the Incompressible Magnetohydrodynamics Equations with Free Surface Boundary
We consider the three-dimensional incompressible magnetohydrodynamics (MHD) equations in a bounded domain with small volume and free moving surface boundary. We establish a priori estimate for solutions with minimal regularity assumptions on the initial data in Lagrangian coordinates. In particular, due to the lack of the Cauchy invariance for MHD equations, the smallness assumption on the fluid domain is required to compensate a loss of control of the flow map. Moreover, we show that the magnetic field has certain regularizing effect which allows us to control the vorticity of the fluid and that of the magnetic field. To the best of our knowledge this is the first result that focuses on the low regularity solution for incompressible free-boundary MHD equations.
Introduction
The goal of this manuscript is to investigate the solutions in low regularity Sobolev spaces for the following incompressible inviscid MHD equations in a moving domain: ∂ t u + u · ∇u − B · ∇B + ∇(p + 1 2 |B| 2 ) = 0, in D; ∂ t B + u · ∇B − B · ∇u = 0, in D; div u = 0, div B = 0, in D, where T (∂D) is the tangent bundle of ∂D, N is the exterior unit normal to ∂Ω t and c ≥ 0 is a constant. The first condition of (1.3) means that the boundary moves with the velocity of the fluid, the second condition of (1.3) means that the region outside Ω t is vacuum, where B · N = 0 on ∂Ω t implies that the fluid is a perfect conductor; in other words, the induced electric field E satisfies E × N = 0 on ∂Ω t . Also, the condition |B|= c on ∂Ω t yields that the physical energy is conserved, i.e., denoting D t = ∂ t + u · ∇, and invoking the divergence free condition for both u and B, we have: u · (B · ∇B) = 0.
Condition (1.4) was first discovered by Hao and Luo [16] when proving the a priori energy estimate for the free boundary incompressible MHD equations with H 4 initial data. Very recently, they proved that (1.1)-(1.3) is ill-posed when (1.4) is violated [17]. The quantity p + 1 2 |B| 2 (i.e., the total pressure) plays an important role here in our analysis. In fact, it determines the acceleration of the moving surface boundary.
History and background
In the absence of the magnetic field B, the system (1.1) is reduced to the free-boundary Euler equations which has attracted much attention in the past two decades. Important progress has been made for both incompressible and compressible flows, with or without surface tension, and with or without vorticity. Without attempting to be exhaustive, we refer [1,5,6,9,18,22,23,24,25,26,28,29,32,33,34,40,41,42] for more details.
On the other hand, the MHD equations describe the behavior of an electrically conducting fluid (e.g., a plasma) acted on by a magnetic field. In particular, the free-boundary MHD equations (also known as the plasma-interface problem) describes the phenomenon when the plasma is separated from the outside wall by a vacuum, whose motion can be formulated as the incompressible free-boundary MHD equations.
Although the MHD equations in a fixed domain have been the focus of a great deal of activities, e.g., [3,4,11,12,13,19,39], much less is known for the free-boundary case. The main difficulty is the strong coupling between u and B (i.e., the appearance of B · ∇B and B · ∇u terms in the first and second equations of (1.1), respectively). In fact, the appearance of the Lorentzian force term B · ∇B destories the Cauchy invariance, which provides good estimates for curl v when B is absent; indeed, one can see this by commuting the curl operator through the first equation of (1.1), which implies 1 Nevertheless, it is remarkable that the magnetic field B yields certain regularizing effect (cf. [38]), which can be derived from the transport equation of B (i.e., the second equation of (1.1)). Such regularizing effect plays an important role to control the full Sobolev norms of curl B and curl u and hence the full Sobolev norm of B and u via the div-curl estimate. We will provide more details on this in Section 1.3.
For the free-boundary MHD equations, the local (in time) well-posedness (LWP) of the linearized equations was studied by Morando-Trakhinin-Trebeschi [27], Secchi-Trakhinin [30] and Trakhinin [37]. For the nonlinear equations, Hao-Luo [16] proved the a priori energy estimate with H 4 initial data and the LWP was established by Secchi-Trakhinin [31] and Gu-Wang [14]. Also, we mention here that in Hao [15] and Sun-Wang-Zhang [35], the authors studied the a priori energy estimate and LWP, respectively, for the free-boundary MHD equations with nontrivial vacuum magnetic field.
In this manuscript, we establish the local a priori energy estimate with u, B ∈ H 2.5+δ with δ > 0 is arbitrary. This agrees with the minimal regularity assumption (i.e., H d 2 +1+δ , where d is the spatial dimension) that one may expect for the velocity field in the theory of the free-boundary incompressible Euler equations (see, e.g., [10,21,22]). In fact, Bourgain-Li [2] proved that the incompressible Euler equations with H d 2 +1 initial data are ill-posed even in the free space R d .
MHD system in Lagrangian coordinates and Main result
We reformulate the MHD equations in Lagrangian coordinates, in which the free domain becomes fixed. Let Ω be a bounded domain in R 3 . Denoting coordinates on Ω by y = (y 1 , y 2 , y 3 ), we define η : [0, T ] × Ω → D to be the flow map of the velocity u, i.e., ∂ t η(t, y) = u(t, η(t, y)), η(0, y) = y. (1.5) We introduce the Lagrangian velocity, magnetic field and fluid pressure, respectively, by v(t, y) = u(t, η(t, y)), b(t, y) = B(t, η(t, y)), q(t, y) = p(t, η(t, y)). (1.6) Let ∂ be the spatial derivative with respect to y variable. We introduce the cofactor matrix a = [∂η] −1 , which is well-defined since η(t, ·) is almost the identity map when t is sufficiently small. It's worth noting that a verifies the Piola's identity, i.e., Here, the summation convention is used for repeated upper and lower indices, and in above and throughout, all indices (e.g., Greek and Latin) range over 1, 2, 3. Denote the total pressure p total = p+ 1 2 |B| 2 and let Q = p total (t, η(t, y)). Then (1.1)-(1.3) can be reformulated as: (1.8) Remark. In above and throughout, the upper index of a represents the number of the rows whereas the lower index represents the number of the columns, i.e., a row column .
For the sake of simplicity and clean notation, here we consider the model case when where ǫ ≪ 1 and ∂Ω = Γ 0 ∪Γ 1 and Γ 1 = T 2 ×{ǫ} is the top (moving) boundary, Γ 0 = T 2 ×{0} is the fixed bottom. We shall treat the general bounded domains with small volume in Section 6 by adapting what has been done in [10]. However, choosing Ω as above allows us to focus on the real issues of the problem without being distracted by the cumbersomeness of the partition of unity. Let N stands for the outward unit normal of ∂Ω. In particular, we have N = (0, 0, −1) on Γ 0 and N = (0, 0, 1) on Γ 1 . In this paper, we prove: Let Ω be defined as in (1.9). Let (η, v, b) be the solution of (1.8) and δ ∈ (0, 0.5). Assume that v(0, ·) = v 0 ∈ H 2.5+δ (Ω) and b(0, ·) = b 0 ∈ H 2.5+δ (Ω) be divergence free vector fields and b 0 · N = 0 on ∂Ω. Let Then for sufficiently small ǫ, there exists a T > 0, depending only on N (0) and ǫ such that holds. Here, P is a polynomial of its arguments.
Remark. We will show that the physical sign condition (1.12) propagates within [0, T ]. In other words, it holds (1.12) 1.3 Strategy, organisation of the paper, and discussion of the difficulties Notations. All definitions and notations will be defined as they are introduced. In addition, a list of symbols will be given at the end of this section for a quick reference. Notation 1.3. We use P = P (· · ·) to denote a generic polynomial in its arguments. Now we can state the strategies we used and discuss the discovery and the difficulty in MHD system.
Gronwall-Type argument and div-curl estimates
The proof of Theorem 1.1 relies on div-curl type estimates of the velocity field v, the magnetic field b and the Lagrangian flow map η. In particular, let N (t) be defined as in Theorem 1.1. Then if vol (Ω) is sufficiently small (i.e., ǫ ≪ 1), there exists a T > 0 such that the estimate . This implies N (t) M 0 by a Gronwall-type argument that can be found in Chapter 1 of Tao [36].
Creation of vorticity by the magnetic field
The vorticity of the conducting fluid cannot be controlled analogously to that in the case of a non-conducting fluid due to the lack of the Cauchy invariance, since its derivation involves the derivative of the Lorentzian force (b 0 · ∂)b, which contributes to higher order terms. In particular, let ǫ µντ be the anti-symmetric tensor with ǫ 123 = 1. We have: where the last term in the second line is equal to is nonzero in general. We remark here that it is the Lorentzian force that causes the strong coupling between v and b. One can imagine that the Lorentzian force twists the trajectory of an electric particle in a magnetic field and produces vorticity even if the initial data is curl-free. However, we can control curl v and curl b from their evolution equation derived by taking the Eulerian curl operator to the first equation of (1.8). This will be dicussed in the following paragraph.
Regularizing effect of b: Controlling curl v, curl b and pressure Q The key to control v H 2.5+δ and b H 2.5+δ is to control B a v H 1.5+δ and B a b H 1.5+δ , where B a denotes the Eulerian curl operator, i.e., [B a X] λ = ǫ λτ α a µτ ∂ µ X α , where ǫ λτ α is the anti-symmetric tensor with ǫ 123 = 1. These quantities are treated straightforwardly for non-conducting fluids (i.e., Euler equations) thanks to the remarkable Cauchy invariance. We, nevertheless, have to control them differently since the Cauchy invariance fails for MHD equations due to the presence of the Lorentzian force term b β a µβ ∂ µ b. Inspired by Gu-Wang [14], one can derive the evolution equation for B a v and B a b. With the help of the following mentioned in Gu-Wang [14], one can rewrite the first equation of (1.8) as Now, one may apply the curl operator B a on both sides of (1.17) and get: which yields an evolution equation after commuting ∂ t and b 0 · ∂ on both sides of (1.18): This, in particular, yields an energy identity for B a v and B a b = B a (b 0 · ∂)η, i.e., 20) and it can be shown that E(t) verifies the following estimates by using Kato-Ponce inequalities (2.3) On the other hand, it is worth pointing out that the control of Q H 3+δ and ∂ 3 Q t L ∞ (∂Ω) (and hence Q t H 2.5+δ ) are both required. These quantities are needed even for the incompressible free-boundary Euler equations, whose a priori energy estimate can be closed by requiring η to be half derivatives more regular than v (see, e.g., [1,21,22]). In the case of a conducting fluid, i.e., MHD equations, we have to use the regularizing effect of the magnetic field (i.e., identities (1.16)) to show that η H 3+δ is still good enough to control Q H 3+δ and Q t H 2.5+δ . In particular, Q t satisfies an elliptic equation that involves b µ 0 ∂ µ a να ∂ ν ∂ t b α as part of its source term, whose H 0.5+δ norm requires η ∈ H 3.5+δ to control. However, this term can be avoided by invoking the identities (1.16) when deriving the elliptic PDE of Q t .
Remark. One may drop the requirement for η H s+0.5 when s > 3.5 using Alinhac's good unknowns thanks to the fact that ∂a ∈ L ∞ . We refer [14,16] for details.
Smallness of the fluid's volume is required: Nonlinear control of curl η
One needs to control curl η H 2+δ (and hence B a η H 2+δ ) to close the a priori estimate. This can be done in the case of a non-conducting fluid using the Cauchy invariance if one assumes ω 0 = curl v 0 ∈ H 2+δ (cf. [22]). This, again, fails for MHD equations. In order to control B a ∂η, one can only hope to use the multiplicative Sobolev inequality and Young's inequality with ǫ to derive the nonlinear estimate, which produces a term ǫ −1 P ( η(0) H 2.5+δ ). Therefore, we require the body of the conducting fluid to have small volume to fight the growth of the vorticity brought by twisting effect of the Lorentzian force (in other words, the strong coupling between b and v), otherwise the Gronwall-type argument no longer holds since it requires ǫ to be sufficiently small. The smallness of the fluid body can be propagated 3 if it holds initially since η is volume-preseving.
Organization of the paper:
The manuscript will be organized as follows. In Section 2 we record the preliminary estimates for the cofactor matrix a and its time derivatives. Also, the well-known Kato-Ponce commutator estimates are summarized as Lemma 2.3 for readers' convenience. Section 3 is devoted to control Q H 3+δ and Q t H 2.5+δ , which is required for the tangential estimate of v. In Section 4 we prove the tangential estimates for both v and b. Finally, in Section 5, we provide the control for the full Sobolev norms of v, b and η using a div-curl type estimate. Also, we show that the physical sign condition (1.12) propagates within a short period by showing that the quantity ∂ 3 Q| Γ1 is 1/4-Hölder continuous in time, which allows us to close the a priori estimates.
List of symbols:
• ǫ: A small positive constant which may vary from expression to expression.
• ǫ: The "height" of the fluid domain Ω, which is also chosen to be sufficiently small.
Preliminary Lemmas
The first lemma is about some basic estimate of the cofactor matrix a, which shall be used throughout the rest of the manuscript.
The next lemma reveals the regularizing effect of the magnetic field b; in particular, the flow map η is more regular in the direction of b 0 . It was also used in Wang [38] and Gu-Wang [14] Lemma 2.2. Let (v, b, η) be a solution to (1.8) with initial data (v 0 , b 0 , η 0 ). Then the following two identities hold: Proof. For (2.1), we multiply a να to the second equation of (1.8) to get 2), it can be easily derived by multiplying ∂ ν η β on the both sides of (2.1) and using a : ∂η = I.
The last lemma records the well-known Kato-Ponce commutator estimates, the proof of which can be found in [20].
Pressure Estimates
In this section we derive the estimates for Q H 3+δ and Q t H 2.5+δ . These quantities are both required in Section 4.
Lemma 3.2. Assume Lemma 2.1 holds. Then the total pressure Q satisfies: and its time derivative Q t satisfies: Proof: Applying a να ∂ ν to the first equation of (1.8), we have: where we have used (2.1).
4) with the boundary conditions
where the second condition can be rewritten as The standard elliptic estimate yields that Bounds for Q 1 : We have: where we used a H 1.5+δ η 2 H 2.5+δ and the multiplicative Sobolev inequality which is a direct consequence of (2.4) and the Sobolev embedding.
Bounds for Q 2 : Invoking Lemma 2.1 (7) and (2.4), we have: (3.10) and similarly via the multiplicative Sobolev inequality. We only write the first term and the others are treated similarly.
Summing up the bounds for Q 1 -Q 3 , then absorbing the ǫ-term to LHS, we conclude the estimates of Q as: Now we start to prove the estimates of Q t . Taking time derivative of (3.4), we obtain: (3.14) with the boundary conditions By the elliptic estimate, we have: Using this and the multiplicative Sobolev inequality the first two terms of (3.16) are treated as: Second, invoking (3.9) and Lemma 2.1 (7), the terms containing Q in (3.16) are treated as: 20) which can be controlled appropriately by the RHS of (3.2) by plugging in the estimate (3.1).
Now it remains to control the terms containing b in (3.16) (the last 6 terms). In fact, all the terms containing b can be controlled with the help of the multiplicative Sobolev inequality (3.18). The terms not containing b t are easier to control: For the terms containing b t , we have to put H 0.5+δ norm on ∂b t when we use the multiplicative Sobolev inequality (3.18), because we only have b t ∈ H 1.5+δ . This can be directly derived by taking time derivative of Therefore, Summing these bounds up, and absorbing the ǫ-term to LHS, we obtain:
Tangential Estimates
In this section, we establish the tangential energy estimate for the incompressible MHD equations.
. Then there exists a T > 0 such that for each t ∈ [0, T ], the estimate holds.
We prove this theorem by estimating v and b separately.
Tangential estimates of v
First, we derive the tangential estimates of v. To control I, we have: Control of I 3 : This is a direct consequence of the Kato-Ponce inequality (2.7), i.e.,
4)
Control of I 1 : We integrate ∂ µ by parts to get: For the second term in the last line of (4.5), we need to integrate 1/2-tangential derivatives by parts and then apply (2.4): (4.7) Summing these up, we have: Plugging this decomposition and the identity (which is obtained by differentiating a : ∂η = I) ∂ m a µ α = −a µ ν ∂ β ∂ m η ν a β α (4.10) into I 2 , we have: Here, R 1 is bounded by P ( η H 2.5+δ ) Q H 1.5 v H 2.5+δ via the multiplicative Sobolev inequality, while the last term in the third line of (4.11) can be controlled by using Kato-Ponce inequality (2.6) as: (4.12) It remains to control I 21 . Writing 2 m=1 S m ∂ m = S − S 0 , we have: It is easy to see the second term in (4.13) can be bounded by v H 2.5+δ Q H 1.5 P ( η H 2.5+δ ). For the first term, we integrate ∂ β by parts to obtain: 14) where the integral on Γ 0 vanishes because N = (0, 0, −1) and a 3 1 = a 3 2 = 0 on Γ 0 . Now, we bound I 211 by the Kato-Ponce commutator estimate (2.7), because we want to move the derivatives on v to a in order to control v. The term on the second line of (4.15) is controlled by (2.4) after integrating 0.5 derivatives by parts, i.e., (4.16) In addition, we apply (2.7) to the term on the third line of (4.15) and get: Therefore, Now we come to control I 212 . We shall compute its time integral, which then allows us to integrate ∂ t by parts to eliminate 0.5 more derivatives falling on v. Since N = (0, 0, 1) and Q = 1 2 c 2 on Γ 1 , we have a β α N β = a 3 α and a µ ν ∂ µ Q = a 3 ν ∂ 3 Q, and so: Invoking the physical sign condition ∂ 3 Q ≤ −ǫ 0 and Sobolev trace lemma, we have: Control of J: Now we start to control J. We first plug the identity (2.1) into J, then write J to be the sum of the highest order term and the commutator, which again can be controlled by Kato-Ponce inequality (2.3) The term J 1 cannot be controlled directly, but it actually cancels with the highest order term in the energy of b. We will see that in the next step.
Tangential estimates of b
We derive the tangential estimates of b in this subsection and then conclude the tangential energy estimates. Taking the time derivative of 1 2 Sb 2 L 2 and invoking the identity (2.1) and Kato-Ponce inequality (2.6), we have: Now we are able to see that J 1 cancels K 1 : Integrating ∂ µ in J 1 + K 1 by parts, we have dS(y) = 0. (4.24) Combining (4.2), (4.21), (4.22), (4.23), (4.24), we derive the tangential estimate as follows:
Closing the estimates
In this section we close our a priori estimate and prove the physical sign condition can be propagated to a positive time if holds for the initial data.
5.1
The div-curl type estimates H 2.5+δ -estimate of v and b: In this subsection we do the div-curl type estimate of v and b to derive the control of full H 2.5+δ norms. Although for Euler equations one can use the Cauchy invariance to give linear estimates for curl v and div v, there is no such analogue for MHD equations. Instead, inspired by Gu-Wang [14], we can derive the evolution equations of curl v to control the curl v and curl b simultaneously thanks to the identity b = (b 0 · ∂)η. Then we apply the div-curl estimate to derive the control of full H 2.5+δ norms of v and b.
The following notations will be adopted throughout: Notation 5.1. Let X = (X 1 , X 2 , X 3 ) be a vector field. We denote the "curl operator" and the "div operator" in the Eulerian coordinate by (B a X) λ = ǫ λτ α a µτ ∂ µ X α , and A a X = a µ α ∂ µ X α , respectively, where ǫ λτ α is the sign of the permutation (λτ α) ∈ S 3 .
Proposition 5.2. For sufficiently small T > 0, the following estimates hold: Proof. The divergence estimates are easy because A a v = 0 and A a b = 0, so: The estimates for curl v H 1.5+δ and curl b H 1.5+δ are more dedicate. Since and so it suffices to control B a v H 1.5+δ and B a b H 1.5+δ . As mentioned in the beginning of this subsection, we will derive the evolution equation for B a v and B a b: Plugging b β a µβ = b µ 0 and b α = (b 0 · ∂)η in the first equation of (1.8), and then applying the curl operator B a on both sides, we have: Commuting ∂ t and b 0 · ∂ with B a on both sides of (5.3), we have: Taking ∂ 1.5+δ derivatives, and then commuting it with ∂ t and b 0 · ∂, respectively, we get the evolution equation of B a v: Taking the L 2 inner product of ∂ 1.5+δ B a v and (5.5), we have: Integrating ∂ ν by parts in the second term on LHS, commuting (b 0 · ∂) with ∂ 1.5+δ B a and then invoking ∂ t η = v, we have: where the boundary term vanishes since b 0 · N = 0 on the boundary. The control of B 3 is straightforward by the multiplicative Sobolev inequality, say, We simplify the commutator term as follows: Invoking the Kato-Ponce commutator estimate (2.3), we can control B 22 as For B 21 , we have (5.11) where we used (4.10) to expand b ν 0 ∂ ν a µτ ∂ µ v α in the second line. Therefore, invoking b = (b 0 ·∂)η again, the L 2 norm of B 21 can be controlled by the multiplicative Sobolev inequality: It remains to control B 1 , specifically, we need to bound F L 2 given by (5.6). The first term is controlled by using Kato-Ponce commutator estimate (2.3). Silimarly as in (5.9), we have For the commutator term in (5.6), we can proceed similarly as in (5.11) to get (5.14) The remaining term in F can be easily bounded by P ( η H 2.5+δ ) b 0 H 2.5+δ v H 2.5+δ via the multiplicative Sobolev inequality.
Combining (5.9), (5.10), (5.12), (5.13) and (5.14), we have Therefore, invoking Lemma 2.1 (7), then absorbing the ǫ-term to LHS, we ends the proof by: For the tangential term, we apply Sobolev trace lemma to get: where the last term in (5.18) can be expressed using the tangential derivative of v by: Combining (5.2) and (5.20), and then absorbing ǫ v H 2.5+δ to the LHS, we have : The estimate of b H 2.5+δ can be derived exactly in the same way as v H 2.5+δ , so we omit the details.
In conclusion, we have proved : H 3+δ -estimate of η: We derive the H 3+δ estimate for η via the standard div-curl estimate: The divergence part is easy to treat owing to the div-free condition A a v = 0, i.e., the Eulerian divergence of v is identically zero.
Now it remains to control A a ∂η. We have: Therefore, it can be controlled as Summing up (5.25) and (5.26), then absorbing the ǫ-term to LHS, we get the control of div η: For the boundary estimate, we have: (5.28) Here we remark that the term ǫ0 2 a 3 α Sη α L 2 (Γ1) is exactly the boundary energy term derived from the physical sign condition in the tangential estimate.
It remains to control curl η H 2+δ , we start with Recall that the i-th component of B a ∂η (resp. (B I − B a )∂η) is of the form ǫ ijk a µj ∂ µ ∂η k (resp. ǫ ijk (δ µj − a µj )∂ µ ∂η k ). So we apply the multiplicative Sobolev inequality (3.9) to get: In addition, using multiplicative Sobolev inequality, Young's inequality and Jensen's inequality, we have: holds for sufficiently small t. Also, and hence (5.34)
The case of a general domain
In this section we show how to adapt the ideas used in the proof on Theorem 1.1 to the case of a general bounded domain with small volume. The physical situation we have in mind is that of a conducting liquid droplet with sufficiently small volume. We shall adapt the idea used in Section 12 of [10] to our case. The goal of this section is to prove: Let Ω ⊂ R 3 be a bounded domain with smooth boundary Γ, and denote by n the unit outward normal to Γ. Let (η, v, b) be the solution of and δ ∈ (0, 0.5). Assume that v(0, ·) = v 0 ∈ H 2.5+δ (Ω) and b(0, ·) = b 0 ∈ H 2.5+δ (Ω) be divergence free vector fields and a µ ν (0)b ν 0 n µ = 0 on Γ. Let Then if diam(Ω) :=ǭ ≪ 1/8, there exists a T > 0, depending only on N (0) and ǫ such that N (t) ≤ P (N (0)) for all t ∈ [0, T ], provided the physical sign condition holds.
Moreover, we must have R ≤ 4ǭ since both Φ and Ψ are volume-preserving diffeomorphisms.
The local Lagrangian map and the cut-off functions: Consider the Lagrangian map η : Ω → Ω(t), and setη = η • Ψ. Then ∂ tη = ∂ t η • Ψ = u •η, where u is the velocity of the moving domain Ω(t). In view of this, if we introducẽ then these new variables verify the incompressible MHD equations in the domain B R (0, 0, 1)∩ {z 3 ≥ 1}. We thus use suitably chosen cut-off functions to produce local estimate, passing to the global estimate by the standard gluing procedure. Let θ be a smooth cut-off function such that 0 ≤ θ ≤ 1 with θ = 1 inB R/5 (0, 0, 1) and supp θ ⊂ B R/4 (0, 0, 1). Therefore, extending all quantities to be identically 0 outside B R/4 (0, 0, 1) and since R ≤ 4ǭ, we may consider the equations and variables defined on the reference domainΩ = T 2 × (0,ǭ). This allows us to adapt the tangential energy estimates in Section 4, but all integrands should carry the cut-off function θ. Also, unlike Section 4, no integral over the lower boundary Γ 0 ofΩ is present since all variables vanish there in view of the way they have been extended.
The energy estimate: First, sinceη(0, z) = (z 1 , z 2 , z 3 + ψ(z 1 , z 2 )), a direct computation yields that at t = 0 we haveã In the proof of Theorem 1.1, for which ψ = 0, we used a(0) − I = O, where O stands for the zero matrix, to produce some small parameters (i.e., Lemma 2.1 (7)) in the energy estimates. We need ∂ψ to be small in order to apply the same argument here. This can be achieved since we may assume, without loss of generality, that ∂ψ(0, 0) = 0, and so the smallness of ∂ψ L ∞ (Γ) can be achieved by the mean value theorem possibly after reducinḡ ǫ, provided that ψ ∈ H 2.5+δ (Γ). We now apply the energy estimates of Section 4 with S· = ∂ 2.5+δ (θ·). (6.4) In order to simplify the exposition, we will omit tildes from all quantities and continue to label η, v, b, a and q, which are only locally defined Lagrangian flow map, velocity, magnetic field, cofactor matrix, and pressure, respectively. We start by differentiating Sv L 2 (Ω) , i.e., =: I + J. (6.5) To control I, we have: Control of I 1 : We integrate ∂ µ by parts to get Here and throughout, R contains error terms when the derivatives fall on θ, which can be controlled by the RHS of (6.16). Now, (6.8) I 113 can be controlled using the Kato-Ponce inequality. To do this, however, each separated term needs to be properly cut-off since the fractional derivatives destroy the compact support. Letθ be a smooth cut-off function such that 0 ≤θ ≤ 1 with suppθ ⊂ B R/3 (0, 0, 1) andθ = 1 on supp θ. The construction ofθ allows us to introduceθ without changing given expressions.
(6.20) There is no problem to control the second term in the first line of (6.20) and I ′ 212 is controlled analogous to I 212 in Section 4. For I ′ 211 , we write The first term can be treated similar to (6.12) by integrating 0.5-derivatives by parts. The second term is equal to The first line can be controlled similar to (6.11), and since so the second line can be bounded by C θ θ a 2 H 1.5+δ θ Q H 2.5+δ θη H 2.5+δ θ v H 2.5+δ .
Control of J + d dt Sb 2 L 2 : This follows from the what has been done in Section 4 except that the cancellation (4.24) holds up to a term of type R, which can still be controlled appropriately.
After covering Γ with finitely many balls, the procedure described above yields the tangential energy estimates near the Γ. We still need to cover the region of Ω not covered by these balls. However, we have no problem to cover this region using finitely many balls with radius r ≤ 4ǭ and again reducing the tangential estimates toΩ. In addition, there are no boundary integrals on either Γ 1 and Γ 0 .
Finally, we need to show that the estimates in Section 3 and Section 5 are still valid in each local coordinate patch. This follows from adapting the estimates in Section 3 and Section 5 to the MHD equations after commuting θ, i.e., T ] ×Ω; a µν b µ b ν = c 2 , Q = 1 2 c 2 , a µ ν b ν N µ = 0 on Γ ; (6.24) We can recover the equations for Q, Q t , B a v and B a b modulo error terms involving derivatives land on θ, but these contribute only to lower order terms. | 2019-04-10T21:14:19.000Z | 2019-04-10T00:00:00.000 | {
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246891733 | pes2o/s2orc | v3-fos-license | FICINO’S DOCTRINE OF LOVE AND BEAUTY AND ITS
This article analyses and compares the doctrines of love and beauty in the relevant works of Plotinus and Ficino, focusing on their allegorical use of the mythology of Aphrodite/Venus and Eros/Amor in their treatises On Love. As a translator and commentator of the collected works of his admired predecessors, Plato and Plotinus, Ficino did not hesitate to slightly bend, or even circumvent, some of their doctrines in his quest for the unification of the Platonic philosophy. Analysing the relevant passages from the Enneads, this article will demonstrate how Plotinus’ emanation theory, as well as his doctrines of love and intelligible beauty, have influenced Ficino’s interpretation of Plato’s doctrine of love, as well as his own exegesis of God’s love and creation.
Introduction
This article focuses on Ficino's creed of love which, typically, displays conjoined religious and philosophical traits. The historical background of his commentary to Plato's Symposium, bearing the title De amore liber, is a banquet, allegedly organised by Francesco Bandini at the instigation of Lorenzo de' Medici, at Careggi in 1468, with the intention of restoring the ritual symposia that used to be held every year on the day of Plato's birth (November 7 th which was believed to be also the day of his death). This Ancient Platonic tradition had been disrupted since Porphyry's time, 1200 years earlier, Ficino writes in the prologue. 1 The tractate, originally written in Latin, was later translated into Italian by Ficino himself. It shows the importance of the work and the author's desire to make it as accessible as possible. 2 It had, indeed, a widespread influence on the surge of similar tractates, following Ficino's example by spreading the doctrine of love. 3 Ficino's text follows that of Plato closely, in the form of seven discourses pronounced by Ficino's friends and colleagues. The work opens with a reverent mention of Plato, father of philosophers, but the content turns out to exceed the nature of commentary, rising to an original meditation on love inspired not only by Plato, but also by other philosophical, theological and mystical texts. It has been observed by modern commentators that Plato's Symposium is not a homogenous philosophical work, since many of its parts express the opinions of sophists and rhetors who do not share Plato's (or Socrates') philosophical beliefs. It would, therefore, be rash to attribute to Plato every statement given in these speeches. 4 Ficino's commentary, on the contrary, unfolds with invariable philosophical earnestness 5 aiming at uniformity of the Platonic doctrine. 6 Plotinus, of course, is prominent among Ficino's sources, and De amore is no exception, although it was published more than twenty years earlier than Ficino's translation of the Enneads. Nevertheless, scholars have made a strong case, proving that Ficino had been working on Plotinus for some thirty years before publishing the Latin Enneads and commentaries to them and, at least from 1460, when he first got access to the Greek manuscript, which he had transcribed, making a habit of marking it with numerous annotations. 7 It appears that Ficino paid particular attention to Plotinus' treatise On Eros (Enn. III 5(50)) of which two versions of Ficino's translation survive. Wolters has convincingly argued that Plotinus' doctrines, and not only the one in the 50 th tractate, but also other tractates, shaped Ficino's doctrine of love which, according to his arguments, should be understood in light of his 2 While this paper was in the making, Ficino's Italian version of De amore first saw light in Slovene translation of Mojca Mihelič, accompanied by a comprehensive study of Igor Škamperle. 3 For these works and their philosophical and literary value, see Panofsky (1972, 144 -146), andŠkamperle in: Ficino (2021, 180 -182), whose review of most notable authors also includes a prolific Croatian humanist Frane Petrić and his Amorous Philosophy (Amorosa filosofia) inspired by Ficino's dialogue. 4 Laurens in: Ficin (2012, XXVII). 5 As observed by Blum in: Ficino (2014, XVII). 6 A recent study on Ficino's hermeneutics, however, observes that Ficino's unitary approach to Platonic texts does not preclude the probability that his De amore was inspired by actual conversations that took place at Florentine Platonic symposia. The same study also points out that the dialogic roles were carefully distributed by the author in a manner that befitted both Ficino's contemporaries and Plato's interlocutors (Robichaud (2015, 115)). 7 Wolters (1986, 192), who also refers to Henry (1948). For evidence of Ficino's early familiarity with Plotinus, see also Corrias (2020, 16 -21). interpretation of Plotinus, even more than through his understanding of the original text of Plato. 8 With these arguments in mind, I shall proceed to the analysis of Ficino's theory of love and beauty: first, I shall compare the mythological symbolism of Venus and Amor in the work of both philosophers, and then I shall observe more closely some passages from the Enneads, the examination of which in some of the cases points to Ficino's deviation from Plotinus, while in others, it displays his profound knowledge and subtle understanding of Plotinus' philosophy that allowed him to attune the latter's doctrines to his own.
The Two Venuses and the Inner Tension of the Soul
The second book of De amore opens with the formulation of the Neoplatonic principle of emanation, according to which everything that steps forth, so to speak, out of the Good, turns back to its source, thus receiving being and formation. 9 Ficino, relying heavily on the Proclean Cylic theory of Causation, 10 marks this process with the three steps of processio, conversio and perfectio, which represent the beginning (Deus creat), the middle (rapit) and the completion (perficit), with reference to the mystical Pythagorean Triad and Orphic conception of the all-embracing Zeus, who is the beginning, the middle and the end (De amore II.1). The second, revertive phase (conversio / Deus rapit) has already been emphasised in the first speech where it is said that the angelic Mind is born, as its dark essence turns toward its source, God, like an eye which is directed toward the light of the sun. This first conversio is actually called "the birth of Love (amoris ortus)" (I.3). 11 On the other hand, the turn back towards the origins (epistrophé) was already an important part of Plotinus' love doctrine: it occurs at the level of the third Hypostasis, the Soul, represented by the heavenly Aphrodite, directing her love (éros) to her father, the source of her 8 Wolters (1986, 189). Between the two colossal enterprises of translating and commenting on the entire opera of Plato and Plotinus, Ficino apparently found ample time to write his own works and to study and translate other Ancient thinkers who shaped his spiritual world. Among these were many post-Plotinian Neoplatonists (Iamblichus, Proclus, Ps. Dionysius among others), which is why some scholars argue that Ficino's Platonism is closer to post-Plotinian Platonic tradition than Plotinus' thought (or Plato's, for that matter); see Celenza (2002, 77 -82). 9 Cf. Plotinus, Enn. V 4(7).2.4 -5. in V 1(10).7.5 -6. V 2(11).1.8 -12. For a detailed commentary on some of these passages, see Bussanich (1988, 7 -54). 10 See Proclus El. Th. 35, although Ficino seems to have followed Ps. Dionysius in his emphasis on the revertive phase of emanation (Gersh (2019, 30)). For a historical overview of this doctrine, see Dodds in: Proclus (1963, 220 -21). 11 The concept of conversion acquires a moral, as well as mystical significance in light of Ficino's conviction that the goal of Platonism is assimilation to God and that it is love that makes us godlike; see Robichaud (2015, 13 -17, 96, 120, and particularly 122 -129) for Ficino's adjustment of Plotinus' ethical doctrine. existence (Enn. III 5(50).2). Her act is presented as a loving contemplation with Eros as her "eye" and the crucial link that keeps the Soul in contact with the intelligible realm. The second Hypostasis -Intellect/Nousdoes not need such an eye, since it contemplates the One/Good within itself. However, even within the Intellect, a similar dynamic can be observed. P. Hadot explains the three "states" of Plotinus' Intellect (being, life and intellection) as the undeployed state (being, esse), the stepping out of itself (life, vivere), and the return to itself (intellection, intelligere). 12 With this trinity of being-life-intelligence, Plotinus enlivened the stately dignity of the self-contemplating Intellect and, moreover, he reaffirmed the basic principle of the Good, giving itself, and thus giving (metaphysical) birth to what comes after it. If intelligere is the key moment of the reversion towards the principle, vivere is the critical moment of the progression, i.e., of the continuance of the emanation, leading to the birth of the Soul and finally to the generation of the cosmos. This double act is comparable to the bending of a bow, for it produces a tension which preserves the status quo, that is the stillness of the intelligible world. While the life is drawn forth, outside, and to generation, the intelligence turns back to contemplation. Strictly speaking, the progression is not opposed by any revertive motion, but by metaphysical rest: for the new-born reality (the Soul) does not aim to return to where it came from (which would disprove the generosity of the Good), but to keep a certain balance by preserving the link to the principle without putting a stop to its progression.
In De amore II.7, the intelligible triad of being, life and intellection is said to reflect in the similar triad at the level of the World Soul: intellection, movement and generation, symbolised by the same triad of gods in both cases: Saturn, Jove and Venus.
From this scheme, it becomes evident that the aforementioned tension (between intelligere and generare, i.e., between contemplation of the higher reality and generation of lower things) is limited to the double figure of Venus. She is, so to speak, a personification of apparently opposing tendencies of turning to the source and proceeding to generation, while Saturn and Jove represent corresponding pairs of esse/intelligere and vivere/movere. She is the focus of duality, which has a rich philosophical, as well as cultural, background.
Ficino's explanation of the two Venuses forms part of his commentary on Pausanias' speech in Plato's Symposium, which introduces two Aphrodites accompanied by corresponding Eroses: the elder one is the heavenly Aphrodite, daughter of Ouranos, while the younger one is the popular Aphrodite, daughter of Zeus and Dione (Symp. 180d-e). Since the speech is essentially about Eros, the details on each Aphrodite come forth gradually and succinctly. One of the reasons may be that ancient readers were well acquainted with the double role of the goddess of love. A similar distinction can be found in the Symposium of Xenophon (8.9-10), who mentions both cults presenting the heavenly Aphrodite as patroness of pure love of souls and beautiful deeds, and the other one as champion of bodily love. Xenophon's differentiation does not diverge from Plato's by much, except that the latter views the Eros of the heavenly Aphrodite as strictly homoerotic, while the lower Eros is more corporeal and directed to women as well. This antithetic presentation of Eros does not exactly match the features of the cults of the two Aphrodites, which show no such clear distinctions. 13 Some studies, therefore, argue against a consistent, unambiguous binary perception of the concept of Aphrodite/Venus and Eros/Amor, since numerous texts display different dichotomies (between love, bodily and spiritual, adulterous and conjugal or, in our case, between intellection and generation): 14 in light of this argument, even the minor difference between Plato's and Xenophon's dichotomy is revealing. Regarding the Renaissance, it seems even harder to speak of an unequivocal symbolism of the heavenly and the popular (or terrestrial) Venus, particularly in art, 15 where Ficino's spiritual influence is sometimes said to surpass even his impact on philosophy. 16 As we shall see, Ficino influenced by Plotinus, availed himself of the duality of Venus and corresponding Amors to make further distinctions. 17 13 Pirenne-Delforge (1988, 147 -151). 14 For the Medieval perception of Venuses and Cupids, see Tinkle (1996, 9 -41). 15 In his studies on the Neoplatonic background of Renaissance masterpieces (Botticelli's Birth of Venus and La Primavera, or Titian's Allegory of Sacred and Profane Love, among others) Panofsky gives an interpretation of the two figures, nude and clothed, based on Ficino's doctrine of the two Venuses, at the same time pointing out the double symbolism of nudity which, in moralistic doctrines, is a sign of bawdiness and debauchery, while its theological meaning often points to the freedom from secular burden and, consequently, represents a higher principle (Panofsky (1972, 151 -60, andId. 1970, 188 -200)). See also Uršič (2002, 186 -189), who defends a singular Venus uniting human and divine, sensual and spiritual, against Gombrich's interpretation (1945Gombrich's interpretation ( , reprd. 1972) of Botticelli's Venus in La Primavera as supra-sensual, divine idea of humanitas. 16 Škamperle (2002, 69). 17 See, particularly De amore VI.5. and VI.8 and his Commentary to Plotinus' Enn. III 5(50).
As goddess of love and beauty, Aphrodite is always accompanied by Eros. In one of his earlier treatises, Plotinus used this companionship to reinterpret the myth of Eros and Psyche, by identifying Aphrodite with Psyche, and conclude that "every soul is Aphrodite". (Enn. VI 9(9).9.31) The complexity of Plotinus' interpretation of this mythological motif is evinced in the treatise On Eros (Enn. III 5(50)), where he uses it as part of his elaborate doctrine of the soul, to illustrate the tension between the third Hypostasis' (i.e., the Soul's) turning back to its principle and progression to the sensible cosmos (Ch. 3). In his study of the treatise, P. Hadot states the problem of Plotinus' allegoresis of the two Aphrodites in the following way: after presenting and rejecting the first possible interpretation, according to which the heavenly Aphrodite symbolises the higher part of the World Soul, which does not turn to this world but dwells separate from it, while the second Aphrodite represents its lower part, which revives and governs the cosmic body, Hadot endorses the second explanation which, outside of the wider context of Plotinus' doctrine of the soul, seems also more acceptable. According to this explanation, the heavenly Aphrodite represents the Soul-Hypostasis, intent on contemplating the intelligible world, while the other Aphrodite represents the World Soul, engaged in the governing of this world. Comparing Ficino's interpretation of this motif in De amore with that of Plotinus, Hadot concludes that the accepted explanation of Plotinus' allegoresis is also more congruous with Ficino's interpretation of the two Venuses. 18 Gersh's study of Ficino's commentary to Plotinus' fifth Ennead provides additional insight into the matter. His conclusions do not disprove Hadot's results, but his analysis of other relevant Ficino's texts neglected by Hadot (particularly his commentary to Enn. III 5(50)) reveals that Ficino's intricate structure of intelligible and psychic realms admits the second (rejected) interpretation of Hadot. 19 In comparing Ficino's and Plotinus' theory of the two Venuses/Aphrodites, we must observe the following divergence, pointed out by the two studies: that, despite the overtly Plotinian character of Ficino's precession and reversion, Plotinus' three Hypostases were subjected to slight modification. As is evident from Ficino's commentary to Enn. III 5 (Chapter 2), the third Hypostasisthe Soulhad been replaced by the Intellectual Soul (see the scheme below), which is the Soul descending directly from the Angelic Mind (the intellectus simpliciter) and existing at two levels: as intellectus quidam (particular intellect), at the higher one, and as anima simpliciter (absolute Soul), at the lower one. What Ficino's Intellectual Soul has in common with Plotinus' third Hypostasis is perfect independence from the sensible world and the fact that it is completely absorbed in the contemplation of the 18 Hadot in: Plotin (1990, 46 -61). 19 Gersh in: Ficino (2017, cxxxivcxxxviii This interpretation, presenting the first as well as the second Venus as two levels of the World Soul (her higher part linked to the Intellect, and the lower one to the body) seems, at first sight, to depart from the above given scheme from De amore II.7, as well as from a corresponding passage in VI.7, where the first Venus essentially represents mens angeli (Angelic Mind), and the second one mundi anima (World Soul). Trying to elaborate, Ficino adds that the First Venus is the intelligence inside the Angelic Mind, and this, as we have seen is the revertive phase of the Intellect corresponding to the Intellectual Soul of the scheme above: a Soul performing the act of intellection while being simply (simpliciter) a Soul, which corresponds to Plotinus' Soul-Hypostasis. This intricate interpretation can only be justified by the double nature of Venus, which first appears at the level of Intellect and then, by the trinitary analogy, resurfaces at the level of the World Soul.
Chapter VI.7 of Ficino's De amore reflects the attempt to unify the Platonic doctrine of love: even though the passage focuses on Diotima's myth of the birth of Eros, Ficino includes in it (as did Plotinus) Pausanias' distinction between the two Venuses (or the double Venus, gemina Venus). According to the scheme of II.7, the heavenly Venus, in this passage, still represents the intelligentia of the Angelic Mind. But, as the discourse in VI.7 continues, Ficino refines the meaning of the heavenly Venus: the intelligence of the Angelic Mind displays two distinct powers, potentia and vis intelligendi. Both Latin words mean "power", but the first one also means potency in the Aristotelic sense of unrealised possibility. 21 This is exactly the 20 The scheme follows Ficino's text, but the English expressions are taken from Gersh in: Ficino (2017 and 2018). 21 These two meanings are united in the Greek word dýnamis. It seems that Ficino tries to distinguish between the passive dýnamis of Venus (potentia intelligendi) and the active one, looking upwards (oculi vis, vis intelligendi).
meaning of the potentia intelligendi, which, according to Ficino, is by its nature formless and dark, and thus represented by Eros' mother, Penia (meaning "poverty"). Ficino is, therefore, explicitly introducing the element of privation at the level of the Angelic Mind. This does not mean that he disregards the essential characteristic, the fullness of the Neoplatonic Intellect, for the unrealised potentiality of Penia is promptly met by the riches of the Angelic Mind, represented by Poros. Nevertheless, Penia stands for the darkness and lack of form inside this potentia intelligendi, which is actualised only with the arrival of the ray of light (radius), represented by Poros. Does that mean that Ficino has departed from Plotinus? Not necessarily: he compares this dark and formless power to the "the eye's power before the arrival of the sun" 22 (vis oculi ante solis adventum), evoking Plotinus' metaphor of the "sight not yet seeing", which refers to the state of undefined desire of the Intellect trying to grasp the simplicity of its principle (Enn. V 3(49).11.4-5). There is another passage from Plotinus narrating the "birth" of the Intellect which, before actually becoming Intellect, "steps out" of the First Principle (= the One) as something other (állo): "This, when it has come into being, turns back upon the One and is filled, and becomes Intellect by looking towards it." (V 2(11).1.7-9). 23 This other shows the qualities, or rather the un-quality and lack of form of the indefinite Dyad and intelligible matter 24 which could be related to Plotinus' interpretation of Penia in III 5(50). Plotinus, however, does not speak of poverty in the intelligible world (the closest he comes to it is with the word "desire" in V 3(49)11.6), or "need" in VI 7(38).33.11) and he is always careful to stress the fullness of the Intellect (III 8(30).11.39). Also, in the tractate On Love, he directly connects Penia to the primary indetermination of Soul, not Intellect, before its turning back to the source (III.5(50).7). As matter, though an incorporeal entity, Penia is not included in Plotinus' hypostatic reality. She is mother of Eros and has nothing to do with the heavenly Aphrodite. It appears, then, that Ficino, while integrating Plotinus' process of emanation into his own metaphysics, took his interpretation of Penia and Poros to a higher level and did not hesitate to place Penia in the intelligible realm. This is evident also from other passages from Ficino's tractate referring to the darkness of the first substance (or essence) of the Angelic Mind (see De amore I.3 and V.11).
Divine Love and Transcendent Beauty
This makes us wonder what the balance of power is between Amor and Venus, which leads us to the question of priority in Ficino's metaphysical hierarchy. In De amore, amor seems to be at the top of this hierarchy. In the fifth speech given by Marsuppini, divine Love is, in a sense, older than the Angelic Mind, which God, the Father of everything, created "because of a certain love for the propagation of his own seed". (V.10) The divinus amor rises above all: not unlike Plotinus' Good, it proceeds to creation non coactus, sed sponte (V.11), and is older than Necessity, in which the Mind creates ideas. Ficino's generative first Love agrees with Plato's definition of Eros as love of "engendering and begetting upon the beautiful" (Sym. 206e), while Plotinus has replaced the first generation with procession (próodos), although he cannot always avoid expressions like "generation" and "creation", either. 25 Nonetheless, he is very careful in his formulation of the generative impulse of the One/Good. To Plotinus, the timeless generation is the result of an inner necessity of the Good, coinciding with its will. We could call it "natural", since it is in the nature of Good to give itself forth. 26 III 5(50), however, does not mention its love, for éros bears the Platonic connotation of poverty, and is therefore banned even from the level of the Intellect: as we saw, the first, heavenly Eros is born with the Soul-Aphrodite. Though it is the primal anagogical force, love is below Good and the Intellect.
There is love in Plotinus' Intellect, but of a different kind: in VI 7(38).35.19-25, it is an intoxication which, although referring to the description of the drunken Poros in Symp. 203b, 27 reflects rather the ecstatic rapture and divine madness described in Plato's Phaedrus (251a-b). Thus, the Phaedrus vocabulary is used by Plotinus to describe the mystical union of the Intellect with its principle. 28 The focus in the passage of VI 7(38) is evidently not on the generative or even less on the deficient trait of love. 29 The two concepts of Platonic love (fullness and deficiency) in Plotinus are also addressed by Ficino in his commentary to Phaedrus. Here also, Ficino puts love at the top of the metaphysical hierarchy, so that the supreme God appears inseparable from his own desire to create. 25 For example, próte hoîon génesis in Enn. V 2(11).1.7. 26 See, Enn. V 4(7).1. 27 -39. 27 See Lacrosse (1994, 97). 28 Lacrosse (1994, 28 -29). 29 In fact, in the treatise On Eros, Plotinus emphasizes this point by distinguishing between Poros' drunkenness and the fullness of the Intellect (III 5(50).9).
Love is also god, that is, the god in whose presence love flourishes. Or rather, he is the highest god himself, for just as he can be called beauty in Plotinus, being the fountain of beauty, he can also be called love as the author of the love pursuing beauty. For when Plotinus puts the will first, he is about to deny not love itself, but assuredly the love that is poor (In Phdr., Summa 12).
The reference to Plotinus is of double interest. Firstly, it addresses the concept of love at the level of the One. Ficino's rather abrupt transition to the will, which "Plotinus puts first", is evidently referring to the treatise, On Free Will and the Will of the One (Enn. VI 8 (39)). This is one of the most controversial of Plotinus' tractates for, at one point, speaking of the One, Plotinus relinquishes his habitual elliptical and apophatic discourse, and indulges in an "incorrect thinking" (Chs. 13-18), which is part of a methodology used deliberately to establish the perfect freedom of the First principle. It is in this context that Plotinus speaks of the love of One for itself: And he, that same self, is lovable and love and love of himself, in that he is beautiful only from himself and in himself. For surely his keeping company with himself could not be in any other way than if what keeps company and what it keeps company with were the one and the same. But if what keeps company is one with what it keeps company with and what is, in a way, desiring is one with the object of desire, and the object of desire is on the side of existence and a kind of substrate, again it has become apparent to us that the desire and the substance are the same (VI 8(39).15.1-8).
These words could easily be applied to the Intellect or intelligible Beauty, where the subject of yearning and thinking coincides with the object. But the context leaves no doubt that Plotinus is speaking about the One/the Good. The love of the One for itself is a daring concept since it exposes the highest Principle to the element of privation. Ficino, distinguishing between the "sated" love and the indigent éros, is clearly aware of this, when he points out that Plotinus denies the love that is poor, and therefore substitutes it with will. 30 But it appears that neither expression fits with what Plotinus had in mind: But he, since he has the highest place, or rather does not have it, but is himself the highest, has all things as slaves; he does not happen to them, but they to him, or rather they happen around him; he does not look to them, but they to him; but he is, if we may say so, borne to his own interior, as it were well pleased with himself, the pure radiance, being himself this with which he is well pleased (VI 8(39).16.9-12).
The expression used here (agapésas) is evidently metaphorical (hoîon hautòn agapésas). It does not express love for anything but itself and this love is clearly turned inwards. Rist has pointed out the development, in this treatise, of the doctrine of divine Eros, arguing that the love of the One for itself is to be understood as love for everything that is immanent in the Good 31 and that, in its natural yearning for It, reaches mystical union with It. 32 However, even if we do conceive the highest Love as turned to that which is in a sense within, it remains ostensibly non-creative and, most importantly, introvertive. The use of the word agapésas does stir interest, but it is impossible to prove that its meaning is much different from the éros used at the beginning of the Ch. 15. 33 Many scholars do not support the traditional distinction between the Pagan Platonic éros as desire, born from privation, and the Christian charity (agápe). 34 The only subtlety of meaning in the agapésas is the idea of pleasure, rather than love, charity or desire, which brings the word agapân closer to the verb hédesthai, used to express Intellect's happiness (see for example VI 7(38).31.5ss.).
Although I remain convinced that the personalised terminology of the 39 th tractate is to be taken as part of Plotinus' method of persuasion (VI 8(39).13.1-5), and therefore metaphorically 35 (Plotinus himself is very emphatic about it), I agree with Lacrosse that, to dismiss it as such, without placing it in the context of Plotinus' discourse on the One, would be superficial. 36 After all, Plotinus took pains to formulate the auto-erotic principle of emanation, and this formulation, together with the paradox of the One "being everywhere and nowhere" reflects, in many ways, the Platonic concept of Eros, as Lacrosse points out. 37 But no matter how we understand the pleasure of the One in Itself, Plotinus' divine éros is not given the 31 In the sense which is elaborated in the 22 nd tractate (Enn. VI 4(22).2 -3). 32 See Rist (1964, 78 -82). 33 See Corrigan and Turner in: Plotinus (2017, 328), who point out the blending of these two expressions (erân, agapân). 34 See Nygren (1953, 205 -207). For the critic of his thesis, see Armstrong (1961), and for the overview of the debate, Lacrosse (1994, 106 -107). For later merging of these two concepts, see Rist (1964, 78 -87). 35 For the detailed argumentation, see the note in Leroux in: Plotin (1990, 355 -358). 36 Lacrosse (1994, 110). 37 Lacrosse (1994, 118). prominent role of Ficino's procreative amor divinus as the driving force of the emanation. Plotinus' procession and reversion are reformulated by Ficino as the supreme Love, followed immediately by the reversive love. One should observe that these two forces do not correspond to the amores that attend the two Venuses, and of which it is the first one that turns back (contemplative love), and the second one that goes forth (generative love). These two amores are, in fact, just a reflection of a force that surpasses them both.
The claim that Plotinus calls the highest god beauty 38 is another challenging interpretation of Ficino that requires further clarification of Plotinus' view on the relation between the Good and the Beauty. In his early treatises, Plotinus did not distinguish sharply between them: "In a loose and general way of speaking the Good is the primary beauty" (see I 6(1).6-7 and 9.39s). In his later works, however, the difference is more pronounced, particularly in the treatise On Intelligible Beauty, where the highest beauty is firmly anchored in the Intelligible world, 39 while the One transcends everything, beauty included. Ficino's reference to Plotinus can be explained by a passage in the 38 th tractate where, speaking of the infinite first Principle, Plotinus calls it "beauty above beauty", and "generator" and "principle of beauty": […] so his beauty is of another kind and beauty above beauty. For if it is nothing, what beauty can it be? But if it is lovable, it would be the generator of beauty. Therefore, the productive power of all is the flower of beauty, a beauty which makes beauty. For it generates beauty and makes it more beautiful by the excess of beauty which comes from it, so that it is the principle of beauty and the term of beauty. But since it is the principle of beauty it makes that beautiful of which it is the principle, and makes it beautiful not in shape; but it makes the very beauty which comes to be from it to be shapeless, but in shape in another way; for what is called this very thing [shape,] is shape in another, but by itself shapeless. Therefore, that which participates in beauty is shaped, not the beauty (VI 7(38).32.29-39).
The next chapter, however, produces a vague reference to the transcendent object of soul's love and intellect's thought: 38 See the quoted passage from Ficino's Commentary to Phaedrus above (Summa 12). 39 See V 8(31).8.5ss., and even more so in V 5(32).12.
[…] if it (sc. the intellect) thinks an individual, it has one intelligible shape; if it thinks all together it has a kind of variegated shape, still in need [and trying to discover] how it should contemplate that which is above that which is all-beautiful and variegated and not variegated (VI 7(38).33.8-12).
The English translation is that of Armstrong, but the Greek original is ambiguous about whether the attributes "all-beautiful" and "variegated and not variegated" are describing the transcendent or the transcended. Modern translations do not agree on this point: the translation quoted above is evidently among those which connect all three attributes to the transcended, i.e. the intelligible beauty, which makes the transcendent the first Good. 40 A different reading of the text ("it is still in need, namely, of considering the Being beyond, the entirely beautiful, variegated and not variegated" in Plotinus, 2018 (ed. Gerson)) suggests that all three attributes should be applied to the transcendent and that the pánkalon, which is at the same time variegated and not variegated, is what the intellect is about to see. If this is the case, can we still speak of the transcendent as the first Principle, the Good? According to Bréhier, who also applies all three attributes to the transcendent, no: this beauty, variegated and not variegated, is still "au-dessous du Bien", 41 and the translation "the Being beyond" in Gerson's edition concurs with that. There is also Hadot's explanation, antithetical to Bréhier's: for him, the pánkalon, whose variety is without variety, represents the Intellect (l'Esprit) who must look beyond it to this other reality. Hadot does not comment on this realité, but it must refer to the Good. In his commentary, though, Hadot introduces two beauties: "la Beauté une et multiple des Formes", which is the beauty of the Intellect, and "la vraie Beauté" (Traité 38, p. 334), which is the object of contemplation of the Intellect in search of the Transcendent.
What seems to emerge from this conflicting interpretation is the concept of transcendent beauty (cf. "the beauty above beauty" in 32.29), not exactly the transcendent Good itself, but somehow transcending the intelligible beauty. To use Plato's 40 It seems to be the prevalent explanation, supported also by Henry and Schwyzer in: Plotinus (1983), as well as Igal in: Plotino (1985), Hadot in: Plotin (1988), Fronterotta in: Plotin (2007), and Radice in: Plotino (2002. Stern-Gillet (2000, 49 and 53), does not seem to have any doubts, either. Slightly different is the translation of Faggin in: Plotino (2000), who applies "variegated and not variegated" to the Good, but leaves out the pánkalon, and that of Harder in: Plotin (1964), who connects the first two attributes to all-beautiful and the not variegated to the Good. 41 In his note to translation (Enn. VI 2 , p. 106).
words, "the power of the Good seems to have fled into the nature of the Beautiful", 42 as this beauty is revealed further in the Ch. 33: These beautiful things, then, must be measured and limited, but not the really beautiful or rather the super-beautiful; but if this is so, it must not be shaped or be a form. The primarily beautiful, then, and the first is without form, and beauty is that, the nature of the Good.
Not pánkalon, but hypérkalon this time is identified as the nature of the Good. What carries this Beauty (kalloné) 43 above the intelligible beauty (kállos) is the fact that it is not only shapeless, but also formless (aneídeon), thus transcending the realm of intelligible Forms (eíde). This makes it more plausible that the "all-beautiful, variegated and not variegated" indeed refer to the beauty of the one-and-many intelligible realm.
Conclusion
This paper has sought to show, through these instances of Ficino's hermeneutics, that his attempt to bring Plotinus' concept of Love and Beauty closer to what he believed to be Plato's doctrine, and which was in many ways his own, sometimes led him to depart, in some points, from Plotinus and his doctrine of emanation. Nonetheless, his reformulation of the Neoplatonic procession remains solidly grounded in the Enneads, to which he keeps referring even in his commentaries on Plato. This not only confirms that his knowledge of Plotinus went alongside his study of Plato and other authors, but also gives full credit to his role as translator of, and commentator on, Plotinus' corpus. | 2022-02-17T16:16:34.527Z | 2022-01-18T00:00:00.000 | {
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245124335 | pes2o/s2orc | v3-fos-license | Integrands of Less-Supersymmetric Yang-Mills at One Loop
We construct a prescriptive, bubble power-counting basis of one-loop integrands suitable for representing amplitude integrands in less-supersymmetric Yang-Mills theory. With the exception of massless bubbles, all integrands have unambiguous, leading singularities as coefficients defined in field theory; for the massless bubbles on external legs, we find two natural choices which lead to different integrands that highlight distinct aspects of field theory. For concreteness, we give the all-multiplicity integrands for MHV amplitudes, and the split-helicity amplitude integrand for six-particle NMHV. The basis we construct is mostly pure and is divided into to separately UV- and IR-finite sectors of fixed transcendental weight, resulting in UV- and IR-finite ratio functions of n-particle helicity amplitudes.
Introduction and Overview
Important recent progress in our understanding of scattering amplitudes in quantum field theory originated from considering the structure of loop amplitudes at the level of the integrand -the unintegrated sum of Feynman diagrams, whose analytic structure is determined by unitarity in terms of on-shell processes. In particular, these investigations at one loop led directly to the discovery of BCFW tree-level recursion relations [1,2], dual conformal-(and ultimately Yangian-)invariance of planar maximally supersymmetric Yang-Mills theory [3][4][5][6], and the correspondence between leading singularities and subspaces of Grassmannian manifolds [7].
The origins of generalized unitarity [8][9][10] are extremely simple to understand: loop integrands, being rational differential forms on the space of loop momenta, can be expanded into a basis of such forms with coefficients that are loop-momentum independent. For any process in any particular quantum field theory and at any fixed loop order and spacetime dimension, the space of all scattering amplitude integrands (arbitrary multiplicity and external particle content) spans a finite-dimensional space of 'master' integrands. Once these integrands are integrated they can be recycled for arbitrary scattering amplitudes of interest in the theory.
A familiar illustration of the power of this idea is the 'no-triangle property' for amplitudes in maximally supersymmetric Yang-Mills and gravity at one loop [11][12][13][14]. Specifically, this means that all amplitudes in these theories are expressible in a basis of 'scalar box' integrals (those that scale like four propagators at infinite loop momentum). This basis was called B (4) 4 in ref. [15], and it is a classic result of Passarino and Veltman [16] that all one loop integrals involving more than fourpropagators can be expanded into those with four or fewer. Thus, at one-loop in these theories, the scalar box integrals suffice for representing all scattering amplitudes.
More generally, the size of the basis required to represent amplitudes in a quantum field theory remains an important and open question. For example, it is known that scattering amplitudes in both the Standard Model and pure Yang-Mills are expressible in terms of the basis of integrands B 0 -integrands that scale like a loopindependent constant at infinite momentum-which is the basis described by OPP in ref. [17,18]; but it is not known whether this is the smallest space of loop integrands needed to express amplitudes in these theories.
In this work, we consider the case of one-loop amplitudes in less-than-maximally supersymmetric (1 ≤ N < 4) Yang-Mills theory ('sYM N '). We show that these amplitudes can be expressed in the space B (4) on-shell scattering states in terms of helicity super-multiplets [20]. In the planar limit, we expect a well-defined notion of the integrand due to the fact that planarity, or equivalently (leading) color ordering, induces a fixed cyclic ordering of the external momenta of the scattering states, which in turn allows us to define unique labels for the loop-variables to any order in perturbation theory. These variables are given either by choosing an origin of loop-momentum space, going to dual coordinates [5], or (in strictly four spacetime dimensions) introducing momentum twistors [21], all of which have played a major role in recent developments for maximally supersymmetric amplitudes, and beyond. One key advantage of the global labels that originated in N =4 sYM arises from multiple different definitions of the integrand, either in terms of a standard diagrammatic representation or via loop-level on-shell recursion relations [22]. For less-supersymmetric amplitudes in the planar limit, these recursion relations should exist, but are associated with various subtleties [23].
One goal of this work is to uniquely define the one-loop integrands for less than maximally (1 ≤ N ≤4) supersymmetric Yang-Mills theory (the pure Yang-Mills case has new features which we leave for future work). The situation is significantly different from the case of maximal supersymmetry because of the presence of poles at infinity as indicated by having triangles and bubbles in the one-loop expansion. We show that the standard cuts considered in the context of generalized unitarity fix the integrand up to massless bubbles contributions. These terms integrate to zero but are nevertheless important at the integrand-level; and we illustrate two choices of contours which can be used to fix their coefficients.
Organization and Outline
This work is organized as follows. In section 2, we review the ingredients of basisintegrand construction and the role of prescriptivity [19]. We describe our particular choice of basis for B (4) 2 in section 2.2, and highlight how it is stratified by its UV/IR structure and its transcendental weight in section 2.2.3.
Because the basis we construct is prescriptive, the coefficient of every integrand is a 'leading singularity' in field theory: i.e. the integral of the amplitude along some particular compact contour (at one loop, always a 'residue'). In less-than-maximally supersymmetric Yang-Mills theory, leading singularities require more information to specify than in N = 4 sYM. We review these ingredients in section 3. In particular, we find that one loop amplitude integrands in sYM N can be represented as a combination of the corresponding amplitude integrands in N = 4 sYM (which have better powercounting), plus corrections involving only those diagrams with so-called 'non-singlet' helicity flow. In section 3.2, we discuss some subtleties that arise in the case of leading singularities associated with massless bubble integrals, and suggest two natural paths to defining a unique integrand.
In section 4, we apply our diagonalized bubble power-counting basis of integrands to write down a closed formula for all-multiplicity MHV amplitudes in section 4.2.
We further illustrate these ideas with a particular six-point NMHV amplitude integrand in section 4.3, and discuss how this representation of amplitudes manifests the finiteness of many observables in these theories before concluding in section 5.
Finally, in appendix A we provide full details for our integral basis, and each basis element's result from loop integration. These results, as well as the all-multiplicity MHV amplitude integrand, are also provided as ancillary files attached to this work.
A Prescriptive, Bubble Power-Counting Basis at One Loop
The fundamental principle behind generalized unitarity [8][9][10] is that loop amplitude integrands A are elements of a vector space of differential forms on the space of loop momenta; as such, they may be expanded into a basis B (large enough that where the coefficients c i are loop-momentum-independent 'on-shell' functions determined by generalized unitarity: i.e. the left and right-hand sides of eq. (2.1) agree on all contour integrals which 'encircle' loop-dependent Feynman propagators. In principle, an arbitrary spanning set of Feynman integrands (rational differential forms involving some number of Feynman propagators and arbitrary functions of loop momenta in the numerators) can be chosen for a basis in (2.1). In this case, the determination of the coefficients c i amounts to a problem of linear algebra: suppose that one has some spanning set of integration contours {Ω j } on which the period matrix were known or determined to be full-rank. Then the coefficients of amplitudes a i would be determined by the system of equations Typically, the cycles chosen to determine coefficients are those involving as many 'residue' contours as possible-those which encircle a number of Feynman propagators, poles at infinity, collinear regions, and so-on. Because these contours enclose physical poles, the periods of amplitude integrands a j defined in (2.3) are called leading singularities [24] and can be determined in terms of on-shell (tree) amplitudes. The story of these coefficients is one with a very rich history. Setting aside the potential computational complexity involved in inverting the period matrix M i,j defined in (2.2), it is worth emphasizing that most seemingly natural choices for bases of master integrands (those involving some Feynman graph's worth of propagators and a spanning-set of 'Lorentz-invariant scalar products' in their numerators) lead to very poor integrals-ones that can deeply obscure many interesting and important features of scattering amplitudes. Thus, it is worthwhile to try and find a good set of integrands for any basis.
Brief Review of Prescriptive Integrand Bases for Amplitudes
A prescriptive integrand basis is one chosen to be cohomologically dual to a spanning set of maximal-dimensional compact contours of integration. That is, a basis is prescriptive provided that there exists a set of compact, maximal-dimensional integration contours {Ω j } such that When this is the case, the coefficients c i of the amplitude integrand (2.1) are leading singularities of field theory because the inversion of the period matrix (2.2) is trivial: Prescriptive integrand bases have been shown to possess many desirable properties. In particular, they often evaluate to pure functions (those satisfying nilpotent systems of differential equations, see e.g. [25,26]), and hence are comparatively easy to integrate. To be clear, prescriptive integrand bases are fairly straightforwardly constructed. Starting from an arbitrary basis of loop integrands B 0 and an arbitrary spanning-set of contour integrals {Ω j }, a prescriptive basis can be obtained according a simple 'rotation' of the basis: It should be clear how important the role of the cycle basis is in the above discussion: different choices of contours {Ω j } can result in strikingly different bases of integrands. Thus, there is relatively little uniqueness here. For our particular purposes in this work, we chose a maximal subset of contours to expose IR and UV divergences, resulting in a basis stratified by divergences. As stressed previously, this choice is by no means unique and one could think about alternative bases inspired by other physical or mathematical properties.
In what follows, we review the elements involved in defining a particular set of Feynman integrands for a basis-as defined by (some proxy for) 'power-counting'. Then we illustrate the kinds of choices made for a dual set of cycles, and how these choices affect the resulting integrand basis.
Defining a Bubble Power-Counting Basis B
(4) 2 As described in ref. [15], one can construct a basis of 'bubble-power-counting' integrands at one loop as follows. Start with any Feynman graph involving some number of p ≥ 2 propagators and consider the vector space of loop-dependent polynomials in the numerator (2.7) That is, [ ] q represents that linear span of all q-fold products of inverse propagators. Thus, the space of B 2 is defined as the linear span of all Feynman integrals with p propagators and a product of p−2 inverse propagators in the numerator. Graphically, if we use to denote the vector space of inverse-propagators times some propagator, then As described in ref. [15], this space is finite dimensional for any fixed spacetime dimension (or multiplicity). In four dimensions, all integrands involving more than four propagators are expressible in terms of those with four or fewer. In particular, the basis B 2 is spanned by the following vector spaces of loop integrands: (2.10) Throughout this work, we always leave implicit the factor of 'd 4 ' in these integration measures. For each set of leg distributions, these spaces of integrands have rank (in four-dimensions) of 20 = 2+18, 6 = 3+3, and 1, respectively. What we mean by this, for example, in is that the 6-dimensional vector space [ ] 1 of loop-dependent numerators for the triangle integrands can be viewed as spanned by 3 'top-level' degrees of freedom and 3 contact terms-spanned by the inverse-propagators appearing in the graph. Similarly, of the 20-dimensional vector space [ ] 2 of numerators for the box integrands, all but 2 can be spanned by contact terms: 4 2 = 6 double-contact terms (with one degree of freedom each), and 4 1 = 4 single contact terms with 3 top-level degrees of freedom each. Labeling only the top-level degrees of freedom for each denominator topology (those numerators not spanned by the contact terms of the integral) , our bubble power-counting basis consists of 2 numerators per box, 3 numerators per triangle, and a single numerator per bubble, denoted by I i A,B,C,D , I I A,B,C , and I A,B , respectively. We may represent each of these integrands graphically as follows: where i ∈ {1, 2} indexes the top-level degrees of freedom of each box, and I ∈ {1, 2, 3} indexes the top-level degrees of freedom of each triangle. To be clear, the sets {A}, . . . represent arbitrary non-empty collections of external momenta flowing into the vertex, with p A := a∈A p a and s A := p 2 A = ( a∈A p a ) 2 . Later on, we will have reason to distinguish between sets of external momenta that are 'massive' (sets consisting of more than one massless leg) from those which are massless. When {A} consists of a single element, we will denote it by a:= {a} = {A}, and similarly for the other momenta labels. More generally, we refer to 'a' as the first label in the set {A}:= {a, . . .}, and so-on. Due to our focus on planar (color-ordered) amplitudes, the sets are endowed with a natural ordering of external legs.
To determine the specific numerators for the basis, we start from a spanning set of contours and fix the precise numerators according to the prescriptivity condition (2.4). It is worth emphasizing how the particular numerators are chosen using these conditions. For example, in the case of box-integrands, two particular numerators are chosen not simply by the condition but also by the requirement that it vanish on all triangle-topology contours of its contact terms (2.14) Thus, of the rank([ ] 2 ) = 2+18 degrees of freedom required to specify the basis numerators n i A,B,C,D , only 2 are fixed by (2.12), while 3 × 4 of the remaining degrees of freedom are determined by (2.13) and 6×1 are fixed by the analogous equations (2.14) for bubble contact-terms. This is what we mean by saying that an integrand basis B (4) 2 is dual to a spanning set of particular cycles. Of course, in order to construct specific integrand numerators, we must specify the contour conditions which define the basis prescriptively as described above. We do this in the following subsection. However, it should be clear that, independent from the precise contour definition, scattering amplitude integrands in this basis will be represented according to To be any more specific, we must specify the contour conditions which define our basis prescriptively.
A Spanning Set of Maximal-Dimension Contours
It is interesting to note that the basis of bubble power-counting integrands in four dimensions can be viewed as B 2 . That is, we may consider the new integrands in B to be those associated with a bubble power-counting basis in three dimensions-merely reinterpreted in four dimensions. This is also motivated by the fact that all the new integrals required have less than maximal transcendental weight when integrated in 4D, but would be of maximalweight in 3D; these weight drops are related to the presence of double-poles when the integrands are interpreted in 4D. Provided the integrands of B 3 are full-weight, they will automatically be diagonal with respect to the integrands in B 2 -that is, they will vanish on all contours involving double-poles.
The basis elements without double-poles-those of B 2 -are easiest to discuss, which is why we start with their defining contours. The basis elements in this category are the chiral boxes I i A,B,C,D as well as the scalar triangle integrands I I=1 A,B,C . All other basis elements have double-poles at infinity and will be considered momentarily in section 2.2.1. A summary of our defining set of contours is also provided in Table 3 of appendix A.1.
The contours defining the chiral box integrands can be represented graphically according to: these are simply the contours encircling the two solutions { * 1 , * 2 } to the quadruplecut equations 2 Only the box integrals have four-propagators to have a non-vanishing contour integral on such a cut, and each box integrand involves a unique set of such propagators; as such, all other basis elements automatically vanish on these contours.
The chirality of box-integrand contours can be seen more clearly in cases where massless corners are present, for which we may indicate the parity of the contour using blue or white vertices. For example, we denote the three-mass box contours as which highlights that these contours involve * 1 = λ a λ X and * 2 = λ X λ a , respectively, and the precise form of λ X and λ X is irrelevant for the moment.
Next, consider the contours for the scalar triangle integrands. Most interesting are the cases where there is at least one massless leg, since the associated dual basis integrands can have IR singularities. For example, we define the two-mass scalartriangle integrals' contours by where the circle is a graphical notation for the collinearity condition a ∼ p a imposed in addition to the triple cut 2 a = 2 b = 2 c = 0. Let us mention that this particular contour is spurious, and thus no scattering amplitude has support here. Furthermore, demanding that the chiral box integrands vanish on Ω I=1 a,B,C guarantees that they are free of this particular collinear singularity associated with IR divergences.
A similar discussion also applies for the scalar one-mass triangle contour Ω I=1 a,b,C (see subsection 2.2.2 for further details). The contour choice in Table 3 for the scalar triangles renders all boxes locally IR-finite as in [27] by demanding that the chiral box integrands vanish in all collinear or soft regions of loop-momentum space. This choice leads to the same numerators that have been described in the context of N =4 sYM in [28].
Defining Contours for Lower-Weight Integrands
The second class of basis integrands and their associated contours involves certain double-poles at infinite loop momentum. These are the objects we turn to now.
The key observation to define a bubble power-counting basis in four dimensions is that B That is, the additional integrands needed, relative to a triangle power-counting basis in four dimensions, are scalar bubbles and triangle integrals with single-inverse-propagator loop-dependence in their numerators which define B 2 ; both of these are naturally defined in three dimensions-and for more than merely pragmatic reasons.
Consider for example the scalar bubble integral. With the appropriate normalization of the numerator in terms of powers of s A , the bubble integrates to a pure weight-one function in either two or three dimensions. Moreover, it is possible to write it as a wedge-product of dlog-differential forms in either case: (for a more detailed discussion, see e.g. [29,30]) where, in the two-dimensional bubble, * a and * a are the two solutions to the maximal cut equation 2 a = 2 b = 0 and the bubble has no pole at infinity, → ∞. The threedimensional bubble is slightly more complicated and has a single pole at → ∞. This can be thought of as a dual conformal triangle in D = 3 where one of the dual points is taken to be infinity [30]. In suitable coordinates (embedding space), infinity is treated on the same footing as any other point which makes this analysis very clear. Here, we refrain from introducing embedding coordinates (see [31]) and work in momentum space directly which leads to the appearance of the two nullvectors q and q normalized by q · q = 1 which are defined by the relations q · p A = q ·p A = 0. (Technically, this is easiest to implement by choosing light-cone coordinates transverse to p A . Furthermore, the dlog form remains valid for massive internal propagators where 2 a,b → 2 a,b −m 2 which will become important for our discussion in D=4.) In D=3, we consider for example the triple cut of the bubble which encircles the two propagators and furthermore encloses the odd combination (parity-even) of simple poles at → ∞ which is clear from the d log form in 3D where one cuts the two propagators 2 a = 2 b = 0 and then encircles the parity-even combination of ·q = 0 and ·q = 0. In three dimensions, this is a leading singularity of the scalar bubble integrand.
In contrast, in four dimensions, the scalar bubble integrand has a double-polesignaling a weight-drop in the resulting integral [32,33]. This is reflected in the fact that the bubble can be written explicitly by decomposing the four-dimensional space of loop-momenta into a three-dimensional subspace and one additional direction, say i orthogonal to the momentum p A (as well as q, q) entering the bubble and to the three-dimensional slice. This effectively means that we can think about the 4D bubble as a 3D bubble where the propagators become massive, with mass m 2 := ( i ) 2 . Since our 3D dlog form was valid for internal massive legs, we find so that the triple-cut residue results in a 'double-pole at infinity': an integrand which is independent of the remaining loop integration parameter.
where i is whatever component of not eliminated in the three integration cycles. Thus, for this integrand the differential of the form 'd i ' looks like a total derivative on the cut. Moreover, this differential form has a double-pole at infinity. Unlike dlog,d i is not scale invariant and thus the coefficient of the double-pole is not uniquely defined. As this example should make clear, the particular component for the final integration, say i , is completely arbitrary: any three components of can be eliminated in the first integrations, always resulting in an integrand of the form d i in the remaining variable. Thus, there is no particular double-pole: there is a three-dimensional (four-dimensional, modulo rescaling) family of such double-poles. Perhaps a more invariant way of describing a bubble integrand in four dimensions would be to start with the fact that in three dimensions, it is purely polylogarithmic: it is a dlog-form. Going from three to four dimensions amounts to appending a trivial d i onto this polylogarithmic three-form.
To be clear, the bubble integral is polylogarithmic on any three-dimensional subspace chosen-which we may denote as ( i ) ⊥ for any component i of . Considering that i := µ · e i µ for some basis vector e i µ , it is clear that we can view the complementary space as the solutions to ·p X = 0 for any p X . For reasons of simplicity, it turns out to be beneficial to take p X to be null. In this construction, we define a three-dimensional subspace of loop momenta according to Noting that the null-space (p X ) ⊥ of p X is defined by ·p X = 0, we see that this can be interpreted more concretely as: Although this three-dimensional subspace depends on p X , we will choose the same subspace for all integrands with double-poles. Thus, when we say that B to be defined as in (2.23) for all integrands and consider contours to be taken over this three-dimensional space .
Illustrations of the Resulting Numerators in the Basis
In order to make some of the abstract definitions of the previous subsections more concrete, we consider a few illustrative examples that highlight all relevant features. The complete list of one-loop basis integrands with bubble power-counting is summarized in Table 4 of appendix A.2. First, we consider the two-mass-easy box integrands (2.25) where we use the kinematic bracket conventions from [34,35] where (a 1 ·a 2 ) α β := a αα 1 αγ aγ γ 2 γβ and a αα := a µ σ αα µ are '2×2' four-momenta, defined via the Pauli matrices. The '[[· · · ]]' object may be more familiar to some readers if written equivalently as 'tr + [· · · ]', are linear in their arguments, and satisfy the following identities The two chiral-box numerators are normalized to unity on the following maximaldimensional cycles where * 1 and * 2 are the two solutions to the maximal cut equations of the box 2 a = 2 b = 2 c = 2 d = 0 and the white and blue vertices in the contour prescription in (2.29) indicates the chirality of the solution at that vertex. In particular * 1 ∼ λ a λ X and * 2 ∼ λ X λ a . Due to the chirality of the solution and the order of the momenta in the brackets of n i a,B,c,D in (2.25), the integrand basis elements are diagonal on the respective contours. In order to claim that our basis is truly prescriptive, it remains to be checked that both integrand basis elements vanish on all other defining contours summarized in Table 3 in appendix A.
First, we should note that the chiral boxes scale at infinity like scalar triangle integrals, i.e. they have at most single poles at → ∞. This implies that these integrands trivially vanish on all contours that involve the instruction of taking a double-pole at infinity. This implies that the chiral boxes vanish on all defining contours for chiral triangles (to be discussed in detail shortly) as well as on the bubble-integral contours. The only remaining question is therefore associated with the defining contours for the scalar triangle subtopologies Ω 1 a,B,C in the language of Table 3. For the example considered above, all triangle subtopologies have one massless leg. Our particular choice of the one-mass scalar-triangle contour involves the collinear limit around the massless corner of the triangle. Fortunately, the chiral box numerators in (2.25) vanish in the collinear limit where a ∝ p a or c ∝ p c due to the properties of [[· · · ]]. Crucially, the fact that these chiral boxes have only single poles at → ∞ together with the fact that they vanish in the collinear regions a ∝ p a or c ∝ p c renders these objects both UV and IR finite. These integrands have been integrated in [28] and for the convenience of the reader we give their result in terms of polylogarithms in Table 5.
A second illustrative example to consider is the one-mass triangle sector . (2.30) The chiral numerators n I=2,3 a,b,C are written in a way to make the collinear and UV properties manifest. In particular, the ordering of momenta in [[p a , b , p b , p X ]] and its conjugated version guarantees that these integrand elements are IR finite in the collinear regions b ∝ p a , p b as well as in the soft region b ∼ 0.
These integrands in (2.30) are constructed to be dual to the following defining contours where the first contour Ω I=1 a,b,C represents the soft-collinear leading singularity that sets b = 0 and uniquely selects the scalar one-mass triangle. (All box integrands are chiral and their numerators guarantee the vanishing in the soft-collinear configuration.) The chiral contours Ω I=2,3 a,b,C warrant some further explanation. This is the first time in our discussion where we have to deal with the double-poles at infinity that are naturally associated with a weight drop at the integrated level. These were discussed abstractly in section 2.2.1 and we would like to concretely give our definition for the chiral one-mass triangles here. The way to think about the chiral contours such as Ω I=2 a,b,C that involve the double-pole at infinity is as follows. First, one projects a into a particular direction which leaves us with a three-dimensional surface for a perpendicular to the above projection constraint. The particular normalization of the projection (2.32) is related to our choice of projection and enters in the overall normalization of our integrand. The remaining three parameters of ( a ) ⊥ are then fixed on the triple-cut Together with the projection condition (2.32), the three on-shell constraints therefore localize all four degrees of freedom of a . There are two different solutions to the constraints which we denote by * 1,∞ and * 2,∞ , where the additional subscript signals that we are interested in the leading behavior of → ∞. Taking into account the proper Jacobian factor J , our numerators evaluated on the leading singularity solutions are unit One additional point worth discussing is the explicit presence of the bubblecontact term + 2 b in the definition of our one-mass chiral triangle numerators n I=2,3 a,b,C . This term is there in order to have the chiral triangles vanish on the massive bubble contour Ω a+b,C , which we define presently.
Consider the generic massive bubble topology Ω A,B . Just as in the chiral triangle sector, we begin by projecting a onto the three-dimensional subspace Next, two additional degrees of freedom are fixed by localizing to the bubble cut 2 a = 2 b = 0. In fact, there are two solutions to these three combined conditions, which we may denote as * 1 , * 2 . (If one parametrizes a in a basis of spinors involving the null momentum p X , these two solutions are chiral and involve loop momenta proportional to either λ X or λ X , as we illustrate more explicitly in section 4.2.) The final degree of freedom is fixed on the double-pole at infinity, with the bubble integral normalized on the parity-even combination of these two evaluations.
The downside of the explicit presence of this bubble contact term is that the chiral triangles are rendered UV divergent. In principle, one could avoid this feature by explicitly removing the bubble contribution. However, this would come at the cost that the resulting basis would no longer be dual to a particular choice of contours, rendering the basis non-prescriptive. This would have the effect that in the representation of an amplitude, the coefficients of bubbles, say, would need to be the difference of bubble-cut leading singularities and whatever pollution arises from the triangle integrals.
Of course, one could start from a prescriptive basis to determine coefficients simply and then rotate into a non-prescriptive one in order to highlight other aspects of interest-such as a better separation between UV and IR divergent integrands. We should note in passing that we have in fact constructed such a possibly preferential basis-one in which the only UV-divergent integrals are the bubbles, and for which all integrals are pure. Such choices, however, are far from unique, and leave open the generally broad questions of aesthetic and technical preferences, and so we leave such potentially illuminating rotations to future work.
Bubble Integrands and Integrals Involving Massless Legs
Most of the integrand and contour definitions are conceptually very simple, although the exact details and choices made required a nontrivial amount of work. There is however, one additional cases that is often neglected: bubbles involving massless external legs: (2.35) In dimensional regularization, this integral is somewhat special in the sense that it is scaleless and integrates to zero in a nontrivial way. UV and IR divergences cancel one another in the form 0 = 1 UV − 1 IR . In traditional generalized unitarity constructions, these terms are neglected at first and a tentative amplitude is computed. Once one separates UV from IR divergences, e.g. by introducing a small mass regulator, one can compare the resulting IR or UV divergences of the tentative amplitude to general expectations and ultimately adjust the coefficients of these massless bubbles to match the expected results. We will come back to this point in more detail in subsection 3.2.
From the viewpoint of prescriptivity and basis-building, however, these integrals pose no subtlety whatsoever: they are defined in exactly the same ways as the massive bubble integrands-except that the collinear condition is imposed on the three-point vertex instead of taking a residue at infinity-thereby highlighting the region of loop-momentum space that is responsible for these integrals' IR divergences.
What is genuinely subtle, however, is the meaning of leading singularities defined on such a contour-which affects the coefficients of these integrands in the representation of amplitudes. We review this issue in some detail in section 3.2, and pose two possible definitions one may take for these coefficients.
3 subspace are all pure, weight-2 functions that are free of any regions of UV-divergence; moreover, only the scalar triangle integrals involving massless legs are IR divergent-all others are locally finite. These general features are summarized in Table 1.
In contrast, all those integrands in the B 2 subspace are weight-one functions when evaluated in 4−2 dimensions (as is natural for having maximal weight in 3D). All but one class evaluates to a pure function. These general features of these integrals are summarized in Table 2.
As discussed above, it is possible to alter the basis of integrands to improve the IR/UV properties of the basis. For example, it is easy to render the integrands I 2,3 a,B,C pure or to make all triangle integrands UV-finite. However, this rotation of the basis would seem to be in conflict with prescriptivity, and make it harder to directly determine the coefficients of an amplitudes in the new basis.
Leading Singularities in N ≤ 4 Super Yang-Mills Theory
Having discussed the integrand basis construction at length, we are now in the position to comment on the second key building block in the generalized unitarity expansion of the amplitude which are the coefficient functions. As discussed in section 2.2.1, all defining contours are of maximal dimension so that the coefficients of our basis integrands are simply leading singularities. In this section, we give details on how to compute these leading singularities in less (than maximally) supersymmetric theories.
The description of on-shell (super-)states for amplitudes in N < 4 super Yang-Mills theory are best implemented by considering the states to be truncations of those in N = 4. This was described in detail in ref. [20], but is worth reviewing. We This scheme makes it obvious that for any amount of supersymmetry, the external states can be labelled as particular instances of those of N = 4-the only difference being in the selection rule for which R-charge labels are allowed among the external states. These selection rules have the effect of requiring that the indices {N +1, . . . , 4} all appear in the labels of some subset of k external states for an N k−2 MHV amplitude. This amounts to a truncation of some N = 4 super-function.
Thus, all processes in an N k−2 MHV amplitude (or on-shell function) must specify precisely k states related by supersymmetry to the (−)-helicity gluon. These are simply 'helicity' amplitudes in the case of 'pure' (N = 0) Yang-Mills theory; but the same n k distinguished labels are required for all component amplitudes for any degree of supersymmetry (other than maximal).
This can be encoded graphically in an on-shell diagram by orienting all its edges. We choose to use an incoming arrow to denote the n k states related to the (−)helicity gluons (incoming at the vertex), and outgoing arrows to denote those related to the (+)-helicity gluons (incoming at a vertex).
For example the three-point super-amplitudes in sYM N would require orientations as in . (3.1) To be clear, these amplitudes may be defined in terms of coherent states as follows: in terms of Grassmann variables η I i for I ∈ {1, . . . , N }. The generalization to MHV amplitudes is extremely natural: is the super-momentum-conserving δ-function and δ 2×2 λ· λ := δ 2×2 n a=1 λ α a λα a encodes overall momentum conservation. More generally, an N k−2 MHV superfunction (such as a leading singularity) in N = 4 super Yang-Mills is related to n k oriented superfunctions in sYM N according to where C represents the k × n 'boundary-measurement' matrix [7] and {i r } label the negative helicity super-multiplets. Just as in the three-point amplitudes given above, any decorated on-shell diagram must be oriented such that each N k−2 MHV tree-amplitude appearing at a vertex has k 'sources'-i.e., incoming arrows.
Decorated On-Shell Diagrams: Singlet vs. Non-Singlet
There is a marked difference between on-shell functions in maximally supersymmetric Yang-Mills and its less supersymmetric cousins. This is primarily a result of the distinction between so-called 'singlet' and 'non-singlet' helicity configurations. In the former case, the R-charges of the external states uniquely determine those of the internal states running through the loop, regardless of the amount of supersymmetry. All such singlet on-shell diagrams are therefore N -independent and therefore equal to (truncations of) N = 4 super-functions and may be immediately recycled. In contrast, when there are oriented loops of 'helicity' in an on-shell diagram, we must sum over all the states in the supermultiplet which clearly depends on N .
A prototypical example of a non-singlet decorated on-shell function is the following four-point box diagram with external states {2, 4} are taken as incoming: For each of the two possible 'helicity' flows through the graph (each involving a sum over states), it is not difficult to determine the corresponding on-shell function by direct computation. In particular, we find: When N = 4, the equation above over-counts the sum over states by 2 as both directions of helicity flow are included in the same coherent state. Furthermore, eq. (3.9) is valid for entire super-amplitudes: replacing the pre-factor (the gluonic component of the MHV tree amplitude) by the superamplitude gives the correct answer for all components such that the R-charges of particles {1, 3} are in the '+' multiplet (related to g + by some number of supersymmetry generators Q I 's). Another example which is directly relevant for the all-multiplicity MHV amplitude presented in section 4 is the generic two-mass easy box cut where the states related to the negative helicity gluons have particle label i, j. It is easy to verify that the only non-singlet configuration in this case is when both i and j are each in a distinct massive corner: On-shell diagrams of either the triangle or bubble type may be computed in an analogous fashion; the structure of the result is depends on whether the cut is singlet or non-singlet. As mentioned in section 2, the evaluation of field theory on triangle and bubble contours involves double-poles at infinity and requires a projection of the loop momentum onto a particular direction. Pragmatically, one can always derive such contour integrals from the double and triple-cuts of standard unitarity. We illustrate this feature for the massive MHV bubble coefficients in section 4.2.
Generalized Unitarity for Massless Bubble Coefficients
For gauge theories with N < 3 supersymmetry, there is an important subtlety associated with loop integrand and cut topologies which define the massless bubble integrals. If only interested in the integrated amplitudes, these coefficients may be ignored as all such integrands integrate to zero (in dimensional regularization). However, if one were interested in disentangling the UV and IR structure of an amplitude, they play an important role. As such, their coefficients can be determined post-integration by the requirement that this behavior is correct (see e.g. [20,36,37]).
To see this subtlety, consider the two-particle, massless cuts of an amplitude. For any N k MHV degree (and any assignments of external helicities), there always exists one singlet and one non-singlet configuration depending on the parity of the three-particle vertex: a , ± a or ± a , a . (3.12) While the singlet cuts are always unambiguous and finite (and in fact always equal to truncated superfunctions of N = 4), the non-singlet cuts are unfortunately always illdefined -as they generally diverge. Thus, there is no obvious meaning to these cuts in field theory, making it difficult to compute the leading singularities corresponding to the massless bubble contours: there always exists some branch of the bubble-cut on which the amplitude diverges. Of course, the massless bubble integrals in our basis have been defined by contours not merely taking the co-dimension 2 residue of the bubble cut, but a contour accessing the double-pole at infinity which starts from the collinear triple-cut in loop-momentum space-the region in which * a = α p a , * b = (1 + α) p a . (3.13) If this contour were viewed as arising as a co-dimension one residue taken along the well-defined (singlet) triple-cut in every case, then because all such cuts are equal to (truncations of) their N = 4 equivalents, no amplitudes would have support on these double poles. This would suggest that every massless bubble coefficient should be identically zero. This is the first option we consider. While this choice for interpretation is ensured to match field theory functionally on all of the well-defined (singlet) massless bubble-cuts, it turns out that it fails to match the conventional UV-structure of amplitudes (as deduced using the logic of e.g. [20,36,37]). In particular, it leads to representations of one-loop amplitudes that exactly misses the standard answer by a multiple of the tree amplitude times the sum of massless bubble integrals.
Perhaps this missing contribution could be attributed to some (however unconventional) renormalization 'scheme'. And it may prove that ignoring all massless bubble contributions turns out to lead to better (more elegant in some way, perhaps) strategies at higher loops. But we must leave such speculation to future work.
However, there is another way to interpret the leading singularities corresponding to these collinear cuts. Namely, it seems natural to associate the collinear configuration as equivalent to a massless bubble on an external leg, as in: (3.14) This interpretation naturally suggests that we interpret theory theory for these contours as being proportional directly to the tree amplitude as in [38]. This reproduces the standard result for one-loop amplitudes' UV and IR structure, and certainly seems like an appropriate 'convention' for defining these bubble coefficients. This is the prescription used in the expressions generated for our concrete examples given in the ancillary files for this work. These kinds of subtleties are much more abundant in pure (N = 0) Yang-Mills theory, the amplitudes of which are known to require worse power-counting in their bases. While we can certainly define a prescriptive basis B 0 to express these amplitudes, the coefficients of tadpoles and constants seem intrinsically ambiguous and for similar reasons. There have been some notable recent proposals for how to deal with tadpoles [39] (see also [40]); however, all these proposals begin from some prior knowledge of the loop integrand-i.e. start from the (literal) sum of Feynman diagrams in some gauge and using some regularization scheme. This does lead to specific coefficients for any integrand in a basis even as ugly as B 0 , but it does not provide a gauge-invariant, cut-level definition of the coefficients in terms of on-shell, tree-level scattering data. (But see e.g. [41] for some interesting ideas in that direction at higher loops that relies on a particular on-shell renormalization scheme.) Naturally, we must leave such questions-important though they are-to future work.
Amplitude Integrands for N ≤ Super Yang-Mills Theory
The derivation of a diagonalized basis of integrands in section 2 has an immediate application: namely, the construction of prescriptive representations of 1 ≤ N ≤ 4 sYM amplitudes. Achieving this amounts to the computation of the coefficient of each basis element-that is, field theory evaluated on the contours defining the basis.
As discussed in section 3.1, there are essentially two cases to consider for each coefficient, depending on the helicity configuration of interest. For a given on-shell diagram, if there is only a single allowed internal helicity flow-i.e., a 'singlet' configuration where the external helicities uniquely specify the internal helicity statesthen the on-shell function is identical for sYM for any N . By virtue of the fact that the N = 4 integrand is free of all poles at infinity, this implies that for all 'singlet' cuts, the coefficient of every basis element defined on contours involving infinite loop momentum necessarily vanishes.
For the 'non-singlet' configurations where there are multiple allowed helicity configurations, sYM for N < 4 can have support on single (and double) poles at infinity and the associated coefficients are generically non-vanishing (and non-trivial).
In this section, we illustrate the procedure outlined above with two concrete examples: the all-multiplicity MHV (A The box coefficients a i A,B,C,D are defined on the two quad-cut leading singularities i.e., field theory evaluated on the two solutions to 2 a = 2 b = 2 c = 2 d = 0. For any singlet configuration, these leading singularities are simply truncations of those defined in N = 4; for the non-singlet configurations, there is a modification resulting from the helicity flow as described above. Regardless of supersymmetry, all one-mass triangle integrands with scalar numerators have coefficients a 1 a,b,C because are defined on the composite 'soft-collinear' residue where one internal leg is set to zero on which amplitudes always have support. Moreover, and just as in maximal sYM, the residue of field theory is always equal to the tree amplitude (as this reflects the only universal IR divergence at one loop); that is, a 1 a,b,C = A n,0 . For similar reasons, the coefficients of all two-mass scalar triangles are always zero: a 1 a,B,C = 0. The non-singlet cuts of amplitudes can generally lead to support on doublepoles at infinity, resulting in non-trivial coefficients for triangles with loop-dependent numerators. For any singlet cuts, these coefficients are all zero. The same is true for the all bubble contours defined on double-poles at infinity. Thus, these coefficients depend strongly on how the helicity-flow at each vertex amplitude of the cut flows into the graph, and varies depending on which of the n k external legs are taken to have 'incoming' helicity.
Exempli Gratia: MHV Amplitude Integrands
We can illustrate how these considerations work in the concrete case of MHV amplitudes (k=2) in N = 1, 2 super Yang-Mills theory. As with maximal supersymmetry, the only box cuts which have non-vanishing support for these amplitudes are Ω 1 a,B,c,D -the (chiral) two-mass-easy contours (and their one-mass degenerations). Of these, most contours admit only a singlet configuration of internal helicity-namely, n,0 . where the final equality follows from the binomial expansion of the exponents in the first line and is valid only for N = 1, 2.
Turning now to the triangle configurations, we may start with the scalar onemass contours, on which all amplitudes have support equal to the tree: (We have neglected to indicate any helicity information from the left-hand-side for the simple reason that every one-mass scalar triangle has the same coefficient, regardless of the helicity configuration under consideration.) For the two triangle integrals elements normalized on double-poles, there are just three classes of leg distributions with non-singlet helicity configurations leading to non-zero coefficients. By directly evaluating field theory on the corresponding contours, we find that these non-vanishing coefficients are: (4.7) Finally, among the massive bubble contours, the only ones with non-singlet helicity flow are those for which {i, j} are on opposite sides of the bubble. These coefficients turn out to be (4.8) The massless bubble coefficients a a,B were discussed at length in section 3.2 and-as emphasized there-we have two options: either a a,B = 2(N −4)A (i,j) n,0 or a a,B = 0. It is worth clarifying how the massive bubble coefficients a A,B in eq. (4.8) may be obtained by a straightforward computation. It is convenient to evaluate field theory on the bubble contour by first computing the two-parameter non-singlet bubble cut, which was in fact given already in [20] and may be written as, A parametrization of a , b which is particularly convenient for the projection onto [[ a , p X ]] = 0 is given by (4.10) The Jacobian of the bubble cut in this parametrization is simply J = β, while the projection condition [[ a , p X ]] = 0 has two solutions, λ a ∼ λ X and λ a ∼ λ X , which correspond to β → 0 and β → ∞, respectively. Our bubble contour prescription amounts to evaluating (4.9) on (4.10), taking the residue at either β → 0 or β → ∞, and extracting the coefficient of the double-pole at α → ∞. We define the bubble leading singularity to be the even combination of these two field-theory evaluations, which are precisely the two terms appearing in (4.8).
The basis of integrands and the collection of non-vanishing coefficients in (4.5), (4.3), (4.7), (4.7) and (4.8), together with a prescription for the massless bubble coefficients, constitutes the MHV one-loop amplitude integrand in the form of (4.1). Combining all terms, one can (numerically) check that the p X dependence drops out of the integrand via a nontrivial cancellation between all terms.
Using the tabulated integration rules found in appendix A.3, we find the n-point MHV integral to be of the form, Here, the expression A (i,j) n,1 is implicitly defined to be the UV-and IR-finite part of the one-loop amplitude divided by the tree amplitude.
It is worth remarking that the while the expression in (4.11) is correct, it is not entirely manifest in our representation. In particular, the expression on the second line does not follow manifestly from the basis we have constructed. Nevertheless, we have explicitly checked its correctness.
Exempli Gratia: a Six-Point NMHV Amplitude Integrand
As another example of prescriptive unitarity with bubble power-counting, we consider the six-particle split-helicity NMHV amplitude integrand with particles {4, 5, 6} to be those related by supersymmetry generators to negative helicity states.
First, it is easy to see that for the particular helicity configuration we've considered, every non-vanishing box diagram is of the singlet type. This implies that the box coefficients are given by extracting the ( η 4 ) 4 ( η 5 ) 4 ( η 6 ) 4 component of the R-invariants appearing in the N =4 superamplitude.
Just as in the MHV example discussed above, the coefficients of the one-mass scalar triangles is always the tree amplitude, A (4,5,6) 6,1-loop . It turns out that the nonvanishing chiral triangle and bubble coefficients, can all be expressed compactly in terms of the following two superfunctions (R-invariants) [56] δ 3×N C 3 · η δ 2×2 λ· λ , where In terms of these two superfunctions, we find that the non-vanishing non-singlet cuts for this amplitude give rise to the following non-vanishing coefficients: X3 Plugging these coefficients into the expansion of the amplitude in eq. (4.1), we obtain the integrand for the six-point split-helicity NMHV amplitude integrand with particles 4, 5 and 6 being related by supersymmetry to negative helicity gluons.
Finite Observables at One Loop
It is widely appreciated that four-dimensional scattering amplitudes for massless particles are problematic due to the presence of long-distance (infrared) divergences associated to low energy (soft) or unresolved collinear radiation, see e.g. [42]. For inclusive enough physical observables such as cross-sections, all such divergences cancel when real radiation effects are taken into account consistently as a consequence of the KLN theorem [43,44] in QED and its generalizations. Another example of an IR-finite observable is the energy-energy correlation function, see e.g. [45,46]. The IR structure of general gauge theories is still an important subject of current study; both formally (see e.g. [47,48]) as well as phenomenologically in the form of efficient IR subtraction schemes for high-precision predictions for collider observables [49][50][51][52][53].
From an amplitudes perspective, it is possible to determine which diagrams can contribute to IR divergences and which ones remain finite. This analysis amounts to investigating all soft and collinear regions of a given diagram, taking into account potential numerator factors that can dampen IR singularities. It turns out that the situation is especially simple for one-loop integrals where one can easily account for all possible singular regions which suffices for the present discussion. For general gauge theories, the infrared structure has been completely understood up to two-loop order by Catani [54] with numerous subsequent progress, see e.g. [55][56][57][58][59][60].
The universality of IR divergences of gauge theory scattering amplitudes at one loop implies that all divergences should be proportional to the tree amplitude. Together with the requirement that UV divergences in a renormalizable gauge theory should be canceled by appropriate counter terms also implies that the one-loop UV divergences is also proportional to the tree-level amplitude. Motivated by this discussion, we can organize the n-particle one-loop amplitude in the following form: A n,1 =:A fin n,1 +A n,0 I UV div +I IR div =:A n,0 A fin n,1 +I UV div +I IR div with A n,1 := A n,1 /A n,0 , (4.20) where we suppress the explicit helicity-labels of the (super-)amplitudes as well as the MHV-degree k. The universality of IR divergences is more general than the specific one-loop example discussed above and is encoded in the following factorization formula (see e.g. [60,61]) for massless parton scattering amplitudes where all IR singularities are factorized in Z n in the form of poles in dimensional regularization = (D − 4)/2. The above equation depends on a factorization scale µ and the running coupling constant α s := α s (µ 2 ). Motivated by this decomposition, it is natural to introduce the IR-finite ratio function where the universal IR singularities cancel. A priori, we can write the ratio of two n-point amplitudes A n to all orders in perturbation theory: In maximally supersymmetric theories, there is only a single independent super amplitude for a given N (k−2) MHV sector and one takes IR finite ratios between amplitudes of different k charge. In this case, the labels 'a' and 'b' denote the respective k-charge of the amplitudes and it is common to always divide by the k = 2 MHV amplitude and denote the resulting ratio function by P (k) n . The IR-finiteness of P (k) n underlies several important features of the integrated results for the maximally supersymmetric theory, including dual conformal invariance [3-5, 62, 63]. These simplifications, together with a number of conceptual and technological advances enabled Dixon and collaborators to obtain function level results to very high loop order, see e.g. [64][65][66].
For the N = 1, 2 supersymmetric amplitudes under consideration, we can furthermore take nontrivial ratios of (super-) amplitudes within the same N (k−2) MHV k sector due to the distinction between the positive and negative helicity gluon supermultiplet and write e.g.
where the ± labels the relevant supermultiplet of particle i. We omit labeling the ratios by the individual helicities of the contributing amplitudes to avoid cluttering the equations and introduced the shorthand notation A (i,j) n for MHV amplitudes to indicate the position of the negative helicity supermultiplets.
All ratios can be expanded perturbatively in the coupling constant g and yield IR-finite quantities at each order in perturbation theory, e.g. up to two-loop order we find where we indicate the loop order of various quantities by an additional subscript. The formulae for the ratio of amplitudes in the same MHV sector follow trivially from the above results. In the presentation above, the various factors of the treelevel amplitudes A n,0 and A (a) n,0 ensure a uniform helicity weight of all terms in the perturbatively expanded form version of the ratio function. It is often convenient to divide out certain helicity-dependence by removing the tree-level amplitude and work instead with A n to define n,1 +O(α 3 s ) . (4.25) At one-loop, the IR and UV finiteness of the ratio function is easy to see. From general expectations (and confirmed by our explicit calculation below), both the UVand IR-divergent parts of the one-loop amplitudes must be proportional to the treelevel amplitudes as in (4.20). Working with the rescaled quantities, we see that the universal factor I IR/UV div cancels in the difference (4.25). Similar arguments also lead to the finiteness of the higher-loop ratio functions.
We may illustrate how this works for the simplest example involving four particles. Before taking the ratios, we give the integrated results for the individual amplitudes (N = 1, 2) in terms of the usual Mandelstam variables s:= (p 1 +p 2 ) 2 ,t:= (p 2 +p 3 ) 2 ,u:= (p 1 +p 3 ) 2 .
In the one-loop ratio function of (4.25), we are supposed to take the difference of the two amplitudes. Both the UV and IR divergences cancel in this difference and we find for N = 1, 2 that where standard Mandelstam invariants s, t, u satisfy s + t + u = 0. As advertised, this result is IR and UV finite, but of mixed transcendental weight. Compared to the individual amplitudes, the ratio is considerably simpler and does not depend on the dimensional-regularization scale µ 2 anymore.
Going to higher point is also feasible by inserting the integral values for each of our basis integrands that are summarized in Table 5 of appendix A.3. At five points, the results depend on five independent Mandelstam invariants which leads to more complicated looking results. Since all ingredients are provided with this work, we refrain from writing explicit results here. In general, however, the fact that these ratio functions are UV-and IR-finite follows directly from the general form (4.11).
General Discussion & Future Directions
In this paper, we computed one-loop amplitude integrands in color-ordered lessthan-maximally supersymmetric (1 ≤ N < 4) Yang-Mills theory ('sYM N ') in the context of generalized unitarity. We constructed a prescriptive bubble power-counting integrand basis, and showed how the coefficients of MHV and NMHV amplitudes can be calculated using contour integrals that are dual to that basis.
While the box, triangle, and massive bubble integral coefficients can be extracted in a standard manner, there is an important subtlety in the case of massless bubbles. This topology is traditionally ignored in unitarity-based approaches due to the fact that scalar massless bubble integrals evaluate to zero in dimensional regularization. In contrast, in this work, it was our primary objective to construct a well-defined integrand. This forces us to specify a prescription for the massless bubble coefficients as well. Here, we have presented two distinct possibilities that appear well motivated from field theory and on-shell function considerations: (a) choose collinear cuts or (b) choose singlet double cuts which are the same for any amount of supersymmetry, including N =4 where these cuts are unambiguously defined. In the first scenario, the massless bubble coefficients are fixed to be tree-level amplitudes. The resulting integrand correctly reproduces both the expected IR and UV divergences upon integration. In the second scenario, we get zero coefficients for the massless bubbles and the integrand has improved behavior at infinity on singlet cuts. While both approaches are justified, each exhibits a different structure for the resulting integrands for amplitudes. We leave it to future work to investigate which of the two directions is preferred from the point of view of defining the unique N <4 sYM integrand beyond one loop.
Having a unique integrand is essential for the formulation of loop-level recursion relations (see e.g. [67]), or attempts to reproduce it as a certain differential form on a positive geometry. Therefore, our work is a crucial first stepping stone for a possible extension of amplituhedron-like geometric objects [68] beyond planar maximally supersymmetric Yang-Mills theory.
As a key extension of our work, it remains to construct a bubble power-counting basis at two-loops, and expand the n-point two-loop integrands in planar N <4 sYM theory in this basis. This would push the computational frontier for integrands in less-supersymmetric Yang-Mills theory, and it would provide valuable theoretical data for further investigation.
Finally, the most difficult and important question is the extension of our work to pure Yang-Mills theory. This requires addressing the problem of tadpole integrals and rational terms.
A Complete Bubble Power-Counting Integrand Basis B (4) 2
Following the general strategy of prescriptive unitarity, constructing a bubble power-counting basis of integrands requires the specification of a spanning set of contours {Ω j }. Once this is done, diagonalization results in a basis such that Ω j I i = δ i,j . In this appendix, we give complete details regarding our choice of integration cycles {Ω j }, the integrands {I i } to which they are dual, and the integrals that result.
A.1 Spanning-Set of Integration Contours Defining the Basis , Ω a,B := a B B * → ∞ (double-pole) Table 3. A complete specification of contours to which the integrand basis B 2 is dual. Table 5. Integration results for all box, triangle, and bubble integrands in B
A.3 Integrals of Basis Integrands in Dimensional Regularization
2 .
B Summary of Results Provided as Ancillary Files
For the interested reader, the results described in this work are available as ancillary files which may be downloaded from the abstract page on the arXiv. Three files are provided: • one loop bubble basis data.m: a plaintext file consisting of the complete bubble power-counting basis of integrands constructed in this work.
• one loop bubble basis tools.m: a Mathematica package file consisting of code useful to analyze and evaluate the data of the preceding file.
• one loop n leq 4 MHV amplitudes walkthrough.nb: a Mathematica notebook which illustrates our results and the functionality of the codebase.
The ancillary file one loop bubble basis data.m provides analytic expressions for the integrand basis as well as their integrated expressions, packaged as follows: • integrands[legList ]: a function which takes as argument a legList of external legs-ordered according to the conventions of this work-and returns the list of diagonalized integrands with bubble power-counting written in terms of dual loop momenta. Alternatively, the more abstract expressions found in appendix A.2 can be generated by using as argument anywhere between two and four numbers (depending on the topology of interest), each of which is either 1 or 2 (indicating either a massless or massive vertex, respectively). In this case, the edges for external momenta are labelled by p The ancillary file one loop bubble basis tools.m contains the all-multiplicity MHV amplitude integrand as well as a variety of useful tools for the analysis and numerical evaluation of our results. We provide a brief summary of some key functionality contained in this package: • completeIntegrandBasis[n , p : 2]: returns a symbolic representation of the complete prescriptive basis of integrands for n-points and p-gon power-counting, for either p = 2, 3.
• symMHVAmplitudeTerms[n ]: returns the n-point chiral box expansion for the MHV amplitude in maximally supersymmetric Yang-Mills theory [28].
Kinematics and Numerical Evaluation
• randomKinematics[n : 6]: defines the global variable Zs to be a randomly chosen set of momentum twistors, the corresponding list of four momenta, stored in pList, as well as the two-component spinors, stored in Ls and Lbs. | 2021-12-14T02:16:17.638Z | 2021-12-13T00:00:00.000 | {
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244579111 | pes2o/s2orc | v3-fos-license | Evaluation of Catchments’ Similarity by Penalization in the Context of Engineering Tasks—A Case Study of Four Slovakian Catchments
: The present study deals with the similarity of catchments, which is a preliminary investigation before performing various water resource analyses and computations regarding other catchments, e.g., catchments’ similarity may be utilized in the context of analogous calculations of river flows in catchments without measured flows. In this paper, the penalization method of evaluating similarity is proposed; this method is appropriate for tasks in which fewer catchments are analyzed for engineering purposes. In addition to the various physical characteristics of the catchment, the “catchment’s calibrability” property is also formulated and evaluated. A methodology that used specific flows from catchments in a case study from Slovakia successfully verified the proposed penalization method. This verification confirmed that physical similarity, as evaluated using the proposed penalization methodology, also helps to identify hydrological similarity, i.e., finding the most similar catchment to a given catchment in terms of the rainfall-runoff process. Such a finding can be helpful, e.g., in the computation of the mentioned flows in ungauged catchments. Determining unmeasured flows can help to solve many engineering tasks, such as various technical calculations during the design of small reservoirs, defining the potential of a given stream for supplying irrigation, flood protection, etc.
Introduction
The similarity of catchments is an intensively researched issue, examining how it can be determined and how it is possible to deduce from the physical similarity of catchments the similarity concerning the formation of runoff from them. A good synthesis of knowledge and an introduction to the topic can be found in [1], in which more than 120 expert international authors participated under the guidance of the leading editors. The formation of runoff consists of the interaction of basic processes: distribution (e.g., interception and infiltration), storage (e.g., in plants, lakes, and soil), and release of water from catchments (e.g., evapotranspiration or river flow) [2]. However, these processes are not often measured or cannot be observed directly with enough precision. Geographical and geophysical features are essential indicators of the hydrological response of catchments [3] and can be used for catchment classification [4][5][6] and the evaluation of their similarity. This is a complicated issue; the dominant factors in runoff generation are different in various parts of the world [7,8], and the identification of the weights or relative importance of various elements of runoff generation is often limited by convoluted effects of multiple variables [9].
Similarity analysis can be used, for example, in determining flows in unmeasured catchments, which in turn is of great importance for solving many engineering tasks of water management. The determination of unknown flows is not addressed in this article; it only serves to introduce a possible context for the utilization of similarity of the catchments.
In most catchments, the flows are not measured, particularly in smaller streams. However, the flows of such streams need to be analyzed to check flood protection, the design of supply irrigation reservoirs, etc. Catchments that lack gauging stations are called unmeasured or ungauged catchments [1].
Rainfall-runoff models can be used for the calculation of these unmeasured flows. However, one needs to calibrate such a model, which is highly problematic in unmeasured catchments, as some period of the measurement of flows is necessary for the calibration. One possible way to create and calibrate a model of ungauged catchment flows involves utilizing a catchment located in a nearby and similar region with measured flows. The chosen region should have comparable climate characteristics, geological conditions, topography, vegetation, land use, and soil types, as such a situation should generally lead to similar runoff. The model is calibrated in this catchment and can be used with limited accuracy in an unmeasured catchment. With this example, we can illustrate that determining the similarity of catchments has very useful applications.
The transfer of information from similar catchments is known as hydrological regionalization [10]. The issue of hydrological regionalization has been the subject of intensive research. A helpful introduction to this topic is Hrachowitz et al. [11] or Guo et al. [12]. This article addresses only a portion of this subject, evaluating the similarities of catchments.
The term hydrological analogy is used in this work as its subject of interest is not larger regions, but rather single unmeasured catchments or small groups of catchments. Many different characteristics fundamentally influence the hydrological response of a catchment (geometric characteristics, topography, soil, land cover, geology, climate, etc.). Similarities between catchments must be evaluated using several such elements. Previous studies have demonstrated the usefulness of clustering in identifying similar groups of catchments. It has been shown to be possible to directly transpose the most similar donor catchment's parameters to the target catchment for catchments in the same clustering group [13,14]. The k-means clustering algorithm was often used in these works. Some of the limitations of this algorithm are the accuracy of the initial random centroid's location and the identification of the optimal number of clusters. To solve this problem, in [15], the authors proposed a similarity analysis method based on the iterative clustering ensemble algorithm with random sampling, which was able to find more potentially similar watersheds than the standard application of k-means. Rao and Srinivas in [16] used a two-step clustering procedure to identify groups of similar catchments by refining the clusters derived from agglomerative hierarchical clustering using the k-means algorithm. The physical similarity approach using clustering and principal component analysis, proposed in [17], differs from the classical regionalization method, which simply transfers a full set of model parameters from donor catchments to the unmeasured target catchments, by finding relationships between physical catchment characteristics and model parameters. In [18], the similarity of catchments with the k-means clustering method was investigated by using the hydrological response units, which are the smallest units that can be thought of as the cells of the SWAT model [19]. Similarity measures based on Kohonen self-organizing maps [20] and copulas have also been proposed [21]. SOMs have some advantages over the k-means algorithm, such as a better visualization in two-dimensional space, into which multidimensional data (for example, a set of different river basin characteristics) are projected. Therefore, they allow a better decision on the number of clusters, which do not have to be input in advance. However, in various works where this method was evaluated, e.g., in [22], comparisons with other methods show that it is only slightly better. Nevertheless, based on the mentioned advantages, the SOM is the more objective strategy in comparison with, e.g., k-means.
Studies on this topic, however, have usually involved testing a larger number of catchments, and so cluster analysis, principal component analysis, Kohonen self-organizing maps, and the like were appropriate. However, in this study, we do not intend to propose tools for analyzing large regions, processing large volumes of data, or studying many catchments. Instead, we aim to offer a penalization methodology for searching for the level of similarity in a smaller group of catchments to complete a practical engineering task (for example, the mentioned detection of unknown flows). From the point of view of solving practical tasks, the use of the advanced algorithms mentioned above is further complicated by several unresolved problems associated with their use, some of which we tried to indicate above.
More emphasis can be placed on engineering know-how-for example, visual inspection based on maps, graphs or direct field evaluation and the consideration of various irregular details when analyzing a smaller number of catchments. Errors in datasets that represent a larger number of catchments may not be as influential in terms of overall evaluation but can frequently lead to incorrect judgment when evaluating only a few catchments. Roughly speaking, when we consider a group of five objects, and a mistake occurs with one object, there is a 20% error. When we make the same error when analyzing 50 objects, the error may not be significant. Therefore, a task involving a smaller number of analyzed catchments differs from a task dealing with larger regions and is the novel contribution of this work.
This paper offers a similarity analysis method for small catchments (under 100 km 2 ) in practical engineering tasks, and its original contributions are as follows: (1) An evaluation model combining several aspects of catchments into a conjunct characteristic of their similarity utilizing a penalty approach (2) A newly proposed catchment characteristic designed to evaluate the overall suitability of a given catchment for tasks related to hydrological similarity, which we call "calibrability" The structure of this paper is as follows: in Section 2, the case study is described; in Section 3, the methods applied in this study are briefly explained; in Section 4, GIS and statistical analyses performed with selected catchments are evaluated and discussed, and Section 5 summarizes the results and offers conclusions.
Study Area
In this case study, a comparison of catchments was tested on four catchments in the Small Carpathians in Western Slovakia ( Figure 1); namely, the catchment of the small mountain stream Parná (catchment size-37.33 km 2 ), measured at the Horné Orešany water gauging station, the catchment of the Trnávka stream using the Buková gauging station (42.96 km 2 ), the catchment of the Vištucký stream using the Modra-Piesok station (9.38 km 2 ), and the Gidra stream catchment using the Píla gauging station (32.9 km 2 ). We will henceforth refer to these catchments by the names of their measuring stations. They were not used to determine similarity, as one catchment is always considered unmeasured. The data on precipitation and temperatures were also obtained from the same institution and used in rainfall-runoff modelling, not in determining the similarity of the catchments, as they are nearby areas. The distribution of flow data is shown in Figure 2. The average monthly precipitation totals and temperatures in the area of interest are provided in Table 1. In a real investigation of catchment similarity aiming to determine the unmeasured flows, the flows in the so-called target catchments are unknown. However, all selected catchments had measured flows in the presented study, meaning that it was possible to evaluate the relationship between their physical and hydrological similarity. To obtain more results, each catchment was alternately considered as unmeasured, and the remaining three catchments were understood as those from which we seek the most similar to this "unmeasured catchment"; so, we de facto examined the mutual similarity of all catchments.
The daily flow data for all the river catchments were obtained from the Slovak Hydrometeorological Institute in Bratislava, Slovakia for the years 1980 to 2017. These data were used to create a rainfall-runoff models and, at the end of the work, to verify the similarity of the catchments determined based on other characteristics of the catchment. They were not used to determine similarity, as one catchment is always considered unmeasured. The data on precipitation and temperatures were also obtained from the same institution and used in rainfall-runoff modelling, not in determining the similarity of the catchments, as they are nearby areas. The distribution of flow data is shown in Figure 2. The average monthly precipitation totals and temperatures in the area of interest are provided in Table 1. They were not used to determine similarity, as one catchment is always considered unmeasured. The data on precipitation and temperatures were also obtained from the same institution and used in rainfall-runoff modelling, not in determining the similarity of the catchments, as they are nearby areas. The distribution of flow data is shown in Figure 2. The average monthly precipitation totals and temperatures in the area of interest are provided in Table 1. As neighboring catchments were investigated, it can be assumed that their properties, i.e., climate, topography, geology, soil cover, land use, the genesis of runoff, etc., were As neighboring catchments were investigated, it can be assumed that their properties, i.e., climate, topography, geology, soil cover, land use, the genesis of runoff, etc., were similar but not identical. Differences are analyzed in the Results section in such a way that in terms of each catchment descriptor, the least similar catchment to the others is always identified.
Methodology
In this work, digital elevation models (DEMs), land use maps, soil maps, and various other information sources were used to analyze the properties of the study area that influence runoff. The characteristics of the catchments investigated are reviewed below.
Various geometric properties influencing a catchment's hydrological response, including the catchment's area, perimeter, stream length, catchment length, and various catchment shape factors such as the form factor, circulation ratio, and elongation ratio, were determined using GIS software. The methods for determining these characteristics are well-known and can easily be found in the established hydrological literature [23][24][25]. For this reason, we present the methodology for their determination in Appendix A.
The topographic characteristics were derived from a DEM using GIS software. QGIS [26] and R software [27] were the primary tools used. The topographic similar-ity was examined using a digital elevation model with a grid size of 20 × 20 m. The characteristics evaluated included altitude, slope, and aspect, among others. Higher altitudes were accompanied by a higher total rainfall, depth, snow cover duration, and reduced temperature. Statistical methods and a hypsometric curve were used to compare the altitudes of the catchments; the hypsometric curve represents the relative area below (or above) a given altitude [28] and describes the distributions of elevations across a catchment. A catchment's slope determines the direction and speed of the runoff [29].
Both the composition of the bedrock and soil properties also significantly influence the hydrological response of a catchment, as these characteristics result in a different rate of rainfall infiltration, percolation, retention of soil moisture, etc. [29].
The parent rock and terrain also influence the formation of the drainage networks of the catchments, which were evaluated based on their density. A digital elevation model and QGIS software were used for mapping the drainage networks and evaluating their density.
Land cover is another crucial factor; it encompasses the percentage of forest soil, agricultural soil, and the built-up area in the catchment. Runoff is usually slowed by forest cover because it influences hydrological processes such as interception, rainfall infiltration, evaporation, and transpiration. Conversely, a built-up area or improperly cultivated arable land accelerates outflow [30]. The areas of the individual land-use types in the examined localities were compared using GIS tools.
The climatic similarity between catchments can be evaluated by comparing factors such as precipitation, temperature, differences in rainfall, and potential evapotranspiration. Quite often, climate has been identified as the most important driving factor for different hydrological behaviours [5,6]. However, when comparing only nearby catchments, as is the case in this study, the climatic differences are usually negligible. As such, this work devotes minimal space to this factor. This work also introduces the catchment characteristic "calibrability", which is the level of precision achieved via the hydrological modelling of flows. Suppose we are investigating the similarity of catchments with the goal of creating a hydrological model for unmeasured catchments. If the flows cannot be modelled with sufficient precision in a measured catchment, transferring the model parameters to another, unmeasured catchment model, would be probably even less successful. The engineers solving the task in which the determination of unknown flows will be used must assess what level of accuracy is still acceptable in a particular task context, i.e., it would be different in the introductory study of an irrigation system design and different in a detailed design of flood protection. In general, however, it can be said that the determination of flow rates below a Nash-Sutcliffe efficiency of 0.5 is insufficient.
In this work, we will therefore use the umbrella term "calibrability" to emphasise the context in which it is used, to stress the importance of evaluating this aspect in searching for a suitable source (analogous) catchment and also because one must decide how to determine this level of accuracy in a particular task. It is necessary to take into account the purpose for which derived flows will be used. In a real-life task, the engineer may choose different indicators for tasks that address low flows and other indicators for solving flood problems. Identifying this characteristic can also be based on graphical methods (for example, the comparison of measured and calculated hydrograms) or on a combination of several indicators. This characteristic is expressed by the Nash-Sutcliffe coefficient in this work [31], since the purpose for which the similarity of the catchments is evaluated is not addressed in it, and it is a frequently used indicator in the hydrological community. As will be shown in the Results section, it served quite well for the given purpose.
Based on the catchment properties (catchment descriptors), this paper offers a practical penalization methodology for identifying the most similar catchments from a few catchments surrounding an ungauged catchment of interest. In this penalization methodology, some space is left to engineering know-how for considering various irregular details. This is both possible and more appropriate due to the smaller number of catchments than in regional studies. Therefore, we proposed testing the process of determining the overall similarity between catchments using a penalization methodology in which a catchment most dissimilar from the other catchments is penalised by one point. The authors think that the determination of the penalty with different values of the penalty coefficient for each characteristic of the catchment exceeds the current know-how in this issue and exceeds the possibilities of a practical study that does not analyse the whole region.
The "hydrological response" view of catchment similarity was verified using specific flows. These are flows in millimetres per time step, i.e., computed from usual units according to the following formula: where Q m3/s is the flow in m 3 * s −1 , Q mm/day is the flow in mm × day −1 , and a is the catchment area in km 2 . We chose these units because the investigated catchments have/can have different areas (so that the flows can be compared). These units are also more advantageous in evaluating the hydrological balance, because evapotranspiration and precipitation are also in mm. The correlation of the specific flows was investigated because the catchments in the presented case study have different areas. In this verification process (Figure 2), the proposed manner of penalization and mentioned unified size of the penalty coefficient was verified. Therefore, the similarity between catchments was evaluated from two points of view, one based on the catchment descriptors (which is an evaluation of the physical similarity) and the other based on the similarity of runoff response. Runoff response of two or more catchments is similar, when flows (time series of flows) have good correlation (e.g., above 0.65). A comparison of these two methods of assessing the similarity of catchments served as verification. In this process, it was verified as to whether both the penalty procedure and the assessment of the hydrological response led to the identification of the same catchment as most similar to the ungauged catchment.
Results and Discussion
This chapter contains catchment similarity assessments from several viewpoints, i.e., according to various characteristics. In the following, the most different catchment from the others in terms of each characteristic is evaluated. This catchment is penalized by one point. Penalization is summarized, and the overall similarity/dissimilarity of the catchments in question is determined at the end of this chapter in Table 6 (firstly, four auxiliary tables follow).
The basic geometric properties of the catchments, including area, perimeter, catchment length, segmentation of the catchment boundary, form factor, circulation ratio, elongation ratio and shape factor, are evaluated and summarized in Table 2. The value of the most different catchment's characteristic is marked in bold and is underlined. The most different catchment is then penalized with one point; these points are summed at the bottom. The perimeter and length of the catchments are not evaluated as they are used in the calculations of other (evaluated) geometric characteristics. Based on Table 2, it can be concluded that the Buková catchment is the most dissimilar in terms of geometric properties, and it is recorded in Table 6, which summarizes all the properties. An analysis of the altitude, slope, and orientation of the catchments slopes was performed using a digital elevation model (Figure 3), as these are the primary topographic attributes that influence hydrological processes. An analysis of the altitude, slope, and orientation of the catchments slopes was performed using a digital elevation model (Figure 3), as these are the primary topographic attributes that influence hydrological processes. (Table 3). Compared to the other catchments, Buková's median elevation was relatively low, and its elevations also in- The lowest and highest altitudes were found in the Buková catchment at 195.3 m above sea level and 738.4 m above sea level, respectively (Table 3). Compared to the other catchments, Buková's median elevation was relatively low, and its elevations also included the highest number of outliers; see Figure 4. Additionally, the Modra-Piesok catchment had the highest median altitude and deviated from the other catchments' altitude conditions. In terms of this characteristic, Buková and Modra-Piesok are the most different from the other analyzed catchments (and are penalized in Table 6). The hypsometric curves of the evaluated catchments are shown in Figure 5, a nondimensional measure of the proportion of the catchment above a given elevation. Hypsometry is used as an indicator of the geomorphic form of catchments. Many researchers have postulated that this is an important characteristic of a catchment's form and is useful for explaining various hydrological or erosion processes [24,32]. It is clear from Figure 5 that the hypsometric curve of the Buková catchment differs the most from the other evaluated catchments (penalized in Table 6). The hypsometric curves of the evaluated catchments are shown in Figure 5, a nondimensional measure of the proportion of the catchment above a given elevation. Hypsometry is used as an indicator of the geomorphic form of catchments. Many researchers have postulated that this is an important characteristic of a catchment's form and is useful for explaining various hydrological or erosion processes [24,32]. It is clear from Figure 5 that the hypsometric curve of the Buková catchment differs the most from the other evaluated catchments (penalized in Table 6). The boxplots of the slopes ( Figure 6) show a similar distribution of values in the Buková, Horné Orešany and Píla catchments; the most frequent slope has a value of around 9°. The Modra-Piesok catchment distribution of slope values shows the smallest standard deviation in the set, i.e., 4.46, and the highest skewness (Table 3), so it was evaluated as the most dissimilar to the rest of the evaluated catchments from this point of view (and it is penalized in Table 6). The boxplots of the slopes ( Figure 6) show a similar distribution of values in the Buková, Horné Orešany and Píla catchments; the most frequent slope has a value of around 9 • . The Modra-Piesok catchment distribution of slope values shows the smallest standard deviation in the set, i.e., 4.46, and the highest skewness (Table 3), so it was evaluated as the most dissimilar to the rest of the evaluated catchments from this point of view (and it is penalized in Table 6).
The catchments' slope aspects, which were obtained through a analysis of their DEMs, are shown in Figures 7 and 8. The working procedures and methodology of these analyses can be found in geoinformatics literature such as the work of Lovelace et al. [33]. Figure 8 shows the so-called radar graph, which summarizes the data on the slope aspect of each catchment. The relative number of cells with a given slope is plotted on each cardinal and intercardinal direction and labelled on the edge of the radar graph. North corresponds to the values of 0 • (or 360 • ), east corresponds to 90 • , south to 180 • , and west to 270 • . Using a relative view enables the comparison of aspects of catchments with different areas. Both the radar plots and the summary in Table 3 led to the overall conclusion that the Modra-Piesok catchment is the most dissimilar in terms of slope aspect. The boxplots of the slopes ( Figure 6) show a similar distribution of values in the Buková, Horné Orešany and Píla catchments; the most frequent slope has a value of around 9°. The Modra-Piesok catchment distribution of slope values shows the smallest standard deviation in the set, i.e., 4.46, and the highest skewness (Table 3), so it was evaluated as the most dissimilar to the rest of the evaluated catchments from this point of view (and it is penalized in Table 6). The catchments' slope aspects, which were obtained through a analysis of their DEMs, are shown in Figures 7 and 8. The working procedures and methodology of these analyses can be found in geoinformatics literature such as the work of Lovelace et al. [33]. Figure 8 shows the so-called radar graph, which summarizes the data on the slope aspect of each catchment. The relative number of cells with a given slope is plotted on each cardinal and intercardinal direction and labelled on the edge of the radar graph. North corresponds to the values of 0° (or 360°), east corresponds to 90°, south to 180°, and west to 270°. Using a relative view enables the comparison of aspects of catchments with different areas. Both the radar plots and the summary in Table 3 led to the overall conclusion that the Modra-Piesok catchment is the most dissimilar in terms of slope aspect. Table 3 includes a summary of the statistical characteristics of the preceding analyses of the catchments' altitudes, gradients, and aspects of catchments. Figure 9 shows the catchments' drainage network, which was obtained using GIS tools (details are in Appendix A). An important parameter in terms of the outflow regime is the density of the drainage network, which was 1.15 for the Buková catchment; and higher in others, i.e., 1.45, 1.53, and 1.71 for Modra-Piesok, Horné Orešany, and Píla, respectively. The Buková catchment can therefore be considered the most different in terms of drainage network. Table 3 includes a summary of the statistical characteristics of the preceding analyses of the catchments' altitudes, gradients, and aspects of catchments. Figure 9 shows the catchments' drainage network, which was obtained using GIS tools (details are in Appendix A). An important parameter in terms of the outflow regime is the density of the drainage network, which was 1.15 for the Buková catchment; and higher in others, i.e., 1.45, 1.53, and 1.71 for Modra-Piesok, Horné Orešany, and Píla, respectively. The Buková catchment can therefore be considered the most different in terms of drainage network. The soils of the catchments investigated consisted exclusively of the loam, sandy loam, and loamy-sand soil textural classes. According to Figure 10, Buková is almost entirely covered by loamy soil, which is less leaky than sandy soils, making it the most dissimilar catchment compared to the other catchments analyzed.
The land use was evaluated in GIS software using a vectorized map. Deciduous forest was the most common type of land cover. Figure 11 shows the high similarity of Horné Orešany, Modra-Piesok, and Píla, which are predominantly covered with deciduous forest and some transition shrubs, i.e., with a land cover that slows down runoff. The Buková catchment is the only one with larger areas of other kinds of land use, mainly arable land and urbanized areas. This catchment also has the least amount of deciduous forest. Arable The soils of the catchments investigated consisted exclusively of the loam, sandy loam, and loamy-sand soil textural classes. According to Figure 10, Buková is almost entirely covered by loamy soil, which is less leaky than sandy soils, making it the most dissimilar catchment compared to the other catchments analyzed. The land use was evaluated in GIS software using a vectorized map. Deciduous forest was the most common type of land cover. Figure 11 shows the high similarity of Horné Orešany, Modra-Piesok, and Píla, which are predominantly covered with deciduous forest and some transition shrubs, i.e., with a land cover that slows down runoff. The Buková catchment is the only one with larger areas of other kinds of land use, mainly arable land and urbanized areas. This catchment also has the least amount of deciduous forest. Arable land and urbanized areas are more susceptible to surface runoff, which significantly influences the hydrological response of the catchment. In sum, Buková is considered the most different catchment; the percentages of the different types of land are shown in Table 4. As with the other catchment characteristics, this assessment is marked by a penalty in Table 6.
Calibrability
The similarity of catchments was analyzed in this work as a preliminary step toward utilizing this factor in various analyses, such as analogous calculations of river flows in unmeasured catchments. A similar measured (donor) catchment to an unmeasured (target) catchment is sought in such calculations. Then, using the donor catchment, one can calibrate a hydrological model and subsequently use it for a target river catchment.
One important indicator of the suitability of a donor catchment is the possibility to satisfactorily accomplish its rainfall-runoff model's calibration. We refer to this donor catchment property as "calibrability". If calibrating the catchment's model satisfactorily is impossible, then the catchment is not a suitable donor, as its model cannot act as a realistic predictor of flows for another catchment.
Calibrating a hydrological rainfall-runoff model means finding its parameters and ensuring the closest possible agreement of the calculated and measured flows on a given stream. Therefore, on the donor catchment, measured flows are required data to accomplish calibration. "Calibrability" was determined in this work using the TUW hydrological model [34]. This model runs with a daily time step and consists of a snow routine, a soil moisture routine, and a flow routing routine; a genetic algorithm was used to calibrate its 15 parameters. The level of precision of the final model (agreement between measured and computed flows) for all catchments, as expressed by the value of the Nash-Sutcliffe (NSE) coefficient, is shown in Table 5. The highest "calibrability" (NSE values of 0.681 and 0.683) was evaluated for the Píla and Modra-Piesok catchments. They are, therefore, suitable candidates for use as donor catchments. The lowest "calibrability" value was seen in the Buková catchment (0.426); an NSE value lower than 0.5 cannot be accepted as satisfactory. Therefore, it can be concluded that the Buková catchment is not appropriate for the hydrological analogy and is also penalized in Table 6 from this point of view.
Overall Assessment of the Similarity of the Catchments
The overall evaluation of the similarity of the catchments was undertaken using penalization. As previously mentioned, this method is more appropriate for a task in which (for engineering purposes) a smaller number of catchments are analyzed, as opposed to a task in which the whole region is analyzed. It is clear from the literature review that a smaller number of works in the hydrological literature have been dedicated to this practical task. For each previously discussed characteristic, the most dissimilar catchment from the others scored one point, as shown in Table 6. In some ambiguous cases, the second least similar catchment was also given a point if it was also significantly different from the others. The penalization results of the individual catchments, together with their overall total, are shown in Table 6.
Horné Orešany, and Píla were not penalized in any category, and the most often penalized (and thus most different) catchment was Buková, with eight points. The climate indicators are not listed in Table 6, as a high similarity level was reported in terms of the climate conditions across all examined catchments.
In a real-life setting, determining the flows of a target catchment would involve one catchment with unknown flows (target) and the evaluation of the best possible choice (the most similar catchment) from potential donor catchments. In Table 6, the best choice of donor catchment when each catchment is considered the target is evaluated.
For the Horné Orešany catchment, the best donor catchment is Píla; the second-best option is Modra-Piesok ( Table 7). The situation is similar when Píla is the target catchment, and Modra-Piesok could also use Píla and Horné Orešany as donor catchments. It would be most appropriate to look for other donor catchments for Buková, the least similar to all the other evaluated catchments.
Verification of Similarity Using Specific Flows
The purpose of this subchapter is to verify the proposed assessment of the similarity of catchments based on their hydrological response.
Catchments with known flows were selected to test the above-described evaluation of the similarity. We did not directly compare flows in the riverbed, but specific flows, as the catchments vary in size. The specific flows are flows per unit area of the catchment (in mm) and are compared in Figure 12a using box plots. Figure 12b shows the same flows, but their logarithms are displayed for clarity, making the similarity/dissimilarity between the catchments more evident. The smallest specific flows are in the Buková catchment; this finding confirms our methodology of examining the physical similarity of the catchments. From that viewpoint, the Buková catchment was also the least similar to other catchments.
The flows of the other catchments are quite similar (as can be seen in Figure 12b; they have close values of their medians, quartiles and outliers). The proposed method of catchment similarity assessment was also verified using flow correlations. The mutual relation between specific flows is expressed in Figure 13 through a plot of the correlation matrix. This matrix makes it possible to see which catchments have the most similar hydrological regimes to others and which are different. A correlation plot shows in the cells above the diagonal the correlation coefficients between the variables (time series of flows), which are identified by the gauging station name on the diagonal of the matrix. The correlation coefficient in a particular cell is between those two variables, which are in the vertical and horizontal directions from the diagonal. This figure serves as a quick overview of the flow data; therefore, mini histograms and mini scatter plots of all pairs of variables (flows) are also presented. The assignment of the variables for these mini plots is similar to in the case of the correlation coefficients. The average correlation coefficient could be easily evaluated for each catchment; it could be used to characterize a given catchment's overall similarity/dissimilarity to others. This average is the smallest for the Buková catchment (0.54), again confirming its dissimilarity. Such catchments should not be used for calculating flows for unmeasured catchments. The low correlation can mean that there are factors in the catchment that influence its runoff that are not present in the other catchments or the given area. Average correlation coefficients with other catchments are as follows: for Horné Orešany, 0.66, Modra-Piesok, 0.60, Buková, 0.54, and Píla, 0.67. The scatterplots under a diagonal in Figure 13 graphically illustrate the similarity/dissimilarity of the catchments; the smaller the dispersion of points around the regression line, the more hydrologically similar a given pair of catchments are.
Conclusions
This case study evaluated the similarity of the catchments in terms of several factors that influence the rainfall-runoff process. Properties such as slopes, altitudes, soils, land use, and "calibrability" help to determine a catchment's similarity in this work. The similarity identified has potential applications in various engineering tasks, e.g., it is useful for determining flows in unmeasured catchments. The overall similarity was evaluated using the proposed penalty method. The proposed method and results obtained were preliminarily verified using the flows from the catchments to detect whether physically similar/dissimilar catchments are also hydraulically similar/dissimilar. The results confirm this hypothesis, and also confirm the correctness of the proposed penalty procedure, meaning that this study's results can, after further extensive testing, be used in practical water engineering tasks.
The authors are aware that the problem in question is a very complex and not yet fully explored area of research. However, even at the current, still open level of research, similar problems need to be addressed in practice. This case study should contribute to a practical solution and does not have the aim of solving complex theoretical issues related to the similarity of catchments and the calculation of flows in unmeasured catchments. For this reason, the authors are considering the further verification of this method for larger areas and using the proposed methodology of identifying the similarity of catchments for engineering tasks in their subsequent works.
Data Availability Statement:
The data presented in this study are partly available on request from the corresponding author. Some of the data are only available from the Slovak Hydro-meteorological Institute in Bratislava on a commercial basis.
The basic geometric characteristics were determined in QGIS [25] software based on the digital map work SVM 50, which was created on the basis of the basic map of the Slovak Republic at a scale of 1:50,000 (ZM50). Thematic maps of watercourses and other water bodies, catchments and DEM were used in this work. The S-JTSK coordinate system is used in these maps.
.2. Catchment Length (LC)
Catchment length is somewhat ambiguously defined in the hydrological literature [35] and can be defined in more than one way-it can be the longest straight line distance between any two points on the perimeter or the greatest distance between the outlet and any point on the perimeter or the length of the main stream from its source (projected to the perimeter) to the outlet, etc. In this work was length of catchment defined as the length of the simplified main stream from its source (projected to the perimeter) to the outlet. Form factor [22] The area of the catchment divided by the square of length of the catchment: Form factor [22] The area of the catchment divided by the square of length of the catchment: Circulation ratio [24] R c = A/A c (A2) A c the area of a circle having equal perimeter as the perimeter of drainage catchment in [km 2 ] Elongation ratio [36] Re = 2 × L C × A 0.5 /π (A3) Shape factor [22] F s = L 2 C /A (A4) Catchment boundary segmentation CBS = P/(2 × (π × A) 0.5 ) (A5) Appendix A.2. The Drainage Network Creation Since the authors of the article had at their disposal the mentioned digital maps SVM 50, including a map of watercourses, this map was used to determine the length of the river network. Using QGIS commands, the total length of the river network was identified. The basic QGIS commands were also used to identify catchments areas. By dividing the length of the river network by the catchment area, the density of the river network is evaluated. If it is necessary to determine the drainage network, it can be generated from a digital terrain model. E.g., SAGA GIS commands integrated into QGIS could be used, specifically the "Channel network" module. This module requires a grid that contains elevation data and various parameters such as the minimum segment length and different algorithm initiation parameters. The output of the command is a grid and vector version of the drainage network.
Appendix A.3. Hydrologic Modelling in Context of the "Calibrability" Evaluation
The flows in every catchment were calculated in R software ecosystem by the TUW conceptual rainfall-runoff model [33], with a genetic algorithm used for calibration [37]. The calibration was implemented based on flow and climate data from time period 1980-2005. The remaining available data for the years 2006 to 2017 were used to evaluate the calibrated model. The results of this testing were used to calculate the flows, which were used to determine the NSE statistics in Table 5. The genetic algorithm population was set to 500, the number of parameters to be determined was 15, and the maximum number of generations was set to 20. The objective function sought to maximize the NSE. | 2021-10-20T15:14:20.286Z | 2021-10-15T00:00:00.000 | {
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54568522 | pes2o/s2orc | v3-fos-license | Isolation of Tibet orbivirus, TIBOV, from Culicoides Collected in Yunnan, China
We isolated a novel virus strain (YN12246) from Culicoides spp. specimens collected at the China-Laos-Myanmar border in southern Yunnan Province. This virus had a cytopathic effect (CPE) on both insect cells (C6/36) and mammalian cells (BHK-21). Electron microscopy revealed the structure of the virions to be spherical with a diameter of 75 nm. Polyacrylamide gel analysis demonstrated that the viral genome consisted of 10 segments of double-stranded RNA (dsRNA), with a distribution pattern of 3-3-3-1. The coding sequences of 9 genome segments of YN12246 (Seg1, Seg3-Seg10) were obtained by high-throughput sequencing and Sanger sequencing. Comparisons of conserved genome segments 1 and 3 (Seg1 and Seg3), encoding the polymerase-VP1 and sub-core T2 protein, respectively, showed that YN12246 groups with the Culicoides-borne orbiviruses. The highest levels of sequence identity were detected between YN12246 and Tibet orbivirus (TIBOV), indicating that they belong to the same virus species (with amino acid identity of 98.8% and 96.4% for the polymerase and T2 protein, respectively). The data presented here confirm that YN12246 is a member of the TIBOV species, which was first isolated from mosquitoes in 2009. This is the first report of the isolation of TIBOV from Culicoides.
Introduction
The genus Orbivirus is the largest of 15 genera within the family Reoviridae, currently containing 22 recognized virus species [1]. Several species are known to cause serious disease in animals, and orbivirus infections have also been documented in association with human disease [2]. The orbiviruses are vector-borne, primarily transmitted by ticks or hematophagus insectvectors (including Culicoides, mosquitoes and sand flies), and have a wide host-range that collectively includes domestic and wild ruminants, equines, marsupials, sloths, bats, birds and humans [3][4][5]. Previous research has shown that oribiviruses can be divided into three groups, namely tick-borne, mosquito-borne and Culicoides-borne, according to evolutionary data [1]. Bluetongue virus (BTV), African horse sickness virus (AHSV) and Epizootic hemorrhagic disease virus (EHDV) are transmitted by adult Culicoides and are regarded to be the most important orbiviruses with respect to economics [3,6]. Although sequence data are available for several of the insect-borne orbiviruses, few of the tick-borne orbivirus species have been analyzed, including Broadhaven virus (BRDV; a Great Island virus species) [7] and St. Croix River virus (SCRV; a St. Croix River virus species) [8]. Comparison of the sequences of homologous proteins has revealed greater genetic divergence between the insect-borne and tick-borne orbiviruses (with 23-38% amino acid (aa) identity) than among the species within either of these groups [8].
The Tibet orbivirus (TIBOV) was first isolated by our laboratory from Anopheles maculatus mosquitoes collected in 2009 from Motuo County in Nyingchi Prefecture, Tibet, China. Molecular genetic analysis revealed that this virus was a new species within the genus Orbivirus [9], denoted the Tibet orbivirus. Although this virus was first isolated from mosquito specimens, molecular evolutionary genetics analysis revealed that it was not closely related to the mosquito-borne orbiviruses, and was instead a better fit for the Culicoides-borne group. Because only one strain (XZ0906) was available for analysis, it was difficult to determine the propagation characteristics of TIBOV.
During a survey of arboviruses in Yunnan Province in 2012, our laboratory isolated a virus strain (YN12246) from a pool of Culicoides spp. specimens, and identified it as a TIBOV strain. Here, we describe the sequence analysis of polymerase (Pol) and T2-subcore-protein of TIBOV. Phylogenetic comparisons with different orbiviruses indicate that TIBOV belongs to the Culicoides-borne orbivirus group. This is the first report of TIBOV isolated from Culicoides.
Sample collection and virus isolation
Culicoides samples were collected in the summer of 2012 in Daluo, Xishuangbanna Dai Autonomous Prefecture, Yunnan province, China. The specimens were frozen to death in a -20°C freezer, then classified and identified on ice, dispensed into cell-freezing tubes, recorded and labeled, stored in liquid nitrogen and finally transported to our laboratory. Specimens were then homogenized, inoculated and blind passaged three times (7 days per cycle) on monolayers of C6/36 and BHK-21 cells in 24-well plates, as previously described [11]. The cells were observed daily for signs of a cytopathic effect (CPE). The culture supernatant was stored at -80°C for further analysis.
Electron microscopy of virions
The culture supernatant from infected BHK-21 cells showing signs of CPE was centrifuged at 8000 rpm for 20 min at 4°C. The resulting supernatant was further centrifuged at 30,000 rpm for 2 h to remove cellular debris. The final supernatant was fixed in 2% glutaraldehyde and processed for negative-stain electron microscopy with 2% phosphotungstic acid.
dsRNA-polyacrylamide gel electrophoresis
Viral RNA was isolated as described previously, and analyzed by polyacrylamide gel electrophoresis (PAGE) [12].
Virus identification by RT-PCR
Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen), and cDNA was synthesized using Ready-To-Go You-Prime First Strand Beads (GE Healthcare), according to the manufacturer's procedure. Samples were subjected to Reverse-transcription polymerase chain reaction (RT-PCR) with specific primers designed to detect the 12th segment of Banna virus (BAV) [13], the 12th segment of Liao ning virus (LNV) [14], the 12th segment of Kadipiro virus (KDV) [15], the 7th segment of Yunnan orbivirus (YUOV) [16] and the 1st and 3rd segments of Tibet orbivirus (TIBOV) [9] (Table 1). Positive samples were sequenced in both directions and the obtained sequences were compared with those available in public databases (GenBank).
Preparation of viral DNA and RNA and Ion Torrent sequencing
Viral DNA was extracted from 200-μL aliquots of virus-infected BHK-21 cell culture supernatants using a QIAamp DNA BloodMini Kit (Qiagen). Viral RNA was extracted from 140-μL aliquots of virus-infected BHK-21 cell culture supernatant using a QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. cDNA was made with a Ready-To-Go kit (GE Healthcare) using random hexanucleotide primers. Samples were then amplified as described previously [9]. Amplification products were sequenced at the National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The Ion Sequencing Kit (Life Technologies) was used with the Personal Genome Machine (PGM) sequencer as described in the Ion Sequencing Kit User Guide [17] (part no. 4467391 rev. B, 04/2011).
RT-PCR was performed to fill in gaps between viral gene sequences obtained with Ion Torrent sequencing using contig-specific primers. Total viral RNA was extracted as described above, cDNA was generated by reverse transcription, and used as a template for complete genome amplification. Next, a set of specific primers was designed to amplify each segment of the viral genome and the amplification products were sequenced using the Sanger method ( Table 2).
Phylogenetic analysis
Multiple sequence alignments were performed using the Clustal X 1.8 software. Phylogenetic and molecular evolutionary analyses were conducted using the Mega 5.05 software. The phylogenetic tree was generated using the neighbor-joining (NJ) method, distance-p model and 1000 replications of bootstrap values. The sequences obtained in this study were compared with those available in public databases, and representative Reoviridae and Orbivirus sequences were used for phylogenetic analysis. The background information of all virus strains used in this study is shown in S1 Table.
Virus isolation
A single pool of Culicoides spp. homogenized supernatants was inoculated onto monolayers of C6/36 and BHK-21 cells, yielding a virus isolate designated YN12246. The supernatant of YN12246 resulted in CPE in C6/36 and BHK-21 cells in successive cell passages. By 48 h postinfection, shrinkage, aggregation and shedding were observed in C6/36 cultures, and rounding, lysis and shedding were observed in BHK-21 cells (Fig 1).
Observation of virus morphology
The supernatant of cultures showing signs of CPE after virus infection were subjected to negative staining with phosphotungstic acid. Using transmission electron microscopy, YN12246 particles were observed to be approximately 75 nm in diameter, spherical, lacking an envelope, and with an obvious capsomere on the particle surface (Fig 2).
Identification of the dsRNA genome structure
Viral RNA was harvested from the culture supernatant of infected BHK-21 cells, and analyzed by PAGE, revealing a 10-segmented double-stranded RNA genome with a migration pattern of 3-3-3-1. Within this pattern, segments 7, 8 and 9 were very weak, but still identifiable, whereas segment 10 was extremely weak (Fig 3).
Virus identification
RT-PCR, using primers designed to target Seg1 (869 bp long) and Seg3 (713 bp long) of the TIBOV genome, generated cDNA of the expected sizes (885 bp for Seg1 and 706 bp for Seg3) from the YN12246 template. However, reactions using primers designed to amplify BAV, LNV, KDV and YUOV all failed to generate PCR amplicons. Sequence analysis showed that Seg1 and Seg3 from YN12246 has a high level of nucleotide sequence identity (94% and 80%, respectively), and a high level of aa identity (98% and 95%, respectively), with TIBOV. Sequence analyses of Seg1 and Seg3 and comparisons with published data, demonstrated that the YN12246 virus from Yunnan is a strain of TIBOV.
Sequence analysis of the TIBOV genome segments
The whole coding sequences of 8 segments of YN12246 (Seg1, Seg3-Seg5, Seg7-Seg10) and partial sequences of Seg6 were obtained by high-throughput sequencing and Sanger sequencing. Differences were observed in both the nucleotide and amino acid sequences of virus YN12246 relative to XZ0906 ( Table 3). The results showed that there are only 4 segments of YN12246 are the same as the mosquito-isolated strain (XZ0906) among the 8 segments available in the nucleotides length of the complete coding sequence. The identical ones are Seg-4, 5, 7 and 9 respectively, and the rest segments are shorter than that of the mosquito-isolated strain (XZ0906). As shown in Table 3, the difference of the nucleotides length between Culicoides-isolated strain (YN12246) and mosquito-isolated strain (XZ0906) is among 9-135 bp, the difference between Seg1 and Seg8 is above 100 bp, the difference between Seg3 and Seg10 is within 100 bp. It means that there is a big difference in the genome length among TIBOV isolated from different vectors. Besides, there is a larger difference (63.6-99.6%) in the homologous aa with each segment between Culicoides-isolated strain (YN12246) and mosquito-isolated strain (XZ0906). Seg1, 3, 4, 5, 7 and 9 of YN12246 exhibited a relatively high aa identity (95.1-99.6%) to their homologues in the XZ0906. Seg8 and 10 are slightly lower than that aa sequence identity respectively for 81.1% and 74%, and the identity between YN12246 and XZ0906 is 63.6% in Seg 6 (partial sequence). The sequences of Seg2 and partial sequences of Seg6 of YN12246 were not obtained by high throughput sequencing and gene specific amplification (see the discussion section). The 1003 bp nucleotide sequences (660-1633) of Seg6 of YN12246 were obtained, and the rest sequences of Seg6 of YN12246 were not obtained (see Table 3). The sequence data were deposited into the GenBank database (accession numbers KP099640 and KP099641 for Seg1 and Seg3, respectively). Whole genome sequence information of representative members of genus Orbivirus, including BTV, EHDV, YUOV, Pata virus (PATV), Palyam virus (PALV), and SCRV, was obtained from NCBI GenBank. The Seg1 and Seg3 sequences were downloaded and compared with those of YN12246.
Phylogenetic analysis
To better understand the taxonomic classification of YN12246, the amino acid sequences of 37 VP1 proteins (S1 Table) covering 14 genera within the family Reoviridae were obtained from GenBank, and used to construct a phylogenetic tree. These 37 virus strains (including different species and different serotypes of one species) readily clustered into 14 evolutionary branches, with YN12246 clustering within the genus Orbivirus branch (Fig 4A). To further determine the genus of YN12246, VP1 amino acid sequences from 29 known orbivirus strains were used to construct a phylogenetic tree specific to this genus (S1 Table). This analysis demonstrated that YN12246 is most closely related to the TIBOV strain XZ0906, with the two strains clustering into the same phylogenetic branch (Fig 4B).
The gene encoding the major sub-core protein (T2) is the most conserved of the orbivirus genes. The T2 protein of Orbivirus is, therefore, useful for classifying members of the genus [8]. T2 amino acid sequences from 26 known orbivirus strains, along with the equivalent region from YN12246, were selected for construction of a phylogenetic tree. This analysis showed that YN12246 clustered into the same branch as the TIBOV strain XZ0906, suggesting that YN12246 from Yunnan is a strain of TIBOV (Fig 4C).
Discussion
The parameters stipulated by the International Committee on Taxonomy of Viruses (ICTV) for the 'polythetic definition' of individual Orbivirus species include the reassortment of genome segments, genome segment migration patterns during 1% agarose gel electrophoresis (AGE), conserved terminal nucleotide sequences, serological cross-reactions, comparison of homologous genome segments or proteins by sequence analysis or cross-hybridization, host and vector range and the nature of clinical signs induced [3]. Recent advancements in molecular biology and sequencing technologies have allowed phylogenetic comparisons to be used as major tools for orbivirus identification, enabling taxonomic classification and the development of diagnostic tests. Sequence data generated for conserved orbivirus genes (e.g. the T2, polymerase or T13 protein genes) have been used previously for phylogenetic comparisons and taxonomic classification [18,19].
The genetic sequences of VP1 and T2 can be used to define new species within the genus Orbivirus. The RdRp protein sequence, encoded by genome segment 1 (VP1), is an important criterion for the identification of members of genus Orbivirus [7,8]. Previous studies had shown that all orbiviruses have more than 30% aa sequence identity with respect to viral RNA polymerase (VP1) [8]. The VP1 proteins from YN12246 and TIBOV XZ0906 share 98.8% aa identity. In contrast, YN12246 VP1 shares between 35.4% and 73% aa identity with VP1 from other representative Orbivirus members. These data indicate that YN12246 belongs to genus Orbivirus. In addition, the sub-core protein (T2) of Orbivirus is used to classify serotypes within the genus. It has also been demonstrated that orbiviruses within a single species group have more than 91% identity with respect to the T2 protein aa sequence [8]. YN12246 and TIBOV XZ0906 share 96.4% aa identity in relation to T2 protein. In contrast, a lower level of aa identity (23.3%- 78.3%) is seen between T2 protein from YN12246 and members of other Orbivirus species.
These data indicate that YN12246 and XZ0906 belong to a single serotype. From the phylogenetic analysis of family Reoviridae and genus Orbivirus, based on the VP1 and T2 protein aa sequences, it is possible to conclude that YN12246 is a member of the TIBOV species. Sequencing on the Ion Torrent PGM sequencer was completed, producing a total of 4,506,288 reads. A total of approximately 800,000 reads (data not shown) was obtained relative to the genus Orbivirus by bioinformatics analysis. RT-PCR amplification was used to close the gaps between contigs for each segment. Ultimately, the whole coding sequences of 8 segments of YN12246 (Seg1, Seg3-Seg5, Seg7-Seg10) and partial sequences of Seg6 (660-1633) were available. Then Sanger sequencing was employed to confirm the sequences using primers newly designed for each of the 9 RNA segments (Table 2). However, the sequences of Seg2 and partial sequences of Seg6 of YN12246 were not obtained. Although several contigs related to Seg2 and Seg6 of orbivirus were acquired, but RT-PCR or sequencing of PCR product all failed. Orbivirus strains. This analysis employed a neighbor-joining method (using the P-distance algorithm) using the MEGA software (version 5.05; www.megasoftware.net). Bootstrap probabilities for each node were calculated using 1000 replicates. Scale bars indicate the number of amino acid substitutions per site. In Fig 4C, because many of the available sequences are incomplete, the analysis was based on partial sequences (residues 356-567 relative to the BTV-1A sequence). Abbreviations and serotypes (or strain name) are shown in the radial tree image of Fig 4. GenBank accession numbers and further details of the sequences can be found in S1 Table ( Previous studies have shown that the VP1 and VP3 proteins (encoded by Seg1 and 3 respectively) of BTV are highly conserved, while the components of the BTV outer capsid, proteins VP2 and VP5 (encoded by Seg2 and 6 respectively), are the most variable of the viral proteins. VP2, in particular, contains neutralising epitopes and controls the interactions between the virus particle and neutralising antibodies, exhibits a relatively low amino acid identity among the different BTV serotypes [20]. Amino acid identities between Culicoides-isolated strain (YN12246) and mosquito-isolated strain (XZ0906) were 98.8% for VP1 and 96.4% for VP3 (T2 protein) but only 63.6% within the VP6 gene (partial sequences), exhibited the lowest homology. On the other hand, the failure to obtain the sequence of Seg2 by high-throughput sequencing indicates a very low homology in the VP2 gene between Culicoides-isolated strain and known orbivirus. Accordingly, Seg2 may be the hypervariable region in TIBOV, further research will be carried out. We will continue to carry out the sequencing work of Seg2 and Seg6, in order to obtain the whole genome sequence of the virus YN12246.
The cumulative genetic information provided by many studies has confirmed that Orbivirus can be divided into three groups, namely Culicoides-borne orbiviruses (such as BTV), mosquito-borne orbiviruses (such as YUOV) and tick-borne orbiviruses (such as Great Island virus; GIV) [1,19]. These studies not only demonstrated that orbiviruses are vector-borne viruses but also elucidated the propagation characteristics of various viruses, and identified evolutionary adaptations to the transmission vector. The result of the T2 protein phylogenetic analysis performed in this study is consistent with previous research. It is interesting to find that YN12246 isolated from Culicoides clusters onto the same branch as the TIBOV strain XZ0906 isolated from mosquitoes, in addition to BTV and EHDV, which are transmitted primarily by Culicoides, but is phylogenetically distinct from YUOV, UMAV (Umatilla virus) and SLOV (Stretch Lagoon virus), isolated from mosquitoes, as well as GIV, BRDV and LIPV (Lipovnik virus) isolated from ticks (Fig 4C). YN12246 and XZ0906 can both be clearly categorized into the Culicoides-borne group of viruses, suggesting that the TIBOV strains isolated from both mosquitoes and Culicoides can be considered Culicoides-borne orbiviruses. Although Eubenangee virus (EUBV), Tilligery virus (TILV), and PATAV were initially isolated from mosquitoes, EUBV has also been isolated from Culicoides, and the evolutionary relationship based on the analysis of the T2 subcore shell protein indicates that these three viruses group more closely with the Culicoide-borne orbiviruses, suggesting that Culicoides may act as their biological vector [21]. There is therefore precedent for orbiviruses originally isolated from one vector to be assigned to a phylogenetic group associated with a different vector.
Viruses within a single Orbivirus species/serogroup usually have similar dsRNA migration profiles in 1% agarose gel electrophoresis (AGE) [3]. The viral RNA migration band pattern, as resolved using PAGE, has been proposed as a criterion for the identification of Orbivirus [22]. YN12246, isolated from Culicoides, has a 3-3-3-1 RNA migration pattern almost identical to that of XZ0906, isolated from mosquitoes [9], suggesting that they belong to the same species. The RNA-PAGE migration pattern of YN12246 is broadly similar to those of EHDV, EUBV, TILV and PATAV [21], indicative of relatively close relationships between the different virus species. The PAGE migration data are therefore consistent with the T2 protein phylogenetic studies.
The indirect immunofluorescence assay (IFA) was applied to detect the IgG antibodies of swine serum samples collected in the virus isolation region against YN12246 strain. The results showed that a special fluorescent reaction appeared between YN12246 virus and swine serum, the antibody positive rate was 14.0% (8/57), while no positive for the serum samples collected from other regions was detected (data not shown). The results suggest that the TIBOV isolated from Culicoides was infectious to pigs, although the results of IgG antibody positive need to be verified by neutralization test.
Both TIBOV XZ0906, isolated from mosquitoes, and TIBOV YN12246, isolated from Culicoides and collected in Yunnan, could replicate in insect cells (C6/36) and mammalian cells . While TIBOV XZ0906 only caused CPE in BHK-21 cells, and not in C6/36 cells, TIBOV YN12246 caused CPE in both cell types. This result may reflect phenotypic differences between strains of one virus species isolated from different vectors. Further study is required to investigate this phenomenon further. In addition, although CPE progressed quickly after viral inoculation of cell cultures, with up to 75% of BHK-21 cells showing CPE after 3-4 days of YN12246 infection, multiple plaque formation revealed a relatively low virus titer. The titer of the TIBOV strain YN12246 was approximately 1.0 × 10 4 -1.5 × 10 4 PFU/mL, similar to the XZ0906 strain (data not shown). This low virus titer might represent a defining characteristic of the TIBOV orbivirus.
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225667951 | pes2o/s2orc | v3-fos-license | Co-gasification of Cassava Rhizome and Woody Biomass in the 1 MWel Prototype Dual Fluidised Bed Gasifier by Gussing Renewable Energy
In the current research, the effect of the mixture ratio by weight of wood chips to cassava rhizome (100%:0%, 75%:25%, and 50%:50%) was investigated on the properties of the product gas produced from the Dual Fluidised Bed gasifier power plant. The DFB gasifier power plant is located in Nongbua district, Nakhon Sawan province, Thailand. The results from this study show that the use of 100% wood chips as a fuel generates high quality product gas as designed. The mixture of wood chips and cassava rhizome in the weight ratio of 75%:25% and 50%:50% also gives satisfactory results: steady operation conditions of the whole power plant process, good quality and quantity of product gas, however, the tar content in the product gas was slightly higher than that of using wood chips alone. The researchers found that cassava rhizome can be used as a fuel mixture together with wood chips in the current DFB gasifier at site to generate heat and electricity. The outcome of this research will create the use of waste cassava rhizome, enormously available around the power plant, as well as the broad application of gasification technology using various biomass feedstock types available in Thailand.
Introduction
Gasification is the thermo-chemical conversion of any carbonaceous fuel to a combustible gas, where the fuel can be in the form of solid, liquid, and gaseous feedstocks such as coals, biomass residues, oils, and natural gases [1]. When air or oxygen is used as the gasification agent, the gasification is actually a partial oxidation process which applies heat to the feedstock at sub-stoichiometric levels of oxygen to that required for complete oxidation. However, when steam is used as the gasification agent, the gasification process is endothermic in overall and thus external heat needs to be supplied. In this research, the gasification technology used is the Dual Fluidised Bed (DFB) gasifier system using steam (H2O) as a gasifying agent. It has been reported that the product gas produced from the DFB gasifier primarily comprises hydrogen (34-45 vol%), carbon monoxide (20-30 vol%), carbon dioxide (15-25 vol%), and methane (8-12 vol%) [2]. The product gas is of high quality with a high calorific value of 12-14 MJ/Nm 3 . This product gas can be utilised in a variety of applications: in gas turbines or engines for heat and power generation, and for further production of hydrogen gas, synthetic natural gas, synthetic liquid fuel, and various chemical products.
The biomass steam DFB gasifer was first invented and developed in a laboratory at the Vienna University of Technology (VUT) by a team of scientists and engineers led by Prof. Hermann Hofbauer and Prof. Reinhard Rauch. The first commercial combined heat and power (CHP) DFB gasification system was established in Gussing, Austria, by the contribution of several parties since 2001 [2]. The Gussing plant uses local wood chips as feedstock to generate 2.0 MWel electricity to the national power grid and 4.5 MWth heat to the households, and businesses of Gussing, since 2002. The Gussing plant was in operation for almost 100,000 hours and it is owned by Biomasse Kraftwerk Güssing GmbH & Co KG, which is a subsidiary of Gussing Renewable Energy International Holding GmbH, Austria.
Gussing Renewable Energy (GRE), a so-called Carbon Recycling enterprise, is the developer of the DFB gasification technology to convert biomass and organic solid wastes to usable energy in a form of high heating-value and valuable gas. The gas product can be used for electricity and heat generation (Combined Heat and Power (CHP), or for other valuable products including but not limited to purehydrogen gas, synthetic natural gas, synthetic liquid fuel, and chemical products (methanol, ethanol, etc.). GRE provides and supports its customers on engineering, procurement, construction (EPC), operation, training of the operators on DFB gasification process, troubleshooting, etc.
GRE has developed and constructed the 1 MWel CHP DFB gasifier power plant in Nongbua district, Nakhon Sawan province, Thailand [3]. The Nongbua plant was designed to operate with various biomass resources such as wood chips, sugarcane leaf, corncob, and other biomass renewable resources. Moreover, a mixture of municipal solid waste (MSW) and biomass can also be fuelled into the 1 MWel DFB gasifier [3].
Thailand is a major producer and exporter of agricultural products. The agricultural or biomass residue generation was estimated to be 134 million tons per annum, of which 53% was used for energy production and other purposes [4]. The amount of unconsumed biomass residues in Thailand is extremely large. The remaining amount of more than 60 million tons per annum is available and should be collected and used for alternative energy to generate electricity, heat or transport fuels, which is equivalent to about 4,000 MW electrical output [4]. The main unconsumed biomass includes rice straw, sugarcane leaves, corn leaves, palm leaves, empty fruit bunch, and cassava rhizome [4].
For Thailand, the use of biomass as fuel for energy production contributes to significantly reduction of reliance on foreign oil and natural gas imports. It also reduces the environmental impact since the use of biomass fuel does not increase the emission of carbon dioxide in the atmosphere (zero carbon emission), and carbon dioxide is recycled into cyclic biomass growth (carbon neutral cycle). The Ministry of Energy under the Thai government has announced and conducted "Strategic Alternative Energy Development Plan (AEDP 2015-2036)" to promote and increase the use of renewable energy in the country from 12% of total energy demand in year 2014 to 30% by the end of year 2036 [5]. Under the AEDP, the government set the target from the end of 2014 to 2036 for the use of agricultural residues or biomass to increasingly produce electricity from 2,452 MWel in 2014 to 5,570 MWel in 2036, to generate heat from 5,144 kTOE to 22,100 kTOE, and to generate biofuels from 1,782 kTOE to 8,712 kTOE [5].
In this research project, therefore, the cassava rhizome is the main focus because it is largely available in Thailand of almost 6 million ton per annum and easily obtainable in Nakhon Sawan province where the Nongbua DFB gasifier plant is located. The cassava rhizome has never been tested or used in the DFB gasifier. It is also found that the price of cassava rhizome is much lower than wood chips and thus reducing the feedstock cost dramatically if cassava rhizome can be used. The objective of the current research, therefore, is to investigate the effect of the mixture ratio by weight of wood chips to cassava rhizome on the properties of product gas produced from the DFB gasifier for electricity generation. Results from this research can be used to determine the optimum weight ratio of wood chips to cassava rhizome for the design and improvement of gasification technology to be more efficient. The outcome of this research will create the use of waste cassava rhizome around the power plant and significantly decrease the feedstock cost. This will result to the broad application of gasification technology using various biomass feedstock types available in Thailand. In the next step, the mixture of wood chips and cassava rhizome with the weight ratio of 25%:75% and 0%:100% will be tested to discover the full replacement of cassava rhizome to wood chips as a single feedstock.
The DFB gasifier and its principle
The principle of the DFB gasifier is presented in figure 1 [2,6,7]. The basic concept is the physical separation of the gasification reaction from the combustion reaction, in order to separate the product gas from the conventional flue gas and thus obtaining a nitrogen-free product gas. The DFB steam gasifier comprises two separated chambersa gasification reactor and a combustion reactor (figure 1 and 2). The overall steam gasification reaction is endothermic, and thus it requires energy to be supplied by the circulation of the bed material from the combustion reaction via the loop seal or siphon. Biomass is fed into the bubbling fluidised bed (BFB) gasification reactor using steam as a fluidising and gasifiying agent. Residual biomass char from the gasification reactor is flowed with the circulating bed material through the inclined chute towards the combustion reactor (see figure 2). The residual biomass char is added energy to the combustion chamber and thus the overall DFB gasifier reactor.
The combustion reactor is operated in a fast fluidised bed (FFB) regime fluidising with air and the reaction take places at about 920C and atmospheric pressure. The two connections, namely loop seal and chute, are fluidised with steam, which effectively prevents the gas leakage between the gasification and combustion reactors and it also allows high solid circulation rate. The temperature difference between the gasification and combustion reactors is determined by the required energy and is controlled by the bed material circulation rate and additional fuel (figures 1 and 2). In the BFB gasification reactor operated at about 820C and atmospheric pressure, the heating rate is fast and thus both pyrolysis and gasification reactions take place simultaneously, which results in low concentrations of volatiles (tars) and cleaner product gas being obtained. Figure 4 illustrates the DFB gasification power plant at Nongbua district.
The process can be described as follows. Biomass or non-biomass feedstocks are transported via a fuel handling system and then fed to the BFB gasification bed. The bed material used in the gasifier is calcined olivine sand (iron and magnesium orthosilicate, (Mg, Fe)2SiO4). The choice of olivine is justified by its hardness required for the use in the fluidised bed and its high catalytic activity in biomass steam gasification. The product gas from the DFB steam gasifier has favourable characteristics of low nitrogen content, high hydrogen content, and thus a high calorific value of about 12-14 MJ/Nm 3 is achieved. The nitrogen content originates mainly from the purge gas in the biomass feeder and product gas particle filter.
The product gas from the gasifier flows to a cooler system, a fabric PTFE filter, and a scrubber before going to the gas engine to produce electricity and heat. The product gas is first cooled by two product gas coolers to reduce temperature from about 820C to 280C. The temperature of the product gas is further cooled down from 280C to 200-220C by mixing with the return flow of the cold clean product gas after the scrubber. This novel process step was patented in Austrain Patent (österreichische patentanmeldung), published by Gussing Renewable Energy International Holding GmbH [9]. Next step, the fabric filter is used to remove almost all of the particulates (char, ash, and fine bed material) from the product gas. The particulates from the fabric filter, which contain mainly char, are transported to the combustion chamber to be used as additional fuel. The last step of the gas cleaning is a scrubber to remove completely all the heavy tars and particulates from the product gas by using biodiesel (rapeseed methyl ester, RME). The scrubber also reduces further the product gas temperature from 200-220C to about 40C, which is then compressed to 300 mbar as required for the gas engine. The used biodiesel is used as additional fuel in the combustion zone and the condensed water in the scrubber is used for steam generation for fluidization of the gasification zone. Finally, the clean product gas is drawn to the gas engine to generate electricity and heat. The sensible heat available in the whole process is from the product gas cooling system, the flue gas cooling system, and the flue gas and hot water from the gas engine. This waste heat can be used in various applications such as district heating system, drying, electrical generation by Organic Rankine Cycle (ORC) process, or to further use for cold water generation by absorption chiller, etc.
For the flue gas generated from the DFB gasifier, its sensible heat is used to superheat the steam and preheat air for the gasification reactor and combustion reactor, respectively. The flue gas is then further cooled down to 160-170C before going to the fabric filter for all particulate removal and to the stack.
Materials
Local Thai wood chips, mainly contain softwood, were used in the current research and it was purchased nearby the Nongbua power plant. Cassava rhizome was also purchased locally from the farmer in Nongbua district and it was chipped and sun dried at Nongbua site. The particle cross section length of the two feedstocks is in a range of 0.5-10 cm. The proximate analysis, ultimate analysis, ash fusion temperature of the wood chips and cassava rhizome was conducted by SGS (Thailand) Limited and the results are given in table 1 and table 2, respectively. In the experiment, the mixture ratio by weight of wood chips to cassava rhizome are set at 100%:0%, 75%:25%, and 50%:50%. To control the feed of each mixture ratio, wood chips and cassava rhizome were filled into two different hoppers and two main screw driver sets, where they were designed for this purpose of multi-fuel feeding. The weight of either wood chips or cassava rhizome was set and controlled by the main screw driver control speed. The main screw driver speed was calibrated based on the fuel bulk density and was pretested beforehand. The wood chips as received with the moisture of about 38-39% was used and it was dried in a dedicated wood chips dryer to about 15% before mixing with the sun-dried cassava rhizome as received with moisture content 15%. The mixture was then fed into the bed of the BFB gasifier. The operation conditions of DFB steam gasifier are outlined in table 3. Table 3. DFB steam gasifier operation conditions.
Fuel feed input (kWth)
3,800 Bed material type Calcined olivine Bed material particle size (m) 300-800 Bed material particle density (kg/m 3 ) 2,800-2,900 BFB reactor temperature varied along the height (°C) 800-860 FFB reactor temperature varied along the height (°C) 870-920 Steam to fuel ratio (kg/kgdry) 0.5 In the experiments, the steam to fuel ratio, which is defined as the ratio of mass flow rate of the feeding steam and moisture in the fuel to mass flow rate of the dry fuel feedstock, was set at 0.5 kg/kgdry. The superheated steam for gasification reaction was produced from the condensation of the wet product gas which was separated from the recycled biodiesel in the sedimentation tank, placed below the tar scrubber column. The condensed and separated water from the sedimentation tank was fed to an evaporator to generate steam. This saturated steam was superheated by the recovered heat from the product gas and flue gas cooling systems. Due to the reuse of water, the fresh soft water used for the entire process is extremely low. The soft water is mainly consumed for the make-up in the cooling tower.
Product gas sampling and analysis
The clean product gas after the scrubber was automatically sampled and analysed by the ABB gas analyser, which is installed at site. The compositions of CO, CO2, CH4, and O2 were online measured, presented, and stored in the SCADA system. The H2 composition was determined by calculation.
Sampling of tar in the product gas
Tar sampling and analysis in the product gas from the gasifier process in this study was performed based on European Standard CEN/TS 15439:2006 Biomass gasification -Tar and particles in product gases -Sampling and analysis.
In the experiments, tar in the product gas was sampled at a sampling point after the product gas was removed from tar in a biodiesel scrubber. To collect the tar samples, a dedicated sampling line for tar analysis was designed and presented in figure 5. In the sampling line, the gas sample was firstly drawn through the trace heating and then through impinger bottles in water bath where tar was condensed and absorbed into the absorbing solution. The stainless steel sampling line was trace heated and insulated and was made as short as possible to avoid tar condensation in the sampling line. The controlled temperatures in the sampling line were set higher than the tar dew point and higher than the water dew In the water bath, each of the first three impinger bottles was filled with 200 mL of pure toluene as an absorbing solution. The last bottle was empty to collect the solution in case of an overflow. The temperature of the water in the bath was maintained at 2C. The moisture in the producer gas was condensed in toluene solution during sampling but this did not affect the measurements of tar because the total volume of the toluene and water is evaporated for tar gravimetric analysis.
The procedure and checklist for tar sampling and analysis were developed by the research group between College of Advanced Manufacturing Innovation under King Mongkut's Institute of Technology Ladkrabang (KMITL), and Gussing Renewable Energy (Thailand) company, and by the support from Prof. Reinhard Rauch from Karlsruhe Institute of Technology in Germany. Figure 6 displays tar sampling at Nongbua power plant and tar evaporation in the lab at KMITL.
Gasifier process operation conditions
During the experiments, it was discovered that the entire gasification process was in a steady operation condition over the test period of this study when pure wood chips and the mixture of wood chips and cassava rhizome were used. As can be seen in figure 7-8, the BFB gasifier temperature was constantly operated between 800 and 860C and its pressure drop was in a narrow range of 110-130 mbar. The FFB combustor was operated at a temperature window of 860-930C (figure 9). The pressure at the bottom of the FFB combustor was varied in a small range from 100-160 mbar, while the pressure at the column middle was 5-30 mbar and column top was between -10 and -2 mbar (figure 10). The DFB gasifier was operating effectively with the addition of the cassava rhizome. Figure 10. FFB combustor pressure at different heights of the reactor over the test period.
In figure 11-13, it was found that the operation of the product gas cooling system, the bag filter, and the scrubber was performed very well and steadily over the test period. As shown in figure 11, the product gas from the gasifier from 820C was cooled down to 400-450C with the first cooler among the two in the cooling system. Then, the product gas temperature was reduced to 250-280C after the second cooler and to 200-220C by mixing with cold recycle gas before passing through the bag filter. Figure 11. Product gas temperature going to cooler 1, and between 2 coolers, after cooler 2, and after mixing with returned clean product gas before passing through the bag filter. Figure 12 and 13 show the product gas outlet temperature and pressure across the bag filter and the scrubber, respectively. These parameters are designed parameters and they need to be controlled at the set points during operation. The results proved that the bag filter and scrubber were operated well as designed. Table 4 and figure 14 show the product gas composition and its lower heating value over the whole test period. The concentrations of H2, CO, CO2, and CH4 are in the same range for pure wood chips, and binary mixture of wood chips and cassava rhizome. The lower heating value of the product gas is as high as 12-13 MJ/Nm 3 , which is comparable with previous studies [10]. There were some peaks of the gas compositions as shown in figure 14, which were expected to be the noise of the instrument. From these results, it is shown that binary mixtures of wood chips and cassava rhizome with the weight percent of cassava rhizome up to 50% are good feedstock in the DFB gasification process. Figure 14. Main gas composition in the product gas from the feed 100%:0%, 75%:25%, 50%:50% by weight of wood chips to cassava rhizome.
Tar concentration in the product gas
The results of tar concentration in the product gas present in this study were averaged from two to four repeated measurements (see table 5 and figure 15). figure 15, it is obvious that the addition of cassava rhizome into wood chips in the feed resulted to slightly higher tar in the product gas. Increasing the feed ratio of cassava rhizome in wood chips from 25 wt% to 50 wt% did not show significant change in the tar concentration. The optimisation of the gasification process is to be performed to reduce the tar concentration.
Conclusion
Cassava rhizome was the first time in this present work to be used in the commercial scale DFB gasifier power plant in Nongbua, Thailand. It is concluded that the addition of cassava rhizome to wood chips with the weight ratio up to 50 wt% gives satisfactory results: steady operation conditions of the whole power plant process, good quality and quantity of product gas. The cassava rhizome can be used to mix with wood chips as a binary mixture to feed to the DFB gasifier plant and the plant operates with high performance.
In the next step, the mixture of wood chips and cassava rhizome with the weight ratio of 25%:75% and 0%:100% will be tested to discover the full replacement of cassava rhizome to wood chips as a single feedstock. The optimisation of the gasification process is to be performed to reduce the tar concentration. | 2020-06-11T09:03:30.769Z | 2020-06-11T00:00:00.000 | {
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14818565 | pes2o/s2orc | v3-fos-license | Quercitrin for periodontal regeneration: effects on human gingival fibroblasts and mesenchymal stem cells
Periodontal disease (PD) is the result of an infection and chronic inflammation of the gingiva that may lead to its destruction and, in severe cases, alveolar bone and tooth loss. The ultimate goal of periodontal treatment is to achieve periodontal soft and hard tissues regeneration. We previously selected quercitrin, a catechol-containing flavonoid, as a potential agent for periodontal applications. In this study, we tested the ability of quercitrin to alter biomarker production involved in periodontal regeneration on primary human gingival fibroblasts (hGF) and primary human mesenchymal stem cells (hMSC) cultured under basal and inflammatory conditions. To mimic PD inflammatory status, interleukin-1 beta (IL-1β) was used. The expression of different genes related to inflammation and extracellular matrix were evaluated and prostaglandin E2 (PGE2) production was quantified in hGFs; alkaline phosphatase (ALP) activity and calcium content were analysed in hMSCs. Quercitrin decreased the release of the inflammatory mediator PGE2 and partially re-established the impaired collagen metabolism induced by IL-1β treatment in hGFs. Quercitrin also increased ALP activity and mineralization in hMSCs, thus, it increased hMSCs differentiation towards the osteoblastic lineage. These findings suggest quercitrin as a novel bioactive molecule with application to enhance both soft and hard tissue regeneration of the periodontium.
Periodontal disease is the result from an infection and chronic inflammation of tooth supporting tissues. Gingival inflammation or gingivitis is the first manifestation, which may lead to soft tissue destruction. In some subjects, this progresses to periodontitis, which is defined by alveolar bone and in severe cases tooth loss 1 . Current strategies for periodontal disease treatment are generally successful in eliminating active disease and some of them have achieved a certain degree of regeneration 2 . To eliminate the active disease, mechanical and antimicrobial approaches 3 are used although the effects of local antimicrobial therapy are modest and mostly temporary, while the use of systemic antibiotics can induce the development of bacterial resistance 4 . Furthermore, these strategies alone are insufficient since periodontal disease is the result of destructive inflammation; thus, if successful, treatment frequently results in a process of gingival fibrosis and limited bone remodelling, rather than in true regeneration of the periodontal tissues 5 . In fact, there is considerable data of successful results in periodontal regeneration using guided tissue regeneration, enamel matrix derivative and platelet-derived growth factor, although the outcomes of such modalities are not always predictable 6,7 .
The ideal periodontal treatment should damp down the inflammatory response to either decrease the excessive production of proinflammatory mediators, destructive enzymes and free radicals and stimulate tissue regeneration, allowing for the restoration of soft tissue attachment and bone formation 8 . Recent strategies for periodontal regeneration include the use of graft materials, barrier membranes, gene therapy or growth factors through topical delivery. However, gene therapy may have undesired host immune reactions or potential tumorigenesis and growth factors are unstable and have short half-life 2 . Hence, there is an urgent need for finding bioactive molecules with both soft and hard tissue healing activities for periodontal tissue regeneration applications.
Natural phenolic compounds, such as flavonoids, are ubiquitous, abundant and offer a range of properties and an array of functions such as antioxidant 9 , anti-inflammatory 10 and antimicrobial capacity 11,12 , among others. Actually, in two independent previous screenings we searched for candidate molecules for soft 13 and hard 14 tissue applications. From both studies, quercitrin was selected as the one showing better performance. Quercitrin decreased extracellular matrix (ECM) degradation, reduced oxidative stress levels, promoted scarless wound healing in human gingival fibroblasts and showed antibacterial properties 13 . Furthermore, it also stimulated the differentiation of mouse pre-osteoblastic cells and inhibited osteoclastogenesis from mouse monocytes, which could prevent bone resorption 14 . Moreover, as a catechol-containing flavonoid, quercitrin could help to control the inflammatory response in periodontal disease progression, since molecules with catechol moieties have shown potent anti-inflammatory effects 15-17 . In the present study, we hypothesized that quercitrin could create a microenvironment suitable for periodontal regeneration due to its positive effects on soft and hard tissue cells. Thus, we set an inflammatory environment, mimicking periodontal disease, and we investigated the effects of quercitrin on primary human gingival fibroblasts and primary human mesenchymal stem cells.
Methods
Cell culture. Three different donors of primary human gingival fibroblasts (hGF; Provitro GmbH, Berlin, Germany) were used (range 19-47 years; male:female ratio 2:1). Provitro assured that cells were obtained ethically and legally and that all donors provided written informed consent. Cells were routinely cultured at 37˚C/5% CO 2 , and maintained in fibroblast growth medium (FGM) supplemented with 10% foetal calf serum (Provitro GmbH), amphotericin (50 ng/ml) and gentamicin (50 μ g/ml).Experiments were performed with hGF between passages 7 and 8 after isolation. Three replicate wells for each donor were seeded in 96-well plates at a density of 7.0 × 10 3 cells per well. Media was supplemented with ascorbic acid (100 μ M; Sigma-Aldrich, St. Louis, MO, USA). Experiments were run in duplicate (n = 18).
Two different donors of human bone marrow-mesenchymal stem cells (hMSCs; Stemcell Technologies, Grenoble, France) were used (20 years; male:female ratio 0:2). Stemcell Technologies assured that cells were obtained ethically and legally and that all donors provided written informed consent. Cells were routinely cultured at 37 °C/5% CO 2 , and maintained in low glucose DMEM GlutaMAX (Life Technologies, Carlsbad, CA, USA) supplemented with 10% stem cell-tested foetal bovine serum (Biosera, Boussens, France), penicillin (100 U/ml) and streptomycin (100 μ g/ml). Experiments were performed with hMSCs between passages 5 and 7 after isolation. Six replicate wells for each donor were seeded in 48-well plates at a density of 8.5 × 10 3 cells per well and grown for 2 days prior to media supplementation without (control) or with additives. Experiments were run in duplicate (n = 12).
Effect of quercitrin on hGF. Quercitrin (PubChem CID: 5280459; Sigma-Aldrich) was dissolved in absolute ethanol and kept at − 20 °C. Stock solutions were further dissolved in culture medium, prior to use. Final concentration of ethanol (1%) was included as vehicle control group in all the experiments.
A dose of 200 μ M quercitrin was used. This dose was selected from previous studies in which different doses of quercitrin (1-500 μ M) were evaluated 13,14 . At confluence (3 days after seeding), hGF were treated for short (1 and 3 days) and long term (14 days) with quercitrin diluted in complete FGM. Quercitrin was added at every media change (3 times per week). The inflammatory stimulus was created by the addition of IL-1β (1 ng/ml). Four different scenarios were set ( Fig. 1A): Treatment for 1 day with IL-1β and quercitrin; treatment for 3 days with IL-1β and quercitrin; treatment with quercitrin for 14 days plus IL-1β during the first 3 days (therapeutic approach); and treatment with quercitrin for 14 days plus IL-1β during the last 3 days (preventive approach).
Cytotoxicity and cell viability assays. The presence of lactate dehydrogenase (LDH) in culture media was used as an index of cell death. LDH activity was determined following the manufacturer's Scientific RepoRts | 5:16593 | DOI: 10.1038/srep16593 Figure 1. Effect of quercitrin on hGF stimulated with IL-1β. (A) Experimental design: the effect of quercitrin (QUER) treatment on hGF was evaluated in four different inflammatory scenarios (stripped pattern); treatment for 1 day with interleukin-1 beta (IL-1β ); treatment for 3 days with IL-1β ; treatment with IL-1β during the first 3 days and culturing the cells for 14 days (therapeutic approach; T); and culturing the cells for 14 days plus IL-1β treatment during the last 3 days (preventive approach; P). (B) Cytotoxicity measured after 1 and 3 days of treatment. Dotted line represents high control (100% cytotoxicity). (C) Gene expression results: cells were treated with quercitrin (grey bars) or without (white bars), following the experimental design. Data were normalized to reference genes, expressed as percentage of control (vehicle), which was set to 100%. Values represent the mean ± SEM of two independent experiments. One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (*) treatment versus control (vehicle); (#) quercitrin versus vehicle in the presence of IL-1β for each time point. instructions (Cytotoxicity Detection kit, Roche Diagnostics, Mannheim, Germany). Results were presented relative to the LDH activity in the medium of cells treated with the vehicle control (low control, 0% of cell death) and of cells treated with 1% Triton X-100 (high control, 100% cell death). Cytotoxicity percentage was calculated using the following equation: Cell viability was measured after 19 days of hMSC culture using PrestoBlue (Life Technologies, Carlsbad, CA, USA) following manufacturer's protocol. Absorbance data was normalised to the vehicle control group (100% viability).
RNA isolation and real-time RT-PCR analysis.
Total RNA was isolated using Tripure (Roche Diagnostics, Mannheim, Germany), according to the manufacturer's protocol. Total RNA was quantified at 260 nm using a nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The same amount of RNA (0.2 μ g) was reverse transcribed to cDNA at 42 °C for 60 min, according to the protocol of the supplier (High Capacity RNA-to-cDNA kit, Applied Biosystems, Foster City, CA, USA). Aliquots of each cDNA were frozen (− 20 °C) until the PCR reactions were carried out.
Real-time PCR was performed for two reference genes, glyceraldehyde-3-phosphate dehydrogenase and beta-actin, and target genes (Table 1). Real-time PCR was performed in a thermocycler (Lightcycler 480, Roche Diagnostics) using SYBR green detection. Each reaction contained 7 μ l of master mix (Lightcycler 480 SYBR Green I Master, Roche Diagnostics), 0.5 μ M of each, the sense and the antisense specific primers and 3 μ l of the cDNA dilution in a final volume of 10 μ l. The amplification program consisted of a pre-incubation step for denaturation of the template cDNA (5 min 95 °C), followed by 45 cycles consisting of a denaturation step (10 s 95 °C), an annealing step (10 s 60 °C) and an extension step (10 s 72 °C). After each cycle, fluorescence was measured at 72 °C. A negative control without cDNA template was run in each assay. All samples were normalized by the geometric mean of the expression levels of reference genes and fold changes were related to the control groups using the following equation: ratio = E target ΔCp target (mean control − sample) /E reference ΔCp reference (mean control − sample) , where Cp is the is the crossing point of the reaction amplification curve and E the effciency from the given slopes using serial dilutions, as determined by the software (Lightcycler 480 software, Roche Diagnostics). Stability of reference genes was calculated using a statistical tool (BestKeeper software, Technical University of Munich, Weihenstephan, Germany).
Alkaline phosphatase (ALP) activity and calcium quantification. Cells were washed with PBS and lysated with 0.1% Triton X-100. ALP activity was quantified by measuring the cleavage of p-Nitrophenyl Phosphate (pNPP; Sigma-Aldrich) in a soluble yellow end product that absorbs at 405 nm. Twenty-five microliters of sample were incubated with 100 μ l of pNPP. In parallel to the samples, a standard curve with calf intestinal ALP (Promega, Madison, WI, USA) was constructed. On the other hand, 200 μ l of sample were incubated with HCl (1 N) overnight, followed by centrifugation at 500 × g for 2 min for the subsequent determination of Ca 2+ content in the supernatant by inductively coupled plasma atomic emission spectrometer (Optima 5300 DV; PerkinElmer, Waltham, MA, USA). Data were compared with CaCl 2 standards included in the assay.
Statistical analysis. All data are presented as mean values ± standard error of the mean (SEM). The
Kolmogorov-Smirnov test was done to assume parametric or non-parametric distributions. Differences between groups were assessed by paired t-test or Wilcoxon test, depending on data distribution. A specific computer program (SPSS version 17.0, Chicago, IL, USA) was used. Results were considered statistically significant at P ≤ 0.05. One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively.
IL-1β induces the expression of inflammatory markers on hGFs.
Here, the inflammatory mediator IL-1β was used to mimic in vitro the inflammatory process in periodontal disease progression. IL-1β is broadly used to induce experimental inflammation and to enhance the proinflammatory response [18][19][20] , imitating the inflammatory pathways activated in response to oral pathogens 21 . The concentrations used here are in the range with the IL-1β levels usually found in patients with periodontitis 22 . The addition of IL-1β for 1 day significantly stimulated the expression of interleukin-6 (IL6), interleukin-8 (IL8) and matrix metalloproteinase-1 (MMP1), reaching a plateau at 1 ng/mL IL-1β (Table 2). These cytokines are key inflammatory mediators in the progression of periodontal disease 21 and the results are consistent with previous reports in the literature 20,23 . Moreover, hGF viability after 1 and 3 days of stimulation with IL-1β remained similar to the controls (Fig. 1B). Based on this data, the dose of 1 ng/mL IL-1β was selected to simulate a periodontitis condition although it should keep in mind that the only addition of IL-1β does not reflect the entire chronic inflammatory process in periodontal disease in vivo, and can only help to investigate in vitro the effects of quercitrin.
Quercitrin decreases the deleterious inflammatory effects on hGFs.
Modifying the local environment to reduce inflammation is one requirement to achieve complete periodontal regeneration 24 . Inducible cyclooxigenase-2 (COX2) and PGE2 production are highly related to periodontal disease 25 . Here, quercitrin treatment was not toxic (Fig. 1B) and effectively inhibited COX2 expression (Fig. 1C) and its functional product PGE2 (Fig. 2) in hGF under an inflammatory stimulus, in agreement with previous observations using different flavonoids 26 . Moreover, previous studies demonstrated that quercitrin also down-regulates inducible nitric oxide synthase, an enzyme highly expressed by inflammatory stimuli that produces the inflammatory mediator NO 27,28 .
The gene expression of different inflammatory markers after quercitrin treatment was also studied (Fig. 1C). Unexpectedly, the upregulation of IL6 and IL8 induced by IL-1β was not reversed by quercitrin treatmentbut for IL8 mRNA levels at short-term (days 1 and 3) although IL8 protein levels remained unchanged among groups (data not shown), contrary to what was previously proved in lipopolysaccharide-stimulated macrophages and in vivo 27,29 . This finding might be due to the stimulation with IL-1β that activates the complex NF-κ B signalling pathway and positive and negative feedback mechanisms that regulate cytokines expression 30,31 , thus, masking quercitrin anti-inflammatory effect. Accumulating evidence suggests that the effects of phenolic compounds are mediated by interactions with signalling pathways 32,33 . In particular, quercitrin has been shown to have an inhibitory effect on activator protein-1 and NF-κ B pathways, which have central roles regulating cell differentiation and inflammation, among other downstream targets 28,34 .
During the progress of gingivitis, the inflammatory process results in connective tissue breakdown as the result of an imbalance of MMPs over the tissue inhibitors of MMPs (TIMPs) 35 . Here, the inflammatory in vitro model decreased collagen III α 1 (COL3A1) mRNA expression and increased MMP1/TIMP1 IL-1β (ng/ml) mRNAlevels (% of control)
Quercitrin increases osteogenic differentiation of hMSCs.
Bone-marrow MSCs were used in this study since they have the capacity to promote the regeneration of alveolar bone, cementum and periodontal ligament 36,37 and that bone-marrow MSCs are comparable to periodontal ligament MSCs in their differentiation capacity and ability to regenerate periodontal bone 38,39 . This study proves that quercitrin does not show cytotoxic effects (Fig. 3) and significantly increases ALP activity in basal and osteogenic media, and mineralization in osteogenic and inflammatory situations (Fig. 4) in hMSCs. No significant differences were observed in calcium content of cells treated with quercitrin in basal media compared to control. Furthermore, under IL-1β stimulation, final calcium content increased while ALP activity decreased (Fig. 4). During osteoblastic differentiation, ALP peaks at the maturation phase and decreases at the mineralization phase. Therefore, in basal conditions data suggests that cells are in early differentiation phases while in the presence of IL-1β and osteogenic media the decreased ALP activity together with the increased mineralization point to a shifted hMSCs differentiation to earlier time points, in agreement with previous reports 40,41 . This might be explained by the increased metabolic activity in the presence of osteogenic media (Fig. 3B). However, quercitrin addition to the IL-1β -stimulated osteogenic group increased the final calcium content almost 20%, while quercitrin addition to the osteogenic group increased it 80%, although calcium accumulation was higher in the inflammatory groups. It has been demonstrated that IL-1β and dexamethasone increase osteoblast mineralization through two different mechanisms, while dexamethasone increases ALP activity 40,41 , IL-1β decreases inhibitory pyrophosphate anions, an atypical biochemical mechanism without differentiating into osteoblasts 42 . The results presented here show that quercitrin increases ALP activity and mineralization of hMSCs, indicating that quercitrin enhanced the osteoblastic differentiation of hMSCs even in the presence of IL-1β , which is supported by a previous study with mouse pre-osteoblasts 14 . Current research in periodontal regeneration is focused in discovering new bioactive molecules, improving stem cell implantation and developing biomaterials that act as scaffolds or as drug delivery systems 43 . Quercitrin might have different applications in periodontal regeneration since it is a bioactive molecule with antibacterial, anti-oxidant and anti-inflammatory properties and it promotes soft and hard tissue regeneration 13,14,28,29 . Besides, quercitrin could decrease the antibiotic administration and increase the safety of anti-inflammatory drugs currently used for periodontal disease treatment 3 . Regarding to bone regeneration, stem cell-based periodontal regeneration is a promising therapeutic option 37 . One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (*) treatment versus control (vehicle); (#) quercitrin versus vehicle; ( §) inflammation groups versus osteogenic groups.
Furthermore, the inhibition of osteoblastic differentiation of bone progenitor cells from the periodontal tissue has been suggested to be dependent on the local environment 43,44 . Here, quercitrin promoted the osteoblastic differentiation of hMSCs even in an inflammatory situation; therefore quercitrin could help hard tissue to regenerate itself. Furthermore, previous results revealed that quercitrin decrease osteoclast formation in RAW264.7 cells 14 , together with the present results, quercitrin might show an anabolic effect on bone formation. Regarding the development of new biomaterials, our group has also used quercitrin to functionalize Ti-surfaces, showing promising results 45 . Therefore, it is hypothesized that quercitrin could be used in other applications to improve periodontal regeneration. For instance, antioxidants added to oral hygiene products improve periodontal disease indexes 46,47 . Thus, we propose the use of quercitrin as a pharmacological agent in the form of toothpastes, mouth rinses or suitable formulations (e.g. hydrogels) for topical application. Future studies should confirm the effects of quercitrin in vivo.
In conclusion, the positive effects of quercitrin on cells from soft and hard tissue under inflammatory conditions are suggested using human primary cultures of gingival fibroblasts and mesenchymal stem cells. Quercitrin decreases the release of the inflammatory mediator PGE2 and partially re-establishes the collagenolytic metabolism induced by IL-1β stimulation on primary human gingival fibroblasts. Furthermore, quercitrin increases ALP activity and mineralization, thus, enhancing human mesenchymal stem cells differentiation towards the osteoblastic lineage. These findings suggest quercitrin as a bioactive molecule that could create a microenvironment suitable for soft and hard tissue regeneration and therefore enhance periodontal regeneration. | 2016-05-12T22:15:10.714Z | 2015-11-12T00:00:00.000 | {
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52539887 | pes2o/s2orc | v3-fos-license | Notes on glottal flow and acoustic inertial effects
This text is a compilation of some of the notes that the author has written during the development of the low-order model"DICO"[2, 8, 10, 11] for vowel phonation and the even more rudimentary glottal flow model [9] for processing high-speed glottal video data. The following subject matters are covered: (i) Incompressible, laminar, lossless flow models for idealised rectangular and wedge shape vocal fold geometries. Equations of motion and the pressure distribution are computed in a closed form for each model using the unsteady Bernoulli's theorem; (ii) The assumption of incompressibility and energy loss (i.e., irrecoverable pressure drop) of the airflow in airways (including the glottis) is discussed using steady compressible Bernoulli theorem as the main tool; (iii) Inertia of an uniform waveguide is studied in terms of the low-frequency limit of the the (acoustic) impedance transfer function. It is observed that the inductive loading in the boundary condition sums up with the waveguide inertance in an expected way; (iv) It is shown that an acoustic waveguide, modelled by Webster's lossless equation with Dirichlet boundary condition at the far end, will produce the expected mass inertance of the fluid column as the low-frequency limit of the impedance transfer function.
Introduction
This text is a cleaned-up compilation of notes that have been written during the development of the low-order model "DICO" [2,8,10,11] for vowel phonation and the even more rudimentary glottal flow model [9] for processing high-speed glottal video data. While it is not possible to include all details and derivations in journal articles, building even a modest model of phonation will require a great number of mathematical and physical considerations, idealisations, and approximations. There exists a number of fora (such as arXiv.org) where complementary material can be presented almost without any limitations, making excuses of obscurity somewhat moot nowadays. Unfortunately, much of the background material of the above mentioned publications is not included here due to shortness of time and other resources of the author. So much for the justification of this text; let's move on. 1 Briefly, the subject of these notes is an air column inside a perfectly smooth, acoustically reflecting tubular boundary. The air column is both translating (when it is considered incompressible) as well as it is an acoustic medium (where compressibility is a prerequisite for the finite speed of sound). The tube, i.e., the flow channel consists of three parts having finite lengths: the subglottal tract (SGT), the glottis, and the vocal tract (VT). The much shorter glottis is positioned between SGT and VT, and it is the only part of the tube boundary that is assumed to be time-dependent. The walls of the tube at the glottis are called vocal folds. As is sometimes required, the SGT may be considered as having been extended from its free end by a piston made of incompressible material (even, perhaps, fluid) that has a much higher density than air. The mass and the dimensions of the piston add to the total (flow-mechanical part of the) mass inertia if they are taken into consideration. The acoustics is considered only in the VT part using Webster's model, hence restricting it to an immobile boundary.
An outline of these notes is as follows: In Sections 2 and 3, incompressible, laminar, lossless flow models are developed for two kinds of idealised glottis geometries: rectangular and wedge shape vocal folds. Equations of motion and the pressure distribution are computed in a closed form for each model using the unsteady Bernoulli's theorem. This is motivated by, and only feasible due to the extremely simplified nature of the glottis geometry. From these equations, the coefficient of inertance shows up for each of the three parts of the flow channel, and their sum -the total inertance -regulates the mass inertial effects in the fluid movement.
In Section 4, the assumption of incompressibility is examined from the point of view of the flow. Constriction areas and thermodynamic state are computed for glottal openings where the compressible steady flow would reach Mach 0.3 (often considered as the upper limit for air flow to be treatable as incompressible) and Mach 1.0. In Section 5, the VT inertance discovered in the flow models of Sections 2 and 3 is associated to acoustics using the uniform diameter acoustic waveguide as a model. It is, in particular, observed that the inductive acoustic termination at the waveguide end (mouth) will add to the inertance of the acoustic system. That the same holds for general acoustic waveguides with nonconstant intersectional areas is indicated in Section 6.
The lossless, incompressible models introduced in Sections 2 and 3 are not as such suitable for simulation modelling of vowels even when combined with VT and SGT acoustics models. The two main reasons are the following: (i) For performance reasons, it is desirable to have some pruning of terms in the equations of motion (see Eqs. (4) and (10)) as well as in the equations of the hydrodynamic pressure components (see Eqs. (6) and (19)) resulting in the aerodynamic force to the vocal folds (not considered in these notes).
Pruning of a term is considered acceptable if its effect can be compensated by tuning of the model parameters in numerical simulations.
(ii) For accuracy reasons, some sort of (unrecoverable) pressure loss terms must be added. Such pressure loss is due to fluid viscosity or various other entrance-exit effects not accounted for by the classical Hagen-Poiseuille's law. Indeed, the pressure head lost due to friction cannot accelerate the column during the glottal open phase, and the flow at the glottal constriction is viscosity dominated right before the moment of closure. Incidentally, the glottal inertance is singular at the moment of closure as well.
To conclude, the underlying story of these notes is the inertance that makes appearance not only in the (incompressible) flow model for the fluid column acceleration but in the equations of the acoustics of the same fluid volume. All acoustic inertia in the proposed system is also flow mechanical inertia but this is not true conversely: The inertia of the piston (i.e., moving tissues during exhalation) as well as the air jet separating from the lips are parts of the flow mechanical loading, only.
The reader of these semi-informal notes should be warned that this text is not, neither will it be, a proper scientific article. Many necessary attributes of scientific articles are missing, including much of the wider scientific context and all references to works of other authors.
Rectangular glottis
In this section, we consider a rectangular glottis where an incompressible, laminar 2 , lossless flow takes place. The length of the SGT, glottis, and VT denoted by L SGT , L G , L V T , respectively. The subintervals [−L SGT , 0), [0, L G ), and [L G , L G + L V T ] denote these parts of the flow channel, and the flow intersection area A(·) is assumed to be time-dependent only on [0, L G ).
An elementary treatment
The velocity, given the time-variant volume velocity
. . . and what a convenient assumption this is!
The corresponding velocity potential, remembering its continuity, is given by The time derivative is given by Unsteady Bernoulli at points x = −L SGT with pressure p s = p s (t) and x = L G + L V T with ambient pressure p amb = 0: or, equivalently, This is exactly the original version of the "lossless model" for the rectangular glottis, given in [9].
A more elegant version
It is desirable to carry out the computation so that the inertance of the full fluid column is treated in an unified manner. We show next that, in fact, where is the total inertance of the subglottal tract, vocal tract, and the interglottal volume. Note that Our assumptions are that the VT and SGT area functions A V T (s) and A SGT (s) have their glottal ends at s = L G , s = 0, respectively, and that leading to stagnation at both of these ends. For the incompressible flow, the velocity is now given by The corresponding velocity potential, remembering its continuity, is given by that is, Denoting the stagnation pressures p s (t) = lim x1→−LSGT p(x 1 , t) and p amb (t) = lim x2→LSGT p(x 2 , t) at the infinitely wide ends of the tube, we get by taking the limits at the both ends by change of variables. Assuming that p amb (t) = 0 is a constant reference ambient pressure level, the result Eq. (1) follows.
where C (T OT ) iner iner is the total inertance excluding the timedependent glottis.
Solving the glottal flow and pressure
By integration of Eq. (1), Assuming that U (0) = 0 (which is reasonable by fixing the opening point in time) we get U (t) = Using again the unsteady Bernoulli at −L SGT < x 1 < 0 and x ∈ [0, L G ], we get Taking the limit x 1 → −L SGT and noting the stagnation to pressure p s (t), we get Thus, the pressure in the glottis x ∈ [0, L G ] is given by Writing U ′ (t) = h (g ′ (t)v(t) + g(t)v ′ (t)) yields the first version of the equations for the pressure: The third term on the RHS relates to the increase of pressure at the narrowing due to deceleration (i.e., when v ′ (t) < 0). Now, it is possible to eliminate the v ′ (t) term from Eq. (6). Inserting Eq. (4) gives for x ∈ [0, L G ) iner v(t) A few concluding remarks are now in order. The first two terms on the RHS of the top row in Eq. (7) are familiar from the steady Bernoulli principle, and one should note that always Ciner(t) < 1 in the second to the last row. One could call this number partial inertance proportion for an obvious reason, and that correction remains there even in a stationary glottis. It is unclear to me what the last term in Eq. (7) stands for but it certainly vanishes for nonmoving glottis. The expression in the brackets can be written as C
Wedge-like glottis
Let us carry out similar computations as in Section 2 but this time for wedge-like vocal folds. We again assume that the length of the glottis is L G , and the glottal part of the airways is the interval [0, L G ]. The smallest opening is denoted by g = g(t) > 0, and the opening at the wide end is denoted by g 0 . The narrow end is always downstream.
The glottal area function is now given by giving for the glottal inertance where we used the fact that Note that the wedge geometry gives a logarithmic singularity in g(t) for C (G) iner (t) at g(t) = 0. Compared to Section 2, the singularity is there stronger since C (G) iner (t) = L G /hg(t) for the rectangular glottis. In any case, it is a general fact in any geometry that the inertance of the glottis (and hence, the total inertance of the fluid column) becomes singular at the moment of closure.
Equation of motion for wedge-like vocal folds
Let us start, again, from the equation of motion for the fluid column. For the incompressible, lossless flow, the velocity is The corresponding velocity potential, remembering its continuity, is given by For −L SGT < x 1 < 0 < L G < x 2 < L G + L V T , the pressure drop satisfies by the unsteady Bernoulli equation that is, Denoting the stagnation pressures p s (t) = lim x1→−LSGT p(x 1 , t) and p amb (t) = lim x2→LSGT p(x 2 , t) at the infinitely wide ends of the tube, we get by taking the limits at the both ends by change of variables. Assuming again that p amb (t) = 0 is a constant reference ambient pressure level, the equation of motion (1) follows by defining the total inertance by C iner (t) = C Obviously These two Eqs. together give the differential equation for the volume velocity that we will use for expressing the glottal pressure distribution: Recalling g(t) ≪ g 0 and omitting the weaker logarithmic singularity, we get the approximation which is quite elegant.
Equation for the flow velocity
Instead of the equations (10) and (11) for the volume velocity U (t), let us turn to the flow velocity at the glottal opening which satisfies a much more involved differential equation. In explicit terms, the wedge glottis without losses gives and solved for the acceleration at the glottis, it gives This together with Eq. (9) yield the equation for motion iner is the total inertance of the nonmoving part of the vocal tract. Note that the first two terms inside the parentheses can be joined as Remark 3.1. Note that the expression C iner (t)hg(t) in the denominator of Eq. (13) has a removable singularity as g(t) → 0 in the sense that the limit at the closing moment = 0. See Eq. (8).
Simplifications based on the wedge geometry
If g(t) ≪ g 0 as is usually the case in the wedge geometry, we get which is directly comparable with the corresponding formula Eq. (4) for the rectangular glottis. Not that the sum in brackets is always nonnegative, and it grows unboundedly as g(t) → 0 right before the closure. So, at the closing glottis when g ′ (t) < 0, the effect of the additional term is to work in the same direction as the stagnation pressure p s (t).
Note that ρLG hg0 in Eq. (14) is the inertance of a tube of length L G with uniform intersectional area hg 0 . Since g 0 ≫ eg(t), that term is insignificant iner as well as to C (T OT ) iner , and we get an even more simplified The further approximation for Eqs. (15)-(15) is to replace C iner (t) given by Eq. (8) by the simplified form (again by g(t) ≪ g 0 ) iner .
One such variant produces from Eq.
where the logarithmic singularity of the glottal inertance is still present in one place.
Finally, removing the glottal opening velocity g ′ (t), we get back to the lossless wedge model in DICO [2,8]. Note that here the glottal gap g(t) is in the denominator in Eqs. (13)-(16) since we did not extend the glottis downstream to a control surface of constant area, right above the glottis.
This differs from the original equation of motion only by the approximation iner ′(t) = 0. What the above reasoning amounts to, is just showing in what approximative sense C that is, by the approximation C where only one term is omitted from Eq. (9).
Glottal pressure for the wedge-like vocal folds
As above for the rectangular glottis, we get from the unsteady Bernoulli principle Thus, the pressure in the glottis x ∈ [0, L G ] is given by (18) The time derivative term on the right must be manipulated as follows: .
Combining this with Eq. (18) yields This equation together with Eq. (10) produces the pressure distribution in glottis, and its first to terms on the RHS of Eq. (19) are plainly the steady incompressible Bernoulli. The third term could be called congestion term (or, water hammer term) as it becomes large and positive upstream the narrowest part of the glottis just before the closure when the volume flow is decelerating. The last term vanishes when g ′ (t) = 0, and for that reason it could be called displacement term.
Since g(t) ≪ g 0 , we may given an approximation where the congestion and displacement terms have been greatly simplified. This equation is best used in conjunction with Eq. (11). A further simplification can be carried out by discarding the logarithmic singularity in the displacement term, or neglecting both the congestion and the displacement terms entirely.
Compressible steady flow
In Sections 2 and 3, the convenient assumption of compressibility was made to derive the equation of motion for the fluid column in the flow channel. Using the equation of motion, the expression for the (hydrodynamic) pressure was derived. In vocal folds models, this pressure produces the aerodynamic forces leading to the movement of the aerodynamic surfaces. 3 To have an educated opinion on this matter, one must explicitly deal with some form of compressible flow not accounted by the acoustic approximation. The challenge here is that the treatment of a general nonsteady (in)compressible flow is not possible using elementary mathematical tools and solutions in a closed form. Thus, we consider only the steady variant of a flow of an ideal gas column. In this case, the modifications due to isentropic thermodynamics are well-known. We give two examples on a steady flow having the physical dimensions resembling the glottal flow.
Generalities on isentropic ideal gas flow
We assume that the usual isentropic relations hold Thus which makes the temperature distribution easier to compute than pressure or density distributions. Actually, it is just the usual equation of state for ideal gas. For the speed of sound, we get A compressible, steady Bernoulli flow inside insulated streamlines is described by The conservation of mass in a tube (i.e., nozzle) whose intersectional area is where V m is the mass flow, considered to be constant of time. We, of course, make the assumption that A(x) is "slowly varying" in the sense that an isentropic, compressible flow can be supported in the entire inner volume of the tube (i.e., tube walls are always streamlines at least in the subsonic part of the nozzle).
Putting these together Now, from the isentropic relations we get Plugging in, we get over the length of the tube. Note that γ+1 γ−1 = 1 + 2 γ−1 . Another form for (23) is given by which leads to the expression for the Mach number The speed of sound is reached at the temperature T (x) = 2 γ+1 T 0 when The condition on the nozzle area function for reaching Mach 1 at x is given by which yields the critical area A sonic . The speed of sound at Mach 1 is given by Of course, the speed of sound c → ∞ as γ → ∞ as well, and hence the Mach number M → 0 for any fixed finite mass flow V m . However, observe that which is a rather peculiar observation since one would expect to have A sonic = 0 for an incompressible fluid. The incompressible steady Bernoulli gives 1 2 v 2 + p ρ = p0 ρ0 which gives an upper limit for the velocity v since p/ρ ≥ 0. This limit corresponds to the limit of the sonic area where the Venturi effect has reached vacuum.
For diatomic ideal gas, the critical "sonic area" is At Mach 1, the speed of sound has been reduced by the factor of 5/6 ≈ 0.913. If the same volume flow was incompressible through the same area, the speed would be v = 2 dl/s/2.05 mm 2 = 97.6 m/s. Considering Examples 4.2 and 4.3, conclude that for glottal opening areas over 2 mm 2 the usual "rule of thumb" value Mach 0.3 holds and the flow can be regarded as incompressible (even neglecting all viscosity and nonsteadyness effects). For human glottis, the area of 2 mm 2 can be considered quite a small opening. Just by halving the opening area to 1 mm 2 , the hypothetical compressible flow already gets supersonic which certainly is counterfactual as far as the glottal flow is concerned. (The adiabatic cooling to 250 K would perhaps freeze the vocal folds, and a pressure drop to half of the ambient would be destructive as well.) Realistic pressure loss effects (such as the Poiseuille law for viscous laminar flow) will check the flow velocity at the narrowest position much before the flow gets supersonic.
Discussion on energy losses in compressible flows
The internal friction due to fluid viscosity leads to a pressure loss that can be modelled by Poiseuille's law for flow channels having circular intersections. Other intersectional geometries, such as rectangular and triangular shapes, can be given analytic descriptions, and also they are known as Poiseuille's law. For even more general geometries, one is compelled to use heuristic approximations of, e.g., hydrodynamic radii or numerical solutions. These variant of Poiseuille's law are derived for an incompressible, laminar, steady flow, too.
When the flow is compressible yet remains isentropic, additional mechanisms for kinetic energy and pressure loss emerge. There is temperature variation (adiabatic heating) at stagnation points. If such heat is conducted to surrounding structures in a lower temperature (in which case the thermodynamic system is not perfectly adiabatic), there is a total energy loss from the fluid at that position. In terms of the compressible, steady Bernoulli flow, the energy loss shows up as an unrecoverable pressure loss. There are reasons to believe that Poiseuille's law alone is not a sufficient description of unrecoverable pressure loss in glottal flow.
A nonexhaustive list of mechanisms that could result in a pressure loss in addition to the viscosity effects: (i) We could have adiabatic heating at a stagnation point or adiabatic cooling in a constriction. In itself, these effects do not (by definition) amount to loss of heat from the fluid in a perfectly insulated flow channel.
In the first case, the area function changes shape in such a way that a streamline actually ends inside the tube. Such a phenomenon would surely take place in a rectangular glottis when the flow meets the glottal wall perpendicularly. Then, the temperature would increase to T 0 (but not higher!) at the stagnation point. If the channel walls at that point are at a lower temperature (driven there, e.g., by the adiabatic cooling due to the flow effect as described above), the heat conduction would actually lead to a loss of energy from the flow. Conversely, the temperature at a constriction may get lower than the wall temperature. Then the heat conduction from the wall would increase the total energy of the fluid. Note that the heat capacity of the tissue walls would be much higher than that of air.
Not that the adiabatic cooling at a constriction is offset by the opposite effect of heat production due to viscosity. It is, of course, strictly speaking wrong to consider adiabatic effects in such a viscous flow.
Based on the figures given in Example 4.3, the temperature variation due to adiabatic heating and/or cooling is about 1 % of the absolute temperature. The effects to the pressure are of the same order due to the thermodynamic equation of state. The heat exchange with the walls seems a very complicated phenomenon, and it is perhaps not an effect that needs accounting for in low-order models.
(ii) There could be a dissipative boundary layer effect transforming kinetic energy into heat by the viscosity of the fluid, not accounted by the Poiseuille's law.
(iii) There could be some form of vortex formation that would ultimately transform kinetic energy into heat via internal viscous losses in the fluid. This would not be a boundary layer effect, nor accounted for by the Poiseuille's law for laminar flow.
(iv) Dynamic effects that would depend on the moving walls, nor observed in model experiments with rigid walls.
It seems likely that properly tuned variants of Poisseuille's law serve well as a first approximation for modelling of the glottal pressure loss. At least, it has a strong theoretical background in the laminar flow regime if the compressibility is not an issue either. The "correction terms" can be motivated by experimental work on physical models (such as the work by van den Berg & al., Fulcher & al., etc.) or deep numerical computations involving Navier-Stokes -based flow models with thermodynamical coupling such as [12]. However, the author has no knowledge of such computational work.
Inertia and termination of a waveguide
In Sections 2 and 3, we derived equations of motion for an incompressible fluid column in a tubular domain where part of the domain boundary was allowed to change as a function of time. We observed that both of these (lossless) models lead to the equation of motion (1) where the only difference is the expression of the glottal inertance term C (G) iner (t); see Eqs. (2) and (8). In both cases, the vocal tract inertance is given by the integral Since acoustics is also about the movement of gas molecules (albeit by rather small distances around an equilibrium position), it is natural to ask if the same expression C iner plays a role in acoustic equations in the same vocal tract volume. The purpose of this section is to answer this question.
For simplicity, let us consider a fluid column of length ℓ > 0 with uniform intersectional area A. The fluid density and the speed of sound are denoted by σ and c. The assumption that c < ∞ means, of course, that the fluid is to some degree compressible.
Generalities
Consider a waveguide of constant intersectional area A and characteristic impedance Z 0 = ρc/A. Its acoustic impedance is given by where we denote the transmission time by T 0 = ℓ/c and the termination impedance by Z L (s). We assume that both Z L (s) and the admittance Z L (s) −1 are analytic, positive real functions on C + . Another form for the impedance is which has the merit of having the numerator and the denominator analytic in C + . There is an immediate conclusion: Proposition 5.1. If the termination load has Z L (s) has a zero at s = kπ T0 , k ∈ Z, then so does have the acoustic impedance Z ac (s). . This observation appears to be of particular value when k = 0 since then it implies the additivity of inertances 4 .
Let us begin by studing the purely resistive termination for observing the qualitative behaviour of waveguide resonances and antiresonances. If Z L (s) = R L , the numerator and the denominator are both entire functions. In this case, the zeroes of Z ac (s) are given by that is, We conclude that sin 2yT 0 = 0, i.e., 2yT 0 = nπ for n ∈ Z. Then cos 2yT 0 = (−1) n , and the equation becomes If Z 0 > R L , i.e., n = 2k, we get for the zeroes If Z 0 < R L , i.e., n = 2k + 1, we get A similar computation with a similar outcome can be carried out for the poles of Z ac (s). If Z 0 = R L , there are no zeroes nor poles as expected.
Remark 5.2. We observe that introducing resistance to termination does not change the resonant frequencies nor antinodal frequencies (as far as we have Z 0 = R L ) but it will add losses to the impedance/admittance system. For acoustic waveguides with nonuniform intersectional areas, the resonant frequencies do depend on the termination resistances. In all cases, the resonant frequencies and the antinodal frequencies are sensitive to inductive or capacitive loading at the termination.
There is one more detail whose statement has some system theoretical interest. Clearly, the impedance Z ac (s) is a positive-real transfer function of an impedance passive system for any positive-real termination impedance Z L (s). Proof. It is clearly enough to show that the function is bounded on such a vertical line where Z(s) is a positive-real analytic function in C + . We first need an observation concerning the hyperbolic tangent, namely that Re tanh (x + yi) = sinh 2x cosh 2x + cos 2y , x, y ∈ R, implying the estimate for all x > 0 since sinh 2x < cosh 2x and |cos 2y| ≤ 1. In particular, Re tanh s > 0 for s ∈ C + . A similar computation shows that leading to exactly the same upper and lower bounds for Re 1 tanh (x+yi) as are given for Re tanh (x + yi) in Eq. (27). We conclude that both Re tanh s and Re 1 tanh s are uniformly bounded from above and below on all vertical lines x 0 + iR ⊂ C + by a strictly nonnegative constant.
it is enough to show that ( 1 Z(s) + tanh s) −1 is uniformly bounded from above on x 0 + iR. Now where s = x + yi, x > 0 and y ∈ R arbitrary.
(ii) Case where Z(s) is bounded on x 0 + iR for some x 0 > 0.
We now write and observe that Z(s) tanh s is uniformly bounded from above on x 0 + iR by using the lower estimate in Eq. (27). We proceed to show that where s ∈ C + and x = Re s.
We proceed to the case where Z(s) is a rational function. Then either Z(s) or 1/Z(s) is proper. A proper transfer function is bounded on some right half plane x 0 + C + , x 0 > 0. The claim of the proposition now follows from the previous two special cases.
Inertial limit
Assume that there is a piston at the input end of the tube moving at the velocity v(t) = v 0 sin kt and acceleration a(t) = v ′ (t). Then the inertial (counter) pressure for 0 < k ≪ 1 is plainly given by the Newton's second law for an (nearly) incompressible fluid is from which for the inertial impedance transfer function (from volume velocity to pressure) we get where C iner = ρℓ/A is the inertance of the fluid column having a constant intersection area A. For a compressible fluid in a column of same dimensions, terminated to an acoustic impedance Z L (s), we get where the characteristic impedance is given by Z 0 = ρc A and the transmission time since tanh 0 = 0.
Definition 5.4. We say that the termination impedance transfer function In this case, the number r iner is called inertial factor.
Obviously, a necessary condition for inertial feasibility is that Z L (0) = 0. For perfectly terminated waveguides Z L (s) = Z 0 we have Z ac (s) = Z 0 which is not an inertially feasible termination.
Let us then compute the value of r iner . We have The last term approaches to 1 as s → 0. By l'Hospital's rule, we get Thus, Using the inertance, we get yet another formula Clearly, r iner ≥ 1 and r iner = 1 if and only if Z L (0) = Z ′ L (0) = 0. Given the termination impedance Z L (s), the inertial factor tells the proportion how much a fluid column of length ℓ must be extended in order it to have the same inertia as a comparable transmission line of length ℓ when terminated to Z L (s).
Example 5.5. Let us consider a commonly used boundary impedance model, namely a resistance and an inductance in parallel. Then R L (s) = sRL R+sL and R ′ L (0) = L. We get Remark 5.6. By Eq. (30), the acoustic inertance of a waveguide can be tuned by external inductive loading to any value larger than C iner = ρℓ/A. In fact, the rational impedance of Example 5.5 is a sufficiently rich class of acoustic terminations for this purpose. However, the inductive loading not only increases the acoustic inertance. It also moves the positions of resonant frequencies of the system. This is inconvenient when the terminated waveguide acts as an acoustic load in a larger system for which both the inertance and the resonant frequencies must be controlled to some predetermined target values.
Remark 5.7. It was already pointed out that the termination of an uniform diameter acoustic waveguide to its characteristic impedance will not result in an inertially feasible acoustic load. This is understandable since such a waveguide appears to be infinity long with infinite mass, and its inertance cannot be expected to have any finite value. In general, acoustically nonreflecting boundary termination of any does not seem to be inertially feasible. However, an absorbing boundary condition may well be a desirable feature in an acoustic (part of a) model.
Inertial proportion
The proportion of the characteristic impedance and the inertive response of a wave guide is called inertial proportion at frequency f , given by where λ = c/f is the wavelength. For low frequencies f , the number 2πC iner f approximates the acoustic impedance |Z ac (2πf i)| = Z 0 |tanh 2πT 0 f i| = Z 0 |tan 2πℓ/λ| of the transmission line of length ℓ, terminated to a short circuit. Thus In the case of the VT, it is typical to use the value ℓ = 0.17 m and c = 340 m/s. The numerical values of P iner (f ) and tan 2πℓ λ for speech relevant frequencies f are given in Table 1. Table 1: Values of inertial proportion of a 17 cm long waveguide with its comparison values. The quarter wavelength frequency of such tube is 500 Hz, corresponding to F 1 .
We conclude that between 100 . . . 200 Hz, the acoustic impedance of a 17 cm long, uniform diameter tube (with Dirichlet boundary condition at the far end opening) may be approximated by the expression Z ac (2πf i) ≈ P iner (f )Z 0 i, Z 0 = ρc/A without making error larger than 15 % in impedance values. This serves as a "rule of thumb" for accuracy whenever we consider the acoustic impedance of a waveguide more than one octave below its lowest resonance (here 500 Hz). Note that the impedance of an infinitely long transmission line is purely resistive ρc/A whereas the inertial transfer function Z iner = C iner s is purely reactive.
Inertial limit from Webster's equation
We concluded above that the inertance C iner of a constant diameter fluid column is given by C iner = ρℓ/A where ℓ is the length and A is the intersectional area. Because mass inertia is an additive quantity, the flow mechanical inertance of the variable diameter waveguide is obtained from this by integrating the infinitesimal contributions ρ dx/A(x); i.e., We proceed to show that the same expression for the acoustic inertance can be concluded from the acoustic waveguide with a varying area function A(x), x ∈ [0, ℓ]. For a mathematical treatment of such waveguides through Webster's partial differential equation, see [3,1,4].
Since there is no possibility of expressing the impedance transfer function of such a waveguide in a closed form in the same manner as in Section 5.1, the argument must be carried out by an a priori technique -studying the partial differential equation rather than its solution. This is always much more difficult, and we only make the computations for the trivial termination transfer function Z L (s) = 0.
We begin by identifying the differential equations for the impedance transfer function of the waveguide using boundary and system nodes introduced in [7,5,6]. Any internally well-posed boundary node Ξ = (G, L, K) (such as the one coming from Webster's horn model) induces an infinite-dimensional system node S = A&B C&D . This system node gives rise to the dynamical system of typė z(t) = A −1 z(t) + Bu(t), y(t) = Cz(t) + Du(t) for t ≥ 0, If Ξ is also impedance passive, then semigroup generator A := L ker(G) of S is maximally dissipative, and the transfer function of S is defined by G(s) := C&D (s−A−1) −1 B I for all s ∈ C + . The transfer function can always be expressed in terms of the original boundary node Ξ as follows: Proposition 6.1. Let Ξ = (G, L, K) be a boundary node associated to the operator node S = A&B C&D whose transfer function is G(·). (i) Then y s = G(s)u for u ∈ U, s ∈ ρ(A), and y s ∈ Y if and only if there exists a (unique) z s ∈ dom (Ξ) such that (ii) Then y s = G ′ (s)u for u ∈ U, s ∈ ρ(A), and y s ∈ Y if and only if there exists a (unique) z s ∈ dom (Ξ) and x s ∈ ker (G) such that In fact, z s ∈ ∩ k≥1 dom L k .
Proof. We use the following relations between the operators in Ξ and S: C&D = K 0 |dom (S), A = L|ker (G), A −1 = L − BG and G(s − A −1 ) −1 B = I. Now the Webster's horn model (35)-(36) for the state z(t) = ψ(t) π(t) takes the form and for all t ∈ R + . We have now constructed a (impedance passive, internally wellposed) boundary node Ξ = (G, L, K) whose transfer function G(s) = Z ac (s) is the impedance of the acoustic waveguide when the far end has been terminated to a Dirichlet boundary condition. We now wish to compute Z ac (0) and Z ′ ac (0), leading to the following result.
In other words, for Dirichlet terminated general acoustic waveguide, the flow mechanical inertance and the acoustic inertance coincide. Thus, Eq. (30) holds for general acoustic waveguides in the special case Z L (s) = 0.
Proof. We must show that Z ac (0) = 0 and Z ′ ac (0) = C iner where C iner is given by (31). The first step in this direction is to solve [ G L ] z 0 = It remains an open question whether Eq. (30) can be generalised to general acoustic waveguides for any termination impedance satisfying Z L (0) = 0 and Z ′ L (0) ∈ R . An educated guess is that this can be done using a same kind but a more complicated form of reasoning as presented in this section. | 2018-09-18T17:42:29.320Z | 2019-11-11T00:00:00.000 | {
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239822587 | pes2o/s2orc | v3-fos-license | Study on the behaviour and comfort of reusable knitting suits of wool blends in the context of the Covid 19 pandemic
Nowadays, humanity is facing serious challenges due to the disruption of the COVID-19 pandemic, affecting dramatically our healthcare system along with the economic structure, social and cultural life. Healthcare personnel treating patients with infections like COVID-19 are exposed themselves to infection. Therefore, it is compulsory to be protected from contaminated body fluids using personal protective equipment (PPE). This equipment made of synthetic fibers proved to be uncomfortable. Therefore, we developed a reusable suit composed of a blend of wool and silk to be wear under full-body protective clothing aiming to improve their comfort and safety. Firstly, we focused on the analysis of some dimensional characteristics: specific mass, porosity, thickness followed by comfort characteristics of knitted fabrics. Accordingly, the test methods comprise measurements for hydrophilic properties, air and water vapour permeability, thermal conductivity and dimensional stability to repetitive washing. After material analysis, a significant number of two-piece suits composed of blouse and trousers were industrially manufactured and delivered to healthcare personnel to be tested. The results are completed and supported with the perception of the healthcare staff involved in the study via a survey. Finally, the results and implications for future research are presented.
Introduction
Nowadays, humanity is facing serious challenges due to the disruption of the Covid-19 pandemic, affecting dramatically our healthcare system along with the economic structure, social and cultural life [1]. Healthcare personnel treating patients with infections like Covid-19 are exposed themselves to infection. Thus, it is compulsory to be protected from contaminated body fluids using personal protective equipment (PPE) [2]. But this equipment proved to be uncomfortable to the wearer in extended use since it consists of synthetic fibers. The current application of textiles in the medical field and past pandemics are well known. Presently, there is a major concern in evolving in the forthcoming future protective clothing based on nonwoven micro-and nanoporous materials. The main benefits of these new materials relay in their, breathability, lightweight, lower price and comfort [1].
Under these circumstances, we take into account the possibility to develop a reusable suit to be wear by healthcare staff in the Covid sections under the full-body protective clothing aiming to improve their comfort and safety. Why is comfort so important? Because better comfort impedes stress and tiredness.
The main objective of the study envisages whether a simple protective cloth composed of a blend of natural fibres wool and silk (95% wool and 5% silk) is appropriate to enhance both ergonomic and physiological comfort. The study was developed by an interdisciplinary team, formed by researchers and engineers from the Textile Industry field, accounting field and healthcare staff from the Covid hospitals.
As a complex issue, ergonomic comfort is considered how well the clothing suits, if it allows the wearer to perform easily all the necessary movements. It is quite simple to estimate by trying the garment on. Physiological comfort involves skin sensory comfort and thermo-physiological comfort [3].
Thermo-physiological comfort refers to a pleasant thermal and humid condition involving both thermo-regulating and moisture-regulating capacity in addition to the thermal insulation capacity of the clothing. All these abilities are related to the environmental and working conditions. Furthermore, thermo-physiological comfort is influenced by the fabric composition and structure and garment construction [4][5][6].
Since the style and cut of the two-piece ensembles conceived by our team do not restrict movements and do not exert any constraints upon the body, the study investigated the parameters representative for their thermo-physiological comfort. Because people using similar clothes, in identical physical ambiance may perceive various comfort degrees, our study was completed with the results of a survey regarding the subjective perception of the comfort level of about 51 volunteers from medical staff.
Material and Methods
Within this experimental part, we focused firstly on the analysis of some dimensional characteristics like mass per square meter, porosity, thickness followed by comfort characteristics of knitted fabrics designed to be used for the suits.
Accordingly, the test methods accomplished comprise measurements for hydrophilic properties, air and water vapour permeability [7], thermal conductivity and dimensional stability to repetitive washing. Since we refer to a reusable ensemble that will be subjected to several washing processes, dimensional stability is also an important aspect, because wool fibre is a precious natural material very sensitive to shrinking.
Materials
Fabric subjected to testing is a blend of wool/silk 95/5% knitted fabric, supplied by SILVANIA WORSTED SPINNING SRL. Why a blend of natural fibers?
Because wool continues to be a unique apparel fibre with a remarkable array of technical features. These properties refer to softness, warmth and coolness, breathability, moisture absorption and buffering resilience, odour absorption, softness, stain and flame resistance, elasticity, biodegradability and recyclability. Due to these excellent attributes, wool can be employed either for a worsted suit or knitted outerwear and/or for technical applications [8,9].
Besides, the adaptability of wool has been revealed by several leading-edge clothing products designed for the sportswear area, including outerwear, mid-layer, and underwear garments, that require superior moisture management [10,11].
Further, the presence of silk in the blend contributes to enhanced softness. Metrimpex apparatus was used. The working parameter was set for a difference in air pressure Δp =5mCOl water and a temperature of T=20 o C. Δp -represents the pressure difference between the two sides of the knitted fabric and T the average air temperature adopted for each season and used for calculations. The recorded values of the air permeability (P a ) were expressed in [m 3 /min m 2 ]. Additionally, air permeability index (i) and airflow passing resistance Rpa [mm m 2 h/kg] have been considered for the assessment. The air permeability index (i) [kg/m 2 h] has been determined with the equation (1) : where: γ -represent the air density [kg/m 3 ]; Pa -air permeability corresponding to the knitted fabric [m 3 /minm 2 ].
The air density shall be calculated with the equation (2): where: γ 0 -air density at temperature 0 °C [γ0=1.293 kg/m 3 ]; t -the temperature of the air passing through the textile material (temperature of 20 °C). . The samples were positioned and sealed above a cup filled with distilled water at a depth of ¾ from the height of the cup. After that, the samples were placed in a standard atmosphere (23 ± 1 0 C and RH 50 ± 2 %) with ventilation in a Pol-Eko-Aparatura oven. The test was carried out for 6 hours. The gravimetric method was used to evaluate the change in mass every two hours. The specimens were weighed on a sensitive Kern balance, model ABT220-4M and the difference between the mass at 2, 4 and 6 hours of vapour exposure and initial mass represents the ability of the knitted fabric to transfer perspiration. Vapour permeability has been also evaluated through water vapour transmission coefficient (WVT) μ[g/m 2 h]. Laundering of textiles is a process designed to remove dirt and/or stain by washing with an aqueous detergent solution. The treatment operation normally includes washing, rinsing, and drying of the material. Dimensional stability refers to the ability of fabrics or knitted fabrics, garments (specimens) to keep the shape and dimensions following domestic maintenance procedures (washing, cleaning, ironing). The change is usually indicated as a percentage of the initial dimension of the textile specimen. From the point of view of the dimensional stability of the knitted fabric, the first washing is of particular importance. Thus, factors like washing temperature, washing agents, treatment duration, and mechanical action exert an important influence upon the washing process and dimensional stability. Accordingly, for washing the knitted fabric suit of 95% wool/ 5% silk, an appropriate program designed for wool clothes was chosen. The washing conditions were as follows: 50 ml of commercial detergent, washing temperature 30 °C, 500 rpm centrifugation, and treatment time 51 minutes. After the washing procedure, the suits have been dried free at ambient temperature.
2.2.10. Softening. Before the testing, the suits were subjected to treatment with a commercial softener for a soft handle. Each person from the healthcare system involved in the study received a suite and a set of information regarding the chemical composition of the textile material, washing guidance and its use.
Results and discussion
3.1. Specific mass (grams per square meter), density and thickness of the knitted fabrics The characteristics of the knitted fabric assessed by standard methods are comprised in table 1.
Evaluation of dimensional stability after home laundering
To better highlight dimensional changes after household washing, 10 washing/drying cycles have been accomplished with measurements taken after each washing cycle. Since the suit should be worn as a mid-layer garment being in contact with the skin, washing after each wear is mandatory. We selected a higher number of washing/drying cycles than the one specified in the standard for the following reasons: the suit is made of knitted fabric that is susceptible to deformation and on the other hand the wool fiber has an accentuated tendency to shrink by repeated washing.
The following references were used to accomplish the measurements: -Length of trousers, front rise and back rise; -Sleeve lengths of the blouse (on the back from the shoulder to the bottom hem) -The body length of the blouse; -Width at the chest of the blouse, both front, and backside. The measurement reveals a change in the length of the trousers after the first wash, this being reduced from 102 cm by 1.3 cm, meaning a shrinkage of 1.27% from the initial length. An increase of the shrinkage was also noticed after the second wash cycle to 1.96%. After this, the length is maintained constant until the washing cycle 9 characterized by shrinkage of 2.94 %.
The same value of 2.94 % was obtained after the 10 th wash cycle. About the dimensional change of the pant in a wet state, an unstable behavior is observed with shrinkage of the material up to the washing cycle 6, after which the material elongation occurs at cycle 7 and 8, followed by shrinkage after cycle 9 and again an elongation after washing cycle 10.
The dimensional changes recorded for the blouse following each washing cycle are presented in figures 3, 4, 5, 6 and 7. Figures 3 and 4 show changes in the sleeve of the blouse after 10 washing cycles. Concerning the changes in the sleeve of the blouse with an initial length of 50.7 cm, a shortening of 0.5 cm was observed after the first wash, corresponding to shrinkage of 0.99%. The shrinkage increased to 1.97% (1 cm) after the second wash and to 2.37% (1.2 cm). after the third wash. This value went on constant for the 4-8 washing cycles after which on the 9 and 10 cycles the shrinkage increased to 3.35% (1.7 cm). Dimensional changes in the wet sleeve show the same variable behavior as the pants, observing several contractions alternated with elongations. Figures 5 and 6 illustrate the changes in the length of the blouse after each washing cycle.
Wc-washing cycles
Wc-washing cycles From the presented figures, it can be observed that the length of the blue has been kept at the same value as the initial length until after the second washing cycle when it has suddenly decreased by 0.5 cm (1.32% shrinkage). From washing cycle 3 to 10 no further change has occurred. As regards the wet length of the blouse, a more stable behavior is observed compared to that of the sleeve or pants, with only 2 slight elongations after washing cycle 7 and washing cycle 10. Finally, analysis of the dimensional stability of the blouse after repeated washings considered the assessment of the width. The values obtained after each washing cycle are given in figure 7. Considering figures 7 it can be observed that the width of the blue has not changed from the initial width (46.5 cm), with the contraction being 0 %. Repeated washings have led to an increase in density in wales direction and compactness of the fabric, thus increasing the number of yarn-to-yarn contact points and the number of contact surfaces between the yarns, all leading to dimensional changes.
Wc-washing cycles
The length of the pants was within ± 2% of the acceptable limits only up to the 9 washing cycle. As regards the blouse, the length of the sleeve exceeds the allowed limit after washing cycle 3, due to the shrinkage of 2.37 %. For the length of the blouse, the shrinkage does not exceed 1.32% even after the 10 th washing cycle. The width of the blouse was constant after all 10 washing cycles.
After each washing cycle, the mass change of the suit was also followed. From figure 8, a mass decrease of 0.83% can be observed after the first wash, reaching a maximum of 1.54% after the 4 th wash and then begins to rise above the initial mass after the seventh wash. This can be ascribed to the fact that after the first washing a removal of impurities and other substances present on the material occurred.
Besides, the increase in mass from washing 8 may be attributed to several successive washing steps that have led to fiber swelling and improvement of the material's hydrophilic features, which makes it easier to regain the humidity from the environment.
Hydrophilic properties
Clothing in general, but particularly the first layer that comes into contact with the skin, should properly dampness the moisture in excess from the undergarment area. This requires that materials designed for the first layer of clothing are hydrophilic, being capable of absorbing or adsorbing moisture and ensuring its migration into or onto the surface of the material.
Average values of five determinations accomplished on different areas of the material have been considered. The results reveal that the knitted fabric in its original state did not absorb any water at all, but its wettability increased with each wash, attaining a wetting time about 30 seconds after the washing cycle 5. This wetting time could be noticed also after the 10 th wash cycle.
For a material to be considered hydrophilic, the wetting time should be approximately 10 seconds. In the present case, no proper hydrophilic feature has been obtained for the analyzed knitted sample. Since the wool and silk fibers are very hygroscopic, the explanation for this result may be ascribed to former treatments applied to the material which generate its hydrophobic behaviour.
Air permeability
Air permeability is an influential determinant in the performance of textile material designed for clothing and technical application. Factors affecting the air permeability include the yarn properties, fabric structure (knitted, woven, nonwoven) and properties such as thickness, density, porosity, together with finishing treatments.
To evaluate the air permeability of the knitted fabric we choose as reference a 100% cotton knitted fabric with similar characteristics designed for an undergarment. According to the values from table2, our suit presents comparable air permeability with reference (60.29 m 3 /min m 2 ) [12]. For a comprehensive evaluation of the air permeability features, another relevant parameter was airflow passing resistance, calculated for undergarments (consisting of 1 layer) by using the values for fabric thickness and the calculated air permeability index. The lower the airflow passing resistance is the better the comfort properties of the analysed suit are.
Water vapour permeability
Water vapour permeability characteristic is one of the crucial factors in determining clothing comfort as it represents the ability to transfer perspiration. To assess the transfer of water vapour by diffusion, the permeability of vapours (Pv) has been used as a direct indicator. The recorded values are depicted in table 3, representing the mass change at every 2 hours of the tested samples. The increasing of the exposure time leads to a more significant mass difference compared to the initial one.
The results suggest the blend's ability to transfer humidity from the body to the outer layer of the suit. Figure 9 presents the water vapour transmission (WVT) coefficient (μ) at 2, 4 and 6 hours for the single-layer knitted fabric samples. A significant increase in the water vapour transmission (WVT) coefficient (~ 4.7) was noticed after 2 hours for the knitted fabric. After four hours, a further increase was observed, but less pronounced, the WTF coefficient being 6.2. From 4 to 6 hours according to the WTF coefficient (6.4) the water vapour transmission was slower. Even so, after 6 hours the material still can transfer moisture.
Thermal conductivity
The thermal isolation of the human body by clothing intends to establish a thermal regime that allows the development of various processes of the body, inside the comfort limits. Besides other functions, the garment competes in establishing a thermal balance among the produced and released quantity of heat. The heat transfer by conduction takes place only in case of a difference between the body temperature and the outside temperature [13].
The defining parameters for heat transfer of the knitted fabric are depicted in table 4. These values reveal a good thermal conductivity with low heat transfer resistance according to the expected values of the end-use of the conceived ensemble. Besides, the heat transfer resistance is even lower than that of the reference (0.0537 m 2 h o C/kcal) [12].
The comfort perception via survey
Conclusions on the wearing comfort of the knitting suits were formulated based on a questionnaire. This questionnaire was answered by 51 medical staff, with an average age between 47 and 48 years (13 men and 38 women). Over 90% of respondents appreciated the suit, consisting of a blouse and pants, as a comfortable one. At the request of explaining the choice made regarding the comfort conferred by the suit, the vast majority were satisfied with the comfort conferred. Only a few responded that it irritates the skin and gives a warm feeling. Almost 90% of the respondents appreciated that this suit ensured good IOP Publishing doi:10.1088/1757-899X/1182/1/012019 11 thermophysiological comfort during all the wearing. More than 90% of the respondents appreciated that from a sensory point of view, the suit gave the feeling of soft/velvety/silky.
Regarding the dimensional changes following washing treatments, at home, almost 40% of respondents reported dimensional changes.
Conclusions
Within the study, we developed a reusable suit composed of a blend of natural fibres (wool/silk 95/5%) addressed to the healthcare staff in the Covid sections. This suit should be worn under the fullbody protective clothing that proved to be uncomfortable, intending to improve their termophysiological comfort. The accomplished investigation methods envisaged whether our suit fulfills or not the termo-physiological comfort properties. The results of the study confirm the fact that the conceived suit can ensure appropriate termo-physiological comfort to the healthcare personnel.
Besides, the results of the research are consistent with the perception of the medical personnel involved in the study via the survey. | 2021-10-26T20:08:04.155Z | 2021-10-01T00:00:00.000 | {
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210054875 | pes2o/s2orc | v3-fos-license | Development and Evaluation of Three Named Entity Recognition Systems for Serbian - The Case of Personal Names
In this paper we present a rule- and lexicon-based system for the recognition of Named Entities (NE) in Serbian newspaper texts that was used to prepare a gold standard annotated with personal names. It was further used to prepare training sets for four different levels of annotation, which were further used to train two Named Entity Recognition (NER) systems: Stanford and spaCy. All obtained models, together with a rule- and lexicon-based system were evaluated on two sample texts: a part of the gold standard and an independent newspaper text of approximately the same size. The results show that rule- and lexicon-based system outperforms trained models in all four scenarios (measured by F1), while Stanford models has the highest precision. All systems obtain best results in recognizing full names, while the recognition of first names only is rather poor. The produced models are incorporated into a Web platform NER&Beyond that provides various NE-related functions.
Introduction
Named Entity Recognition is the task of identifying named entities in text (Nadeau and Sekine, 2007), which is often used as a first step in question answering, information retrieval, anaphora resolution, topic modeling, etc.The first Named Entity set had 7 types (Grishman and Sundheim, 1996): organization, location, person, date, time, money and percent expressions.Sekine et al. (2002) proposed a NE hierarchy which contains about 150 NE types.
There are three categories of NER systems: 1) The rule-based (RB) (Krupka and Hausman, 1998;Friburger and Maurel, 2004); 2) the Machine Learning (ML) based (Finkel and Manning, 2009;Singh et al., 2010); and 3) hybrid methods (Jansche and Abney, 2002).The ML-based methods can often be "black boxes", in comparison with RB techniques which are easy to interpret.Yet, ML-based methods are state-of-theart.Such example is a Stanford Named Entity Recognizer (Manning et al., 2014), which can be trained for many languages.Other notable NER platforms include GATE (Desktop application that enables NER across many languages and domains),1 OpenNLP (rule-based and statistical NER),2 spaCy (Honnibal and Montani, 2017) (module written in Python, used for advanced NLP)3 and many others.
For Serbian, thus far a rule-based and lexicon-based NER system was developed -SRPNER (Krstev et al., 2014).Its development started with the recognition of a NE class present in all NE schemes, personal names (Krstev et al., 2005), while the recognition of other main NE classes was subsequently added.In the next Section we present briefly this system and how it was used to produce the corpus of newspaper texts annotated with personal names -the gold standard.Section 3 describes NER systems based on Machine Learning methods that were trained on the corpus derived from the gold standard, while the evaluation and discussion of results are presented in Sections 4 and 5, respectively.In Section 6 we present a web platform that enables use and evaluation of these systems.Finally, some directions for future work are given in the last Section.
1061 2 SRPNER and the Gold Standard
Rule-Based NER for Serbian
The first NER system for Serbian was a rule-and lexicon-based system developed several years ago.It has been designed to recognize the main classes of NEs: 1) numerical expressions (measurement and money), 2) temporal expressions (date and time, and 3) name expressions (personal, geopolitical and organization names).
The system was designed in a form of the cascades of Finite-State Transducers (FST) in which every transducer recognizes and tags a certain class of NEs (Friburger and Maurel, 2004;Maurel et al., 2011).Each transducer rely in its work on the results of previous transducers and on edictionaries of Serbian (Vitas and Krstev, 2012).E-dictionaries play an important role specifically in the recognition of name expressions, since, beside general lexica, they contain many proper names, both personal and geopolitical.The system is modular which means that steps can be omitted and can change order; however, it performs best when used in predefined way since in each step the disambiguation of some names is performed.
SRPNER presented in (Krstev et al., 2014) recognizes 11 classes of NEs: dates (moments and periods), time (moments and periods), money expressions, measurement expressions, geopolitical names (countries, settlements, oronyms and hydronyms), and personal names (one or more last names with or without first names and nicknames).The presented evaluation results for the recognition of all mentioned NEs obtained on a sample of newspaper texts were F 1 = 0.96 (R = 0.94; P = 0.98).The system also recognized titles, roles and functions of persons when they accompany personal names.
Since this first results, system has been continually improved by adding new NE classes (organization names) and new sub-classes (e.g. for geopolitical names: regions, super-regions and city counties).In addition, the e-dictionaries of Serbian were also continually improved and enhanced, and that by itself contributes to better performance of SRPNER.
The new version of this system recognizes more variations for naming persons: distinguished persons and first names alone.Moreover, system distinguishes names used for men from those used for women (Krstev et al., 2015).Presented results show that the system was more successful in rec-ognizing names of men than women.
The output of the system are texts with XML tags for recognized entities inserted in them.Since the system allows embedded NEs, recognized names of persons can be components of other NEs, e.g.organizations.
The Preparation of the Gold Standard
The system presented in the previous section was used for the preparation of the gold standard -a large text sample annotated with personal names dubbed GOLDPERS.The sample consists of short news published on the Web in the period 2009-2016 by 4 Serbian daily newspapers (Politika, Danas, Blic, Novosti), one news portal (B92) and one weekly magazine (Bazar).The sample consists of 321,127 tokens (simple running words).
The forms of personal names taken into account and their tagging are presented in Table 1.The gold standard was produced following these steps:4 • Each text was annotated using SRPNER; • Tags that did not refer to personal names were deleted; • The remaining tags were evaluated as correct, partially correct (overlapping), not correct (not a name); • The missing tags were inserted, and typos that led to incorrect tagging were corrected.For some texts this process was repeated from one to four times which yielded "four levels" of gold standard.Between these repeated runs the development of SRPNER continued, as well as the enhancement of e-dictionaries of Serbian.
3 Training Different NER Systems
Named entities in sentences are annotated using tags listed in Table 1.These tags contain different levels of information: name type, role, gender.We wanted to examine the recognition of NEs on different level of details.Therefore, on the basis of the gold standard, we developed its four versions by 3. We split each of these four versions of the gold standard (namely PERS_{1, 3, 4, 9}) into training and test sets (containing 8, 151 and 895 sentences, respectively).We named this gold test set STUDENTS-GOLD.
As we wanted to have an independent text of the similar structure and content we prepared an additional set of news articles from Danas daily journal.This was one of the source journals for the STUDENTS-GOLD, but for this new sample, we have randomly chosen recent news (from year 2018, that is 2 years after the most recent news in GOLDPERS).This set of articles containing 860 sentences was tagged with SRPNER and manually corrected, thus producing the second test set DANAS-GOLD.The distribution of NE tags in the training set, and both test sets is given in Table 4.
We used four versions of the gold standard to train two different Named Entity Recognition systems: SPACY NER (Subsection 3.2) and STAN-FORD NER (Subsection 3.3).Trained models for Serbian are available on NER&BEYOND platform, which is presented in Section 6.
spaCy NER
spaCy (Honnibal and Montani, 2017) We used it for training NER on our four versions of GOLDPERS.We coded a Python script that transforms each sentence into a training sample, represented as a list of triplets.6For example, for the sentence "srpski reditelj Aleksandar Saša Petrović" (Serbian director Aleksandar Saša Petrović), the corresponding triplet representation for the PERS_4 model would be: (0, 14, "ROLE"), (16, 39, "P ERS_F U LL") where the first and the second element represent the start and the end character offset, while the third element represents the NE itself.Each of the four models were trained in 10 iterations, using 0.5 value for the drop-out parameter.These trained models can be inspected online.7
Stanford NER
STANFORD NER (Manning et al., 2014) it is based on Conditional Random Fields (Lafferty et al., 2001).For training this model, 8 we had to transform our texts into CoNLL02 IOB format (namely, "inside -outside -beginning") with conll extension (Sang, 2002).For this purpose, we used XML → CoNLL converter available within NER&BEYOND on-line tool.An example of this format is given in Table 5.
Evaluation
In order to evaluate three NER system for personal names in Serbian, we need to have the output results in the same format.After running SPACY NER on a text, an output is provided in BRAT format with ann extension.This format is similar to the one for spaCy training, described in subsection 3.2.For the example given in the same sub- 8 Training Stanford NER, https://nlp.stanford.edu/software/crf-faq.shtml#a section, an output file has the following content: T1 ROLE 0 14 srpski reditelj T2 PERS_FULL 16 39 Aleksandar Saša Petrović After running STANFORDNER on a text, an output is provided in already mentioned CoNLL02 format.We used CoNLL02 → BRAT converter available within NER&BEYOND online tool.
Finally, for both SPACY NER and STANAFORDNER output files, we applied ANN + TEXT → XML converter offered by Gemini, also available within NER&BEYOND online tool.An output of SRPNER is already an XML file with marked named entities, as is the gold standard explained in Section 2 and illustrated in Table 3.
We evaluated three NER systems using the open source Gemini tool, described in Section 6, that offers various options for files comparison (Feng, 2018) where exact overlapping of NE annotations is subsumed (both annotation labels are the same) or weighted, where partial overlapping is taken into account, but with some weighted value to measure overlapping segment.To indicate alignment type, one can choose among the two options: the first option is greedyMatching, where the matching of annotations in the first and second files is done with a greedy algorithm that tries to match the closest annotations first.The second option is maxMatching, where the matching of annotations in the first and second file is done optimally using a maximum matching algorithm in bipartite graphs.An annotation in the first file will correspond to at most one annotation in the second file, and vice versa, in both cases.We run 2 × 3 × 4 evaluation rounds: two test sets, three NERs and four models per each.All trials were run with strict matching type and max-Matching alignment type.
To indicate the chosen score type to evaluate the correspondence between one annotation from the first file and one annotation from the second file, calculation of precision P , recall R and Fmeasure in Gemini comes in three different flavors: weak an annotation of the first file will be considered as corresponding to an annotation of the second file if they intersect on at least one character; strict an annotation of the first file will be considered as corresponding to an annotation of the second file if they start and end exactly at the same characters; weighted the match is scored by the ratio of the number of characters common to both annotations divided by the total number of characters covered by at least one of the two annotations.
Figure 1: The evaluation of SPACY NER, SRP-NER and STANFORD NER on STUDENTS-GOLD
Discussion
The results of three NER systems, four models and two test texts are presented in while SRPNER achieved the highest F 1 measure in all cases.STANFORD NER and SRPNER performed better on both test texts with models that use less tags (PERS_1 and PERS_2), while SRP-NER performed significantly worse only for the model with 9 tags.Contrary to our expectations, all NER systems for all models achieved better results for the independent test text DANAS_GOLD than for the test set randomly chosen from the gold standard.Namely, a number of news in GOLD-PERS that come from 6 different sources were produced at the same time period, and thus involved same persons.However, that did not influence results favorably towards STUDENTS_GOLD test text.
All measures are for all NER systems and models highest for weak calculation, followed by weighted, the strict calculation giving the lowest result.However, as displayed in Figure 1 for the STUDENTS-GOLD test set and in Figure 2 for the DANAS-GOLD test set the mutual relationship between three NER system remains the same.
We also compared performance of all three NER systems by each named entity type (Figure 3).Results for all three models distinguishing entity types show that all three systems perform poorly in recognizing first names only.For STAN-FORD NER and SPACY NER it can be explained by the considerably smaller number of these tags in training texts compared to other tags (see Table 4).As for SRPNER one can presume that developers devoted less effort to this entity type occurring only occasionally in newspaper texts.Similarly, in all experiment settings, the recognition of full names was better than the recognition of last names only.Again, the number of last name tags was smaller than the number of full name tags (Table 4).A rule based system SRPNER makes use of a personal name context in cases of disambiguity which tends to be less specific in the case of the use of a last name only.
One can also note that when using the model PERS_4, SRPNER system performs best in recognizing the role entity.Between other two systems, STANFORD NER achieves better recall and SPACY NER slightly better precision.
The chart for model PERS_9 shows that all systems according to F 1 measure recognize better masculine names than feminine names regardless of entity types.Feminine names show grater variety of forms than masculine names, especially when only last names are used; moreover, they occur significantly less than masculine names in newspaper texts (as pointed in (Krstev et al., 2015) there is approximately one feminine name per 7 seven masculine names) and confirmed in our training sets (Table 4).
We compared the performance of SRPNER with its previously reported results.In (Krstev et al., 2005) results for the recognition of personal names were P = 0.97, R = 0.86, F 1 = 0.91.One notes that all measures are higher than those obtained when using the GOLDPERS (see rows PERS_1 in Table 4), which can partly be due to the inclusion of first names only into the present version of SRPNER.On the other hand, results obtained for the recognition of roles with GOLD-PERS (F 1 = 0.87 for STUDENT and F 1 = 0.86 for DANAS) were higher than those presented in the same paper (F 1 = 0.83).The same goes for the recognition of last names only: the previous result was F 1 = 0.78, while the use of SRPNER on the gold standard yielded F 1 = 0.86 for STU-DENT and F 1 = 0.85 for DANAS.
To the best of our knowledge, STANFORD NER and SPACY NER were used for the first time for the recognition of personal names in Serbian texts.Ljubešić et al. (2013) used STANFORD NER to build models for Croatian and Slovene.When they used distributional similarity to improve results, on texts coming from different sources they obtained the following results: for Croatian P = 0.91, R = 0.93 and F 1 = 0.92, higher than STANFORD NER for the model PERS_1, and for Slovene P = 0.82, R = 0.87 and F 1 = 0.84, comparable with STANFORD NER for the model PERS_1 (Table 6).One should note, however, that their test set contained a smaller number of personal names (approximately one third of number of personal names in our test sets).BRAT, CoNLL02 and XML.BRAT (Stenetorp et al., 2012) is a web-based tool 9 for text annotation, i.e., for adding notes to existing text documents.It is designed for structured annotation, allowing embedded annotations, which are especially convenient for NER.Annotations are external, so for each text file, an additional annotation file contains annotation data described in Section 4. CoNLL02 is a two-column format, also described in Section 4.An example of XML file whit tags interpreted as NEs is given in Table 3 7 Future Work For the upcoming research, we plan to apply the procedure we used for personal names to other NE classes (organization, location, event, temporal, quantitative, etc) and to experiment with other ML NER methods and tools with an ultimate goal to produce a successful hybrid system.The important next step is the enhancement of our newspaper corpus with other types of text (Wikipedia articles, domain texts, literary texts).The literary texts would be particularly important for improving the recognition of first names.Finally, another intended step is Entity Linking (EL), i.e. disambiguation of recognized named entities to a knowledge base, such as Wikidata, DBpedia WordNet and BabelNet.Such example would be automatically assigning Wikidata URL that points to a biography of a famous person to the corresponding named entity detected in text.
Figure 2 :
Figure 2: The evaluation of SPACY NER, SRP-NER, and STANFORD NER on DANAS-GOLD Jiang et al. (2016) compared 4 NER systems, two of which were STANFORD NER and SPACY NER, for English.Their test set consisting of Wiki articles contained approximately the same number of personal names as our both sets -around 900.Their results were for STANFORD NER P = 0.72, R = 0.87 and F 1 = 0.79, and for SPACY NER P = 0.73, R = 0.73 and F 1 = 0.73.Our results are comparable in the sense that they also show that STANFORD NER achieves the best recall, while SPACY NER tends to have the more balanced precision and recall.
Figure 3 :
Figure 3: Evaluation of SRPNER, SPACY NER and STANFORD NER on two test sets, by each named entity type
Table 4 :
. It is possible to select matching type: strict, Number of NE tags vs. number of different name forms in the training set and in test sets STUDENTS-GOLD (S) and DANAS-GOLD (D)
Table 5 :
CoNLL02 IOB format -the beginning of the sentence I was fascinated by Sonja Savić and her transformation, Anica Dobra and her charm...
Table 6 :
The comparison of strict precision, recall and F 1 between NER systems, models and test sets.
(Manning et al., 2014)2017), a free, open-source library for advanced NLP tasks in Python.This portal offers automatic annotation of texts in English, Spanish, German, Portuguese, French, Italian, Dutch and Serbian;StanfordNER module provides Named Entity annotation using STANFORD NER models(Manning et al., 2014), which are available for Serbian, English and German with different levels of details, e.g.number of NE classes.Serbian model is developed withing presented research, while English and German are integrated from Stanford repository; NER statistics module is developed for analysis of annotated text co llections in BRAT, that can be automatically downloaded via BRAT 9 BRAT, https://brat.nlplab.orgweb interface.Various statistics related to distributions of named entities and attributes can be computed, including frequencies of annotated entities, classes, attributes per document and collection; Gemini tool allows comparison of two text annotation files and provides different alignment scores.It is possible to compare a pair of XML files, a pair of files in BRAT for mat and one XML file against a file in BRAT format.The first file is the output of a NER system and the second file represents a gold standard.10 | 2019-11-28T12:18:34.269Z | 2019-01-22T00:00:00.000 | {
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11469104 | pes2o/s2orc | v3-fos-license | Parcellating Cortical Functional Networks in Individuals
The capacity to identify the unique functional architecture of an individual’s brain is a critical step towards personalized medicine and understanding the neural basis of variations in human cognition and behavior. Here, we developed a novel cortical parcellation approach to accurately map functional organization at the individual level using resting-state fMRI. A population-based functional atlas and a map of inter-individual variability were employed to guide the iterative search for functional networks in individual subjects. Functional networks mapped by this approach were highly reproducible within subjects and effectively captured the variability across subjects, including individual differences in brain lateralization. The algorithm performed well across different subject populations and data types including task fMRI data. The approach was then validated by invasive cortical stimulation mapping in surgical patients, suggesting great potential for use in clinical applications.
INTRODUCTION
The human cerebral cortex is organized into areas based on distinct features such as cytoarchitecture or topography (e.g., 1 -4 ). These brain areas contribute specialized functions that interact as part of distributed networks 1 , 5 -7 . Recent advances in non-invasive neuroimaging techniques, especially the emergence of functional connectivity MRI 8 , 9 , have made it possible to explore the functional organization of regions and networks in the living human brain 10 -15 . Initial work has revealed a number of complexities including aspects of organization that respect traditional notions of brain areas, as well as network organization that has organizational properties that span and split areas. Further, there are individual differences in organization that are distributed non-uniformly across the cortex. Obtaining functional atlases at the level of the individual person is a critical step towards understanding the anatomy-function association in the human brain and the stability of this relationship across individuals 16 .
The capacity to identify the unique functional architecture of an individual's brain is particularly important for personalized medicine. Clinical and imaging studies, including those employing invasive functional mapping techniques, have demonstrated marked interindividual variability in the organization of different functional systems of the brain 17 -19 .
Localizing functional architecture in a particular subject is therefore a fundamental requirement in clinical procedures such as surgical planning 20 and brain stimulation therapies 21 , 22 . However, non-invasive functional mapping techniques are generally limited in accuracy and reliability at the single-subject level 23 . To date, precise functional mapping in individual patients still heavily relies on invasive measures.
Individual-level functional mapping is also essential for the investigation of variations in human behavior and cognition. Functional imaging studies of individual differences commonly use regions of interest (ROIs) defined by anatomy or by population-averaged fMRI studies 24 . To improve specificity, individual-level ROIs can be defined using a taskbased functional localizer 25 (see 26 ). Recently, increased effort has been devoted to developing methods for parcellating functional networks in individual subjects based on resting-state connectivity 14 , 16 , 27 -30 . An individual-level functional parcellation can be used not only as the "localizer" for specific functions, but can also provide a basis for crosssubject alignment according to functional characteristics, instead of macroscopic anatomical landmarks, to improve group-level analyses.
Achieving individual-level precision is thus a major goal of neuroimaging. Specifically, to be clinically useful, a non-invasive functional mapping technology must fulfill the following criteria: 1) it should have high reproducibility within subjects; 2) it should be sensitive to functional differences between subjects; and 3) it should match results derived from invasive cortical stimulation, currently considered the gold standard for individual-level functional mapping. Based on these criteria, here we develop a novel approach for individual-level functional parcellation based on functional connectivity, which can be applied to either resting-state fMRI data or spontaneous activity extracted from task fMRI data. Test-retest reliability of the parcellation and its sensitivity to individual differences were evaluated on multiple datasets. Validity of the network parcellation was then examined in a group of surgical patients who underwent invasive cortical stimulation.
The parcellation strategy is described below (see Figure 1).
Step 1-A functional cortical atlas consisting of 18 networks was first estimated based on 1,000 healthy subjects 10 and projected onto the individual subject's cortical surface using the FreeSurfer software (see ONLINE METHODS). The individual subject's blood oxygenation level-dependent (BOLD) fMRI signal time courses were then averaged across the vertices that fell within each network. These atlas-based network time courses were used as the "reference signals" for the subsequent optimization procedure.
Step 2-The individual subject's functional MRI signal at each vertex was then correlated to the 18 "reference signals" derived from the previous step. Each vertex was reassigned to one of the 18 networks according to its maximal correlation to the "reference signals". A confidence value was also computed as the ratio between the largest and the second largest correlation values. For example, if a vertex had the strongest correlation with the "reference signal" of network A with a correlation coefficient of 0.8, and had the second strongest correlation with the network B with a correlation coefficient of 0.4, then the confidence that this vertex belongs to network A was 0.8/0.4=2. After all vertices were reassigned to one of the 18 networks with a certain confidence level, in each network the BOLD signals of vertices with a confidence value greater than a preselected threshold (e.g., >1.1) were averaged and termed the "core signal". Several parameters were computed for each network, including the pre-estimated inter-subject variability in functional connectivity 31 and temporal signal-to-noise ratio (SNR), which were normalized and averaged across the vertices where the confidence values exceeded the given threshold.
Step 3-For each network, the "core signal" derived from Step 2 and the original "reference signals " derived from Step 1 were averaged in a weighted manner. Before averaging, the "core signal" was multiplied by the weighting parameters computed in Step 2, including inter-subject variability, SNR, and the number of iterations. The resulting signal estimate was used as the new "reference signal" for the next iteration. This weighting strategy ensured that the original atlas-based "reference signal" was weighted less compared to the "core signal" in regions of high inter-subject variability and regions of high SNR, and gradually reduced its weight as the iteration proceeded. Using these new "reference signals" which incorporated both the individual subject's information and the information of the population atlas, the brain vertices were further reassigned to one of the 18 networks.
Step 4-Steps 2 & 3 were iterated until the algorithm reached a pre-defined stopping criterion, e.g., the procedure was stopped if network membership remained the same for 98% of the vertices in two consecutive iterations or if it reached a predetermined number of iterations.
Maps are Reliable and Capture Inter-subject Variability
The parcellation technique was first applied to a longitudinal dataset consisting of 23 subjects who were scanned five times within a period of six months (Dataset I). During the iterative search, the boundaries of the functional networks were gradually refined according to the connectivity patterns estimated in individual data but guided by the population-atlas (see Supplementary Fig. 1 for an example showing the intermediate results after each iteration). In general, vertices in the primary visual and sensorimotor regions showed relatively stable network membership assignment over the iterations. However, vertices in the association cortices showed greater adjustment during the optimization process.
Within each subject, the resulting functional atlases converged to be visually consistent across the five sessions, both in the primary sensorimotor regions and the higher order association regions ( Figure 2). Quantitative analyses indicated high intra-subject reproducibility across the five sessions (mean Dice coefficient = 83%). At the same time, functional maps varied substantially across different individuals (mean Dice coefficient = 67%), especially in the higher-order association regions. These results indicate that the iterative parcellation technique is able to obtain reliable functional networks within the same person, and can reflect the network distribution differences between individuals (see also Supplementary Fig. 2 for the maps of three subjects who demonstrated high, median, and low reproducibility across sessions). Most critically, each individual brain had unique features.
Parcellation Is Widely Applicable to Different Data Types
To objectively examine the performance of the iterative parcellation, quantitative analyses of the test-retest reliability and sensitivity to individual differences were performed in a population independent from Dataset I that was involved in the algorithm development. MRI data of 100 unrelated subjects publicly available from the Human Connectome Project (HCP; Dataset II) were used for this replication purpose. This cohort was substantially different from Dataset I in terms of age, data acquisition length, ethnicity, scanner type and scanning protocol. Each subject performed two resting-state fMRI (rs-fMRI) sessions and seven task fMRI (tfMRI) sessions (see ONLINE METHODS). The two rs-fMRI sessions of each subject were conducted on two separate days; thus, they could be employed to evaluate the test-retest reliability of the network parcellation.
Intra-subject reliability and inter-subject variability were both measured using the Dice coefficient after each iteration (Figure 3a). Because the algorithm was initialized with the population-based atlas, intra-subject reliability was 1 and inter-subject variability was 0 at the beginning. As the iterative procedure progressed, inter-subject variability increased while intra-subject reliability decreased, but both stabilized after several iterations (see also Supplementary Fig. 3 for the spatial distributions of vertex-wise reliability and variability after different numbers of iteration).
The iterative parcellation technique showed good generalizability in this independent dataset. Functional maps derived from the two rs-fMRI sessions were highly consistent within subjects (see Figure 3b for the maps of three randomly selected subjects, results of the 100 subjects can be downloaded from: http://nmr.mgh.harvard.edu/bid/download.html).
Comparing two rs-fMRI sessions of the same subject, the Dice coefficient was 82.4% ± 3.2%. Critically, the maps also demonstrated substantial inter-subject variability. Between any two individuals, the Dice coefficient was only 60.5% ± 2.8% (corresponding to intersubject variability of 39.5%). The intra-subject consistency of network membership was significantly higher than the inter-subject consistency (unpaired two-tailed t-test, t(5048) = 91.0, p < 0.001, Figure 3c).
An important question is whether the iterative parcellation technique can be applied to the task fMRI data that are widely available. Given that numerous task fMRI datasets already exist 32 and task fMRI is routinely performed for preoperative mapping in many hospitals, the practical value of this iterative parcellation technique will be greatly enhanced if this technique can be directly applied to task data. To test this possibility, the task-based fMRI data of the 100 HCP subjects were bandpass filtered (0.01 -0.08 Hz) and processed in the same way as the resting-state data. Parcellation can be derived from the data of a single task but is less reliable due to limited data acquisition length (see Supplementary Fig. 4 for the parcellation maps derived from single-task data, see ONLINE METHODS for the data acquisition length of each task). The data of different tasks were therefore concatenated within each subject to increase the amount of data per subject and to minimize the impact of any specific task design on the connectivity estimates 33 , 34 . For each individual subject, iterative parcellation was performed on the concatenated task fMRI data, as well as on the concatenated resting-state data (see Figure 3d for the maps of three exemplary subjects). Parcellation results based on the task fMRI data and the resting-state data were similar (Dice coefficient = 81.7% ± 4.0%). The consistency between the rest-and task-based parcellation maps was as high as the reproducibility between two resting-state sessions (paired two-tailed t-test, t(99) =1.76, p = 0.08; see Figure 3c). These results suggest the feasibility of obtaining whole-brain functional atlases of individual subjects from task fMRI data.
Brain Lateralization Is Reflected in Network Parcellation
Hemispheric lateralization is an important organizational principle of the human brain and a potential marker of individual differences in brain development 35 . Here we quantified the laterality of network distribution in individual subjects. For each network, a laterality index (LI) was computed based on the count of vertices in the left hemisphere and the count in the right hemisphere (see ONLINE METHODS for the definition of LI). Among the 18 networks that resulted from the iterative parcellation, we identified two networks that demonstrated strong asymmetry. The most left-lateralized network (LI = 0.22 ± 0.08, positive LI values indicate left-lateralization) included the inferior frontal gyrus and the temporal parietal junction -traditional language regions (Figure 4a). Among the 100 subjects, only a few subjects demonstrated atypical right lateralization of this network (see Figure 4a for the histogram of LI). The most right-lateralized network (LI = −0.13 ± 0.09) included the insula and the angular gyrus -traditional ventral attention regions 36 . Lateralization of these two networks showed moderate test-retest reliability (see Supplementary Fig. 5). To directly examine the relationship between the left-lateralized parcellation network and language function, we mapped the regions showing activation (at a z-threshold of Z > 1.96, corresponding to uncorrected, two-tailed p-threshold of p < 0.05) during a story comprehension task 37 . At the group level, 71.2% of the vertices in the leftlateralized parcellation network fell within the regions showing language-related activation (Figure 4b), suggesting that this left-lateralized network is related to language function.
Finally, we investigated the effect of handedness on functional network laterality in 52 lefthanded and 52 matched right-handed individuals (Dataset III). These subjects were matched in terms of age, gender, ethnicity, education, fMRI data acquisition, data quality and other parameters (see Supplemental Table S1 for the matching criteria and participant demographics). Iterative parcellation was applied to each individual subject to identify the 18 networks. Again, the language-related network and the ventral attention-related network showed the strongest lateralization in both groups. Compared to left-handed subjects, righthanded subjects showed a trend for stronger lateralization in the language-related network (mean LI 0.20 vs 0.16, unpaired two-tailed t-test, t(102) = 1.9, p = 0.057), and significantly stronger lateralization in the ventral attention-related network (mean LI −0.14 vs −0.07, unpaired two-tailed t-test, t(102) = 3.1, p = 0.003, Figure 4c).
Comparing Parcellation Networks with Task fMRI
The reliability of task-evoked response in individual subjects is affected by many factors some of which extend to analysis of resting-state networks as well as other factors that are preferential to task fMRI 38 . Many studies have used task fMRI activation maps to validate or evaluate the accuracy of results derived from resting-state fMRI. Here, we quantified the intra-subject reliability of task fMRI activation maps and the functional networks derived from the iterative parcellation. For this investigation, two brain functions that are routinely examined in preoperative mapping, motor and language functions, were assessed in the 100 HCP subjects. The hand motor network and the language network of each individual subject were localized by conventional task-evoked responses and by iterative network parcellation.
Task-evoked responses were estimated using single task runs and showed a range of low to high reliability across two runs within the same subject. Reliability was evaluated using the Dice coefficient across a variety of thresholds (from Z = 1.96 to Z = 10.0 in 0.1 increments). The maximum reliability was 40.4% (when Z = 6.76) for the motor task and 34.4% (when Z = 1.96) for the language task. Iterative parcellation was then performed on short resting-state data segments, with length matched to the motor and language task runs (i.e., 3m 34s and 3m 57s, respectively). Compared to the task-evoked responses, the iterative parcellation yielded higher reproducibility across two runs (paired two-tailed t-test, t(99) = 11.2, p < 0.001, for the hand motor network; paired two-tailed t-test, t(99) = 21.9, p < 0.001 for the language network), with a Dice coefficient of 66.6% ± 10.2% for the hand motor network and a Dice coefficient of 61.5% ± 9.1% for the language networks. While the task data analyzed here reflect only a subset of possible tasks and range of data quality that could be explored, it is notable that the present iterative parcellation approach performed comparably and in many individuals better than traditional task-based analysis.
Validation Using Electrical Cortical Stimulation (ECS)
To further validate the results derived from the iterative parcellation approach, we employed a clinical dataset consisting of eight surgical patients who performed a battery of motor tasks in MRI prior to surgery (Dataset IV). Resting-state data were also collected in six of the eight patients. Their hand and tongue sensorimotor regions were localized using ECS, which is the current gold standard for preoperative functional mapping. This unique dataset provided an opportunity to evaluate the clinical applicability of the iterative parcellation technique. Parcellation was performed in each individual patient based on the motor task fMRI data that were bandpass filtered (0.01-0.08 Hz) and processed in the same way as in the 100 HCP subjects. The hand and tongue regions were also mapped using the traditional task activation approach for comparison.
Sensorimotor regions identified by ECS were used as references (Figure 5a), where the red dots on the ECS maps indicated negative electrodes (no symptoms related to the sensorimotor cortex were reported when stimulated) and the yellow dots indicated positive electrodes. Motor and sensory regions identified by traditional task activation showed low consistency with the ECS maps ( Figure 5b). In contrast, the sensorimotor regions identified by iterative parcellation were more consistent with the ECS maps (Figure 5c), suggesting that the iterative parcellation technique was valid and could serve as a prescreening method for ECS (see Supplementary Fig. 6 for the results of all eight subjects; the subject shown in Figure 5a was Patient 2 in Supplementary Fig. 6). In addition, a parcellation map of multiple functional networks with confidence values greater than a predetermined threshold (e.g., 1.1) can provide a rough estimate of the regions of interest for invasive cortical stimulation (Figure 5d), potentially shortening the stimulation procedure.
To objectively assess the potential of our parcellation technique in preoperative mapping, the sensitivity and specificity of the hand and tongue sensorimotor maps in 8 surgical patients were statistically measured across different confidence thresholds. Sensitivity and specificity of the task fMRI were also computed by varying the t-value thresholds of the task activation.
In addition, we masked the task activation maps using the pre-central and post-central gyri labels generated by FreeSurfer to improve specificity. This operation mimics the procedure of human experts, who usually disregard the noisy activation responses outside of the regions of interest. Receiver operating characteristic (ROC) curves were then plotted for the iterative parcellation algorithm (Figure 5e, green curve), traditional task-activation mapping alone (purple curve) and task-activation masked with anatomical labels (red curve). The iterative parcellation technique significantly outperformed the other two task-based methods and showed significantly larger area under the curve (AUC, p = 0.008 and p = 0.015, Wilcoxon rank sum test; AUC of iterative parcellation = 0.91, AUC of task fMRI = 0.76, AUC of task fMRI masked with anatomical labels = 0.78).
The iterative parcellation was then applied to the pure resting-state data in six patients
DISCUSSION
In this study we present a novel approach for parcellating functional networks across the cerebral cortex in individuals based on functional connectivity. Each individual brain had unique features. Parcellation networks were reproducible within subjects across multiple scans and could capture inter-individual differences in functional organization, including variability in brain lateralization. We found that this approach can be applied to various populations and can be extended to task fMRI data. Using invasive cortical stimulation as the gold standard, the sensitivity and specificity of iterative functional parcellation were evaluated in surgical patients and compared to that of conventional task fMRI. Our results indicate that the individual cortical parcellation technique can correctly localize functional networks in individual subjects and has potential for use in clinical applications.
Revealing Individual Variability in Brain Organization
Inter-individual variability in human brain organization has long been studied 39 . However, systematic in vivo research on the variability in the functional organization of the human brain, especially in the patterns of connectivity, has just begun. Variability in functional connectivity has been related to individual differences in human behavior and cognition, such as IQ, musical ability and reading ability 24 . Brain changes associated with neurological and psychiatric disorders are also reflected by variations in functional connectivity 40 .
Recent explorations of resting-state functional connectivity in healthy humans have suggested that association regions (including the language, executive control, and attention networks) present with particularly strong variability that may relate to individual differences in behavior 31 , 41 . Substantial inter-individual variability in functional organization calls for imaging techniques that can precisely capture the functional characteristics of each subject. To enable functional analyses at the individual-level, Carddock et al. parcellated rs-fMRI data into functionally and spatially coherent regions-ofinterest (ROIs) that tended to be equally sized 30 . Arslan et al. proposed a cortical parcellation method based on spectral graph theory and were able to obtain reliable results at the group level. However, inter-subject variability was underestimated and the method aimed to identify a group-wise parcellation that can represent each subject in the group 42 . Goulas et al. parcellated the lateral frontal cortex using a module detection algorithm and demonstrated inter-subject variability in these modules; however, intra-subject reliability was not evaluated at the same time 29 . Using a region growing method, Blumensath et al.
mapped functional networks in individual subjects with high reproducibility 28 and found that functional connectivity network boundaries might overlap with task activations. These technical developments are important and merit future validation, especially based on invasive measures. A precise parcellation technique with high sensitivity to individual variations will facilitate discovery of meaningful biomarkers for cognitive ability or disease states, and will provide increased statistical power for investigating behavioral or genetic associations.
Implications for Clinical Intervention
An individual-level functional atlas has strong implications for clinical practice, especially for surgical planning and brain stimulation that depend on precise functional localization. Current preoperative mapping with task-based fMRI suffers from poor signal-to-noise ratios, limited test-retest reliability and limited overlap with analogous maps derived from invasive cortical stimulation 43 , 44 , causing many to question its clinical utility. For example, based on a meta-analysis of 63 published studies, task fMRI has only a moderate (~50%) withinsubject test-retest reproducibility 38 . In the present study, limited reproducibility was also observed between the two runs of task fMRI data in the HCP subjects. Whereas this was partially due to the limited acquisition length of the task runs and variability in data quality, the iterative parcellation based on the same amount of data were significantly more reliable. In addition, the iterative parcellation can be directly applied to the bandpass filtered task fMRI data and produce functional maps comparable to maps based on pure resting-state data (see Figure 3d & Figure 5e). In a small group of surgical patients, we found that sensorimotor networks could be localized with higher accuracy by the iterative parcellation than using conventional task fMRI.
The advantage of the iterative parcellation over conventional task fMRI may be explained by the different amount of variance in the BOLD signal they use for mapping. Task-evoked activity accounts for only a small percentage of the total variance in the functional MRI signal and therefore provides less stability, as the practical limits of scanning burden constrain the amount of task data that can be acquired, especially in patient populations. Variance utilized in task activation mapping can be estimated based on the variance explained by the hemodynamic task model. In the eight surgical patients reported in the present study, task-related activity in the motor regions of interest defined by ECS accounted for only 32.5% of the total variance in the functional MRI signal. Prior work has shown that coherent spontaneous activity does not disappear during task paradigms, but continues 45 .
Our parcellation approach utilizes the spontaneous activity for mapping, which may account for the major portion of the variance in task fMRI BOLD signal 45 .
To render this parcellation strategy useful in mapping the language and memory networks in patients, further optimization and validation are necessary. Nevertheless, our preliminary observations indicate that parcellation can reliably identify a strongly left-lateralized network overlapping with the regions activated by a language task, and a right-lateralized network that is located in traditional ventral attention regions. Additionally, lateralization of these networks may relate to handedness. These observations suggest that this iterative individually-tailored parcellation captures a large portion of the individual variability present in the organization of cerebral networks.
Improving Cross-subject Alignment for Group Analysis
Establishing the functional correspondence between subjects is a prerequisite for group-level functional imaging analyses. Although the association between brain anatomy and function is not fully understood and can vary across individuals, most fMRI processing tools align individual subjects to a common template based on anatomical features such as global morphology or landmarks identified by structural MRI 46 , 47 . Functional networks are likely to be misaligned if they are not tightly linked to the macroscopic anatomy. For example, aligning subjects for the investigation of language function can be particularly challenging because the distribution of the language network is known to be highly variable and can even be found in different hemispheres in different individuals. Substantial inter-subject variability in functional regions was found even after carefully aligning the data based on curvature which largely removed macro-anatomical variability 19 . Some recent studies have attempted to align subjects based on functional characteristics. By incorporating the intersubject signal correlations into a cortical registration algorithm, Subuncu et al. brought functionally similar regions into correspondence during a movie-watching task 48 . However, this strategy relies on consistent task activations across subjects. Robinson et al. developed a novel method that is capable of aligning subjects using a wide variety of characteristics including both structure and function 49 . They demonstrated strong increases in the cluster mass of task activations when subjects were aligned based on resting-state functional connectivity compared to curvature-based registration. The development of functional network parcellation using resting-state connectivity 10 , 11 , especially parcellation at the individual level 14 , 27 , may offer a complementary connectivity-based functional localizer for group-level analyses. A parcellation as described in the present study can provide a set of functional landmarks for cross-subject registration and lead to novel strategies of brain image alignment.
Limitations and Future Directions
There are several technical limitations of this study that deserve mention. First, the number of networks was selected according to specific technical criteria instead of relying on biological considerations 10 . The fixed number of networks may not be appropriate for all individuals, especially for patients who have experienced functional reorganization due to diseases. In some patients certain networks may become completely absent. For example, dramatic reorganization of the tongue motor area was observed in one of our patients due to encephalomalacia (see Patient 5 in Supplementary Fig. 6). This change in functional organization has led to misalignment of the hand motor networks in the parcellation, where the hand network spread to lower portions of the post-central gyrus. Thus, additional improvement and validation of this iterative functional parcellation method are required in order to apply it to patients with distorted anatomy. For example, in patients with localized lesions (limited to one hemisphere), the iterative functional parcellation could be performed in the healthy hemisphere without distortion, as well as in the cerebellum if no lesions are observed. The functional properties in the affected hemisphere could then be estimated based on its functional connectivity to the healthy cerebral hemisphere or the unaffected cerebellum.
Secondly, we parcellated the cortex into a relatively small number of networks, which can reduce the sensitivity to subtle changes of functional networks, such as those due to learning or other experiences. Future development of the parcellation technology should aim at mapping functional networks with finer spatial resolution and determining the number of networks more flexibly in different subjects. A possible strategy is to initiate the iterative parcellation from a population-based atlas with large number of networks, and gradually adjusting the number of networks by merging networks with similar time courses (e.g., r > 0.5). Once a merger occurs, the iterative parcellation can be restarted with the reduced number of networks. This strategy flexibly adjusts the number of networks based on an individual subject's data and can accommodate the need for identifying small networks. Iterative parcellation with this flexible strategy can also achieve high reproducibility (see Supplementary Fig. 7 for an example). Alternative strategies are also possible such as estimation of regions based on local transitions in connectivity properties 13 -15 .
Finally, functional maps derived from fMRI can be influenced by various confounding factors. For example, spatial specificity of the functional connectivity maps can be influenced by the signal in macroscopic veins, and signal correlations can be overstated within or between highly vascularized regions 50 . Thus, inter-subject variability observed in functional connectivity patterns can also be confounded by variations in vascular anatomy. While it is difficult to quantify the exact contribution of vascular variation, the high intersubject variability in functional connectivity observed in the association cortex, especially the variability in hemispheric lateralization, is unlikely to be dominated by vascular variations, but contributions of vascular anatomy to the topographical maps studied here will be critical.
Participants and Data Collection
Four separate fMRI datasets obtained with different imaging parameters were employed in the current study.
Dataset I-The first dataset consists of twenty-five healthy subjects (age 51.8 ± 6.99, nine female, two left handed) enrolled as a control dataset in a longitudinal fMRI study on stroke recovery. Participants were screened to exclude individuals with a history of neurologic or psychiatric conditions, as well as those using psychoactive medications. Each subject underwent five scanning sessions within 6 months (7, 14, 30, 90 and 180 days from enrollment). All participants performed two or three resting-state runs per session (6 m 12 s per run) to estimate intrinsic functional connectivity. After quality control, 23 subjects who had at least two good runs (tSNR > 100) in each session were included in this study (mean = 2.02 runs). This dataset has been previously reported 31 . MRI data were acquired on a 3 Tesla Siemens TimTrio system (Erlangen, Germany) using the 12-channel phased-array coil supplied by the vendor. Structural images were acquired using a sagittal MP-RAGE threedimensional T1-weighted sequence (TR All HCP subjects were scanned on a customized Siemens 3T "Connectome Skyra" scanner. Structural images were acquired using the 3D MPRAGE T1-weighted sequence with 0.7 mm isotropic resolution (FOV = 224 mm, matrix = 320, 256 sagittal slices in a single slab, TR = 2,400 ms, TE = 2.14 ms, TI = 1000 ms, flip angle = 8°). The scan parameters of the rs-fMRI data were: TR = 720 ms; TE = 33.1 ms; flip angle = 52°; FOV = 208 × 180 mm; slice thickness = 2.0 mm; 72 slices; 2 mm isotropic voxels, multiband factor = 8; echo spacing = 0.58 ms; Bandwidth (BW) = 2290 Hz/Px; time points = 1200. The task acquisitions were identical to the resting-state fMRI acquisitions in order to provide maximal compatibility between task and resting data. 12 s per run). All data were collected on matched 3T Tim Trio scanners (Siemens, Erlangen, Germany) using a 12-channel phased-array head coil. Images were acquired using the gradient-echo echo-planar pulse sequence (TR = 3,000 ms, TE = 30 ms, flip angle = 85°, 3 × 3 × 3 mm voxels, FOV = 216 and 47 slices collected with interleaved acquisition with no gap between slices). Whole brain coverage including the entire cerebellum was achieved with slices aligned to the anterior commissure-posterior commissure plane using an automated alignment procedure, ensuring consistency among subjects 54 Table S1.
Dataset IV-The fourth dataset included eight surgical candidates (age 19.5 ± 5.0; five female; one left handed) with intractable epilepsy. This was a subset of patients from a recently published study of cortical mapping using gamma activity recorded from subdural electrode grids 56 . The experiment included a preoperative fMRI scan, surgical implantation of subdural electrode grids and direct electrical cortical stimulation (ECS) using these grids.
No seizures were observed one hour before or after the fMRI or ECS in all patients. The locations of the electrodes and how long they would stay implanted were determined solely by clinical criteria. Written consent was obtained from each patient or their guardians and the experiments were approved by the Ethics Committees of the Second Affiliated Hospital of Tsinghua University. MRI data were collected on a Philips Achieva 3.0 Tesla TX whole body MR scanner using an 8-channel SENSE head coil. Structural images were acquired using a sagittal magnetization-prepared rapid gradient echo T1-weighted sequence (TR = 8. Two types of functional runs were collected from the epilepsy patients: task activation runs (all eight subjects) and resting state runs (six of eight subjects). All eight subjects performed five motor task activation runs. Each run consisted of one type of self-paced movement (left hand, right hand, left foot, right foot, or tongue) consistent with standard preoperative mapping paradigms. Each run was 144 seconds long and consisted of six 12-second task blocks interleaved with six 12-second rest intervals. Patients performed motor tasks according to the instructions presented on the computer screen using the Psychophysics Toolbox in MATLAB (MathWorks, Inc.). Six subjects also underwent two resting-state runs (360 s each run), during which they were asked to fixate on a crosshair in the center of the screen. These pure resting state runs were collected for comparison purposes with the maps created based on the task runs.
After an adequate number of seizures had been recorded, bedside ECS mapping was performed to identify the sensorimotor cortices 56 . Using an Ojemann Cortical Stimulator (Integra Life-Sciences), trains of 60-Hz biphasic pulses lasting for 2 -5 seconds were delivered to selected pairs of electrodes. The current intensity of the stimulation started at 2 mA and was gradually increased until patients showed or reported symptoms related to the sensorimotor cortex or the stimulus strength reached 15 mA. Each stimulation involved a pair of electrodes; thus, both electrodes were considered positive when a hand or tongue movement or sensory was produced.
Data Processing
Dataset I-Resting-state fMRI data of the 23 subjects in this longitudinal dataset were processed using the procedures previously described 10 , which were adapted from 8 and 57 .
The following steps were performed: (1) slice timing correction (SPM2; Wellcome Department of Cognitive Neurology, London, UK), (2) rigid body correction for head motion with the FSL package 58 , 59 , (3) normalization for global mean signal intensity across runs, and (4) bandpass temporal filtering (0.01-0.08 Hz), head-motion regression, whole-brain signal regression, and ventricular and white-matter signal regression. The BOLD frames were not censored based on head motion but all runs included in the present study showed temporal SNR > 100.
Structural data were processed using the FreeSurfer version 4.5.0 software package. Surface mesh representations of the cortex from each individual subject's structural images were reconstructed and registered to a common spherical coordinate system 46 . The structural and functional images were aligned using boundary-based registration 60 within the FsFast software package (http://surfer.nmr.mgh.harvard.edu/fswiki/FsFast). The preprocessed resting-state BOLD fMRI data were then aligned to the common spherical coordinate system via sampling from the middle of the cortical ribbon in a single interpolation step 10 .
FMRI data of each individual were first registered to the FreeSurfer template which consisted of 40,962 vertices in each hemisphere. A 6-mm full-width half-maximum (FWHM) smoothing kernel was then applied to the fMRI data in the surface space. The smoothed data were then down-sampled to a mesh of 2,562 vertices in each hemisphere using the mri_surf2surf function in FreeSurfer software.
Dataset II-The "minimally processed" fMRI data of the HCP subjects were used, which had been preprocessed in the HCP pipeline using FSL (FMRIB Software Library), FreeSurfer, and the Connectome Workbench software 37 , 61 -63 . The preprocessed data were projected to the FreeSurfer template with a mesh of 40,962 vertices in each hemisphere. The following steps were then performed: 1) demeaning and detrending across each run, 2) bandpass filtering (0.01-0.08Hz), 3) head-motion regression and whole-brain signal regression and 4) smoothing with a 6 mm FWHM smoothing kernel in the surface space. The data were then down-sampled to a mesh of 2,562 vertices in each hemisphere using the mri_surf2surf function provided by FreeSurfer. For connectivity analyses, the task fMRI data were processed in the same way as the resting-state data. To map the brain regions activated by the seven tasks, fixed-effects analyses were performed using FEAT 61 ; see 37 for details.
Dataset III-Resting-state fMRI data of the 52 pairs of left handed and right handed subjects were preprocessed identically to the first dataset.
Dataset IV-For parcellation analysis, bandpass filtered task fMRI and pure resting-state fMRI data of the surgical patients were preprocessed identically to the first dataset. Conventional task-evoked activation maps in this dataset were estimated using the general linear model. Regressors of no interest included motion correction parameters and low frequency drift. The task-induced BOLD response was modeled by convolving the hemodynamic response function with the experimental design. Intracranial electrodes were registered to the cortical surface using our in-house software 56 to enable the comparison between the ECS maps and the functional parcellation. A post-implantation CT scan was obtained within 24 -48 h after the implant surgery for localization of the electrodes. The post-implantation CT images were registered to T1-weighted MRI images using a mutualinformation-based linear transform 56 . Due to postoperative edema, electrodes extracted from the post-implantation CT images may appear off the surface reconstructed from the pre-surgical MRI. Our in-house tool allows us to manually adjust the locations of electrodes according to the 3D shape of the cortical surface. MRI surface vertices within a 6 mm radius of the positive electrodes were defined as positive. This resulted in an ECS map on the surface that can be directly compared to the map obtained from the functional parcellation.
Population-based Functional Atlas
A functional network atlas was estimated based on 1,000 healthy subjects 10 and projected onto the individual subject's cortical surface using the FreeSurfer software. The original atlas included 17 networks where hand sensorimotor areas were not separated from other areas. Given the common need to map hand areas in surgical patients, we identified the hand sensorimotor areas from this atlas based on activations in a hand motor task 64 . As a result, this population atlas consisted of 18 networks and would serve as the initial guess of the functional network organization in an individual subject's brain.
Evaluating Test-retest Reliability and Inter-subject Variability of the Maps Derived from the Iterative Parcellation
Intra-subject test-retest reliability of the parcellation results was computed using the Dice coefficient after projecting the parcellation results back to each individual subject's cortical surface. This can be simply computed as the percentage of vertices that were assigned to the same network in two sessions. To assess the reliability of the parcellation technique at the group level, Dice coefficients were then averaged across all subjects. Inter-subject variability was computed on the FreeSurfer surface template (2,562 vertices in each hemisphere) based on the Dice coefficient between any pair of subjects and then averaged across all pairs.
Comparing the Task fMRI and Iterative Parcellation with the ECS Findings
For the patient dataset (Dataset IV), the results of different mapping modalities were projected to each patient's cortical surface for comparison with the ECS findings. Taking the ECS findings as references, the sensitivity and specificity of the activation map and the network parcellation were quantified. Sensitivity was computed by dividing the number of true positives (fMRI positive vertices that were also positive in the ECS maps) by the number of true positives plus false negatives (i.e. total vertices positive in the ECS maps). The specificity was computed by the number of true negatives (fMRI negative vertices that were also negative in the ECS maps) divided by the number of true negatives plus false positives (i.e. total vertices negative in the ECS maps). ROC curves were obtained by calculating the sensitivity and specificity across a wide range of different thresholds. The area under the curve was computed for each subject and compared across methods using a Wilcoxon paired non-parametric test.
Estimating Functional Lateralization
Lateralization was computed for each network derived from the iterative parcellation.
Vertices that belonged to a specific network were separated into left-hemisphere and righthemisphere portions. A lateralization index was then computed based on the following equation: (1) Where V L is the count of vertices in the left hemisphere, V R is the count of vertices in the right hemisphere.
Visualization and Statistics
The iterative parcellation was performed on the FreeSurfer fsaverage4 template and the resulting network labels were upsampled to each individual subject's own cortical surface using the mri_surf2surf function. The labels were then merged into a single "annotation" file using the write_annotation function provided by FreeSurfer. The parcellation results were visualized in each individual's cortical surface using FreeSurfer.
No statistical methods were used to pre-determine sample sizes but our sample sizes are larger than or similar to those reported in previous publications 31 , 37 , 65 , 66 . Within each dataset, no randomization or blinding was employed to separate subjects into different groups. Two-tailed t-test was used for all comparisons in this study except for the experiment shown in Figure 5, which used Wilcoxon rank sum test. For the t-tests, data distribution was assumed to be normal but this was not formally tested.
Code Availability
The code of the iterative parcellation algorithm is available from the corresponding authors upon request.
A supplementary methods checklist is available.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material. Parcellating the functional networks in an individual subject's brain using an iterative adjusting approach. The technique includes the following steps: 1) A population-based functional brain atlas was registered onto the individual subject's cortical surface using FreeSurfer. The individual subject's BOLD signal time courses were then averaged across the vertices that fall within each network. These atlas-based network time courses were used as the "reference signals" for the subsequent optimization procedure.
2) The individual subject's BOLD signal at each vertex was then correlated to the 18 "reference signals". Each vertex was reassigned to one of the 18 networks according to its maximal correlation to the "reference signals". A confidence value was also computed as the ratio between the largest and the second largest correlation values. After each vertex was reassigned, the BOLD signals of the high confidence vertices (e.g., >1.1) in each network were then averaged and termed the "core signal". 3) For each network, the "core signal" derived from Step 2 and the original "reference signals " derived from Step 1 were averaged in a weighted manner. Specifically, the "core signal" was multiplied by the weighting parameters derived from inter-subject variability and SNR, as well as the number of iterations. The averaged signal was used as the new "reference signal" for the next iteration. Using these new "reference signals", the brain vertices were further reassigned to one of the 18 networks. 4) Steps 2 & 3 were repeated until the algorithm reached a pre-defined stopping criterion.
Figure 2.
Iterative brain parcellation is highly reproducible within subjects and captures differences across subjects. Twenty-three healthy subjects underwent five resting-state scanning sessions within six months. The functional organization of the individual subject's brain was parcellated into 18 networks using the data of each scanning session. The parcellation networks of three subjects that showed the highest reproducibility across sessions are displayed so that inter-subject variability can be appreciated. The functional maps of different subjects differed substantially, especially in the higher-order association areas (see also Supplementary Fig. 2 for maps of three subjects that showed the highest, median, and lowest reproducibility. Maps of all 23 subjects can be downloaded at: http:// nmr.mgh.harvard.edu/bid/download.html). Quantitative analyses of intra-subject reliability and inter-subject variability based on the HCP subjects. (a) One hundred subjects from the Human Connectome Project (the "Unrelated 100") were employed for validation purposes. Intra-subject reliability and intersubject variability of the parcellation maps after each iteration are plotted. Standard deviations are represented as shaded regions around the curves. As the iteration progressed, inter-subject variability increased, while intra-subject reliability decreased (see also Supplementary Fig. 3 for spatial distributions of reliability and variability after each iteration). (b) The networks of three exemplary subjects are displayed. Maps of all 100 subjects can be downloaded at: http://nmr.mgh.harvard.edu/bid/download.html. (c) Parcellation based on the resting-state fMRI demonstrated high intra-subject reliability and high inter-subject variability. Comparing two rs-fMRI sessions of the same subject, on average 82.4% ± 3.2% of the vertices were assigned to the same networks. Between any two individuals, on average only 60.5% ± 2.8% of the brain vertices were assigned to the same networks. Error bars are mean ± SD. The intra-subject consistency of network membership was significantly higher than the inter-subject consistency (unpaired two-tailed t-test, p<0.001). The iterative parcellation technique was also applied to the concatenated task data of the 100 HCP subjects. Parcellation results based on task data and resting-state data demonstrated an overlap of 81.7% ± 4.0%, suggesting that whole-brain network parcellation could also be obtained from an individual subject's task data. (d) Networks derived from the concatenated task data are shown for three exemplary subjects. Compared to left handed subjects, right handed subjects showed a trend for stronger lateralization in the languagerelated network (p=0.057, unpaired two-tailed t-test) and significantly stronger lateralization in the ventral attention-related network (p = 0.003, unpaired two-tailed t-test). Error bars are mean ± SEM. Sensorimotor networks identified by individual brain parcellation showed good correspondence with functional regions localized by invasive cortical stimulation. (a) The hand and tongue sensorimotor regions of eight surgical candidates were mapped using multiple approaches for comparison. Sensorimotor regions identified by ECS were used as the gold standard. The red dots on the ECS maps indicate negative electrodes, while the yellow dots indicate positive electrodes. (b) Sensory and motor areas identified by traditional task activation showed low consistency with the ECS maps. (c) The hand sensorimotor regions identified by iterative parcellation based on the concatenated task fMRI data were consistent with the ECS maps. The map shows the vertices with high confidence values (>1.2). (d) Individual brain parcellation may serve as a prescreening method for ECS. The map shows the network membership of vertices with high confidence values (>1.1). Iterative parcellation can provide a rough estimate of the regions of interest for cortical stimulation, potentially shortening the stimulation procedure. (e) The sensitivity and specificity of the hand and tongue sensorimotor maps in 8 surgical patients were statistically measured across a wide range of thresholds for five different mapping approaches. The results are displayed in ROC curves, including the iterative parcellation technique using task fMRI of eight subjects (green), the iterative parcellation technique using pure resting-state fMRI of six subjects (black), directly projecting the populationbased atlas to each individual subject (blue), traditional task-activation mapping alone (purple) and task activation masked with anatomical labels generated by FreeSurfer (red). | 2017-05-06T03:53:10.080Z | 2015-10-23T00:00:00.000 | {
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17612899 | pes2o/s2orc | v3-fos-license | Dose-Dense Epirubicin and Cyclophosphamide Followed by Weekly Paclitaxel in Node-Positive Breast Cancer
Background. Adding taxanes to anthracycline-based adjuvant chemotherapy has shown significant improvement in node-positive breast cancer patients but the optimal dose schedule has still remained undetermined. Objectives. The feasibility of dose-dense epirubicin in combination with cyclophosphamide (EC) followed by weekly paclitaxel as adjuvant chemotherapy in node-positive breast cancer patients was investigated. Methods. All patients were treated with epirubicin (100 mg/m2) and cyclophosphamide (600 mg/m2) every two weeks for four cycles with daily Pegfilgrastim (G-CSF) that was administered 3–10 days after each cycle of epirubicin and cyclophosphamide infusion which followed by (80 mg/m2) paclitaxel for twelve consecutive weeks. Results. Sixty consecutive patients were analyzed, of whom 57 patients (95%) completed the regimen and no case of toxicity-related death was observed. Grade 3/4 hematologic toxicity was uncommon and the most common grade 3/4 nonhematological adverse event was neuropathy disorders. Conclusions. Dose-dense epirubicin and cyclophosphamide followed by weekly paclitaxel with G-CSF support is a well-tolerated and feasible regimen in node-positive breast cancer patients without serious complications.
Introduction
Anthracyclines are the most effective drugs in the treatment of breast cancer, and the addition of a taxane to an anthracycline-containing regimen, after or concurrently with anthracycline treatment, appears to provide significant benefit, particularly in node-positive cases [1][2][3] and the combination of paclitaxel with anthracycline has been reported as an active regimen in improvement of diseasefree survival and overall survival [4,5]. A large number of adjuvant taxane studies have been reported. The CALGB 9141 and NSABP B-28 trials demonstrated that doxorubicin and cyclophosphamide (AC) plus paclitaxel (PAC) is superior to four cycles of AC alone, which in turn has equivalent efficacy to six cycles of cyclophosphamide plus methotrexate and 5fluorouracil (CMF) [6,7].
A randomized multicenter phase III study was conducted by Polyzos et al. to compare the sequential docetaxel followed by epirubicin/cyclophosphamide combination with that of epirubicin, cyclophosphamide, and 5-fluorouracil (FEC). The sequential docetaxel followed by epirubicin/ cyclophosphamide adjuvant chemotherapy regimen resulted in improved five-year disease-free survival (DFS) in women with axillary node-positive early breast cancer [8].
Also, higher doses (dose intense) and more frequent administration of these drugs (dose-dense) were well tolerated and correlated to disease-free survival in breast cancer [9][10][11]. One method for increasing dose intensity in high risk patients in order to achieve the most benefit of maximum dose intensity is reducing the conventional drug dose intervals (dose-dense regimen).
Clinical trials suggested that paclitaxel was more effective and less myeoltoxic taxane than docetaxel and the lowdose weekly paclitaxel might be superior to higher doses given less frequently in both metastatic and adjuvant setting [4,5].
The cancer and leukemia Group B trial 9741 compared different sequential schedules of doxorubicin, cyclophosphamide, and paclitaxel given either every 3 weeks (conventional) or every 2 weeks (dose-dense) with systemic granulocyte colony-stimulating factor (G-CSF) support in the dose-dense arms. The dose-dense regimens significantly prolonged both disease-free survival and overall survival without increasing toxicity [11].
In a published trial, women with early breast cancer received four cycles of doxorubicin and cyclophosphamide every 3 weeks postoperatively and then they were allocated to weekly taxane versus 3-weekly taxane and paclitaxel versus docetaxel in a factorial design. They concluded that treatment with doxorubicin and cyclophosphamide followed by weekly paclitaxel is associated with improved disease-free survival and overall survival in comparison with paclitaxel given every 3 weeks [5].
Previous clinical trials and meta-analysis showed that epirubicin is effective as doxorubicin but with less cardiotoxic and myelosuppressive effects [12,13].
Since the optimal schedule of administration of epirubicin and cyclophosphamide plus paclitaxel and sequencedependent toxicity have not been elucidated yet, further clinical trials with higher sample size are suggested.
We conducted this trial to evaluate the safety and feasibility of dose-dense epirubicin and cyclophosphamide followed by weekly paclitaxel as adjuvant chemotherapy in nodepositive breast cancer patients.
Patients were excluded if they had even one of the following: T4 stage, inflammatory breast cancer, ductal carcinoma in situ (DCIS), prior history of any other cancer or anticancer therapy, other significant medical conditions (most notably cardiac, neurologic disorders), sensory or motor neuropathy of severity greater than WHO grade 1, pregnant or breastfeeding patient or inadequate contraception, or any other condition that was considered to make the patient ineligible for this study by the investigators. The study was performed in accordance with the declaration of Helsinki and written informed consent was obtained prior to participation in the study. The study protocol was reviewed and approved by institutional review board of the Shohadaye-Tajrish Hospital.
Patient Assessment.
Eligible patients who had given consent were invited to attend the assessment to provide baseline data as follows: full medical history and physical examination, hematology and biochemistry assessment (such as renal and liver function tests), hormone receptor status, chest radiography and/or computed tomography (CT) scan, electrocardiogram and echocardiography, abdominal and pelvic ultrasound or computed tomography (CT) scan, bone scan, and other evaluation based on symptoms of patients.
Treatment Plan.
All patients received epirubicin (100 mg per square meter of body-surface area, given by slow intravenous push during a period from 5 to 15 minutes) and cyclophosphamide (600 mg per square meter in 300-400 cc normal saline solution by intravenous infusion from 30 to 60 minutes) every two weeks for four cycles. Followed by weekly paclitaxel was given as a 1-hour intravenous infusion via 300 cc normal saline solution at a fixed dose of 80 mg of per square meter for twelve cycles. Granulocyte colonystimulating factor (G-CSF) 300 microgram daily was administered in all patients on days 3-10 of each course of epirubicin and cyclophosphamide.
Premedication for EC consisted of a 5-HT3 serotonin receptor antagonist (e.g., granisetron or ondansetron) and dexamethasone intravenously. Standard premedication with glucocorticoids, H1 and H2 receptor blockers (e.g., promethazine, clemastine, and ranitidine) were given before paclitaxel administration. Actual body weight was used for bodysurface area calculations. A complete blood count with leukocyte differential was performed before each chemotherapy treatment. Patients were seen every week during treatment for history and physical examination and assessment of performance status and toxicity.
Dose
Modification. Treatment was given on day 1 of every cycle if absolute neutrophils count (ANS) and platelets were ≥1.5 × 10 9 /l and ≥100 × 10 9 /l, respectively. Unless doses in the subsequent cycle were reduced, doses in the current cycle were administered according to protocol. In case of grade 4 nonhematologic toxicities (excluding nausea, vomiting, and alopecia), treatment was delayed by up to one week, and complete blood count and toxicity grading were repeated weekly. Patients requiring a treatment delay of more than three weeks were removed from the study or treatment continued after patients' adequate recovery.
After chemotherapy completed, radiation therapy was conducted following the last cycle of chemotherapy and after recovery from any toxicity, according to standard institutional dosing guidelines and techniques. Patients whose tumors expressed either (or both) the estrogen or progesterone receptor-positive were placed on a 5-year course of tamoxifen 20 mg/day. Postmenopausal patients were offered aromatase inhibitors as an alternative to tamoxifen.
Evaluation of Toxicity.
Toxicity for each cycle was assessed before the commencement of the following cycle and was graded using the National Cancer Institute Common Toxicity Criteria (NCI-CTC version 3).
Patients were followed by history and physical examination within 15 days and one month after last infusion, and this follow-up was processed every 2-month intervals for the first year of chemotherapy completion then every six-month interval for years 4-5. Each visit included a complete blood count, along with hematologic studies and chemistries (liver and renal function tests), chest X-ray, and ECG. Computed tomography scan of the chest, abdomen, and pelvic and a bone scan (or both) were considered if clinically indicated abnormal laboratory values at the discretion of the physician. After treatment completed, echocardiography was carried out in all patients. Mammography was performed on the remaining breast(s) annually.
Statistical
Methods. The objective of the study was to evaluate the toxicity of EC plus paclitaxel in node-positive breast cancer and to answer the trial aims, 60 eligible patients were required. Descriptive methods were applied for all the variables. Statistical analyses were carried out using SPSS software version 16 and < 0.05 was considered significant.
The primary endpoint was the incidence ( ) of grade 4 toxicity. The study was designed as a one-stage three-outcome phase II study, in which H0 was > 50% and HA was < 25%. Under these assumptions and with and errors rate of 5% each, 60 patients were assigned to reject a toxic treatment (with >50% grade 4) and accept a nontoxic treatment (with <25% grade 4) with a probability >90%. If <15 grade 4 toxic events occurred, the treatment was to be considered tolerable. If >30 grade 4 toxic events occurred, the treatment was to be considered intolerable. If 16-29 grade 4 toxic events occurred, the study was not conclusive. Table 1. The mean age of the patients was 49.6 years and 58.3% of the patients were <50 years old. The median number of examined lymph nodes was 11 (range 2-37). 58.6% of the patients had one to three positive lymph nodes, 27.8% had four to nine positive nodes, and 13.6% had ten or more positive nodes. Median tumor size was 3 cm and in 28.3% of the patients, size of tumor was less than ≤2 cm in maximum diameter. The tumor was positive for both of estrogen and progesterone receptors in 60% and positive for HER2 in 39.7%.
Toxicity. The treatment was generally well tolerated and
Chemotherapy cycle was completed in all patients except three patients (5%). Nine patients (15%) have undergone any grade 4 of adverse events that two of them went off the study after the second infusion of paclitaxel cycle due to grade 4 paresthesia concomitant with muscular pain and/or diarrhea, which was not tolerable by patients and did not further receive paclitaxel. Besides, two patients experienced paresthesia after last infusion of paclitaxel. Treatment delayed in 10 patients (16.7%). The cause of delay was nausea, diarrhea, neuropathy, and skin-nail disorders. No decrease in dose was required. Also, one patient withdrew after the tenth infusion of paclitaxel cycle due to hyperosmolar diabetes which was not related to study treatment.
Six patients (10%) were hospitalized due to adverse events such as paresthesia, skin-nail disorders, arthralgia, dehydration, nausea, vomiting, and diarrhea. However, there were no cases of toxicity-related deaths.
Hematological and nonhematological toxicity data are summarized in Table 2.
None of the patients experienced cardiac toxicity or sign of heart failure during treatment and follow-up as initial and posttreatment echocardiography in all patients was normal.
As a consequence of the regular assessment of blood counts, except one patient who experienced grade 3 neutropenia, none of the patients suffered from grade 3/4 hematologic toxicity; however, there was a nearly high rate of grade 1/2 neutropenia (35%), but it was asymptomatic and almost did not modify the treatment plan (there was only one case of grade 3 neutropenia). Likely, it was the reason for the use of G-CSF in all patients. Grade 1/2 anemia was common (75%) as well. During weekly paclitaxel, sensory neuropathy was a common adverse event 25 (41.7%). Another remarkable nonhematological toxicity grade 3/4 was 14 cases of skinnail disorder (23.4%). High fraction of patients suffered from muscular toxicity; 12 cases suffered (20%) from myalgia; and 14 cases suffered from (23.3%) arthralgia. Eight patients (13.3%) developed total alopecia.
Follow-Up.
At the time of the analysis, the median follow-up period was 27 months. 91.6% of patients were disease-free. Four systemic relapses were observed and three of them are dead.
Discussion
This trial was designed to assess safety and tolerability of dose-dense epirubicin and cyclophosphamide followed by weekly paclitaxel as adjuvant chemotherapy in node-positive breast cancer patients and it was found that this chemotherapy regimen was well tolerated in terms of low hematologic toxicity which is most likely due to G-CSF support. Also, there were not any cases of cardiac toxicity and nausea and vomiting were controlled easily. The results are consistent with studies which confirmed the feasibility of dose-dense regimens and benefits of weekly paclitaxel [5][6][7][8][9]11].
Results from the recent studies have demonstrated that accelerated epirubicin or doxorubicin with cyclophosphamide given at 2-week interval with G-CSF support could be well tolerated as same as given schedules over standard 3-week intervals in early breast cancer with fewer grade 3/4 neutropenia [9].
Citron et al. compared standard 3 weekly and accelerated 2 weekly schedules of concurrent doxorubicin and cyclophosphamide followed by paclitaxel or sequential doxorubicin, paclitaxel, and cyclophosphamide. They found that grade 4 neutropenia was more frequent in the standard 3 weekly schedules than the accelerated regimens (33% versus 6%, < 0.0001) [11].
Hamid Reza Mirzaei and colleagues conducted a trial of dose-dense epirubicin and cyclophosphamide followed by docetaxel. 55% of patients suffered from grade 4 toxicity and most common grade 3/4 toxicities included neurosensory, arthralgia, and skin toxicity [14].
There was more nausea and vomiting with the EC/T regimen compared with the other two arms. The rate of febrile neutropenia was the highest in the CEF arm. Cardiac toxicity as reflected by symptomatic congestive heart failure was low but was the highest in the CEF arm [15]. As in our trial, there was more peripheral neuropathy with the taxane-containing regimens.
Mamounas et al. conducted a trial of doxorubicin and cyclophosphamide every 3 weeks for four cycles compared to doxorubicin and cyclophosphamide followed by four additional 21-days cycles of paclitaxel. Most common grade 3 or greater toxicity during paclitaxel therapy included neurosensory toxicity, neuromotor toxicity, arthralgia and/or myalgia, and febrile neutropenia in 15%, 7%, 12%, and 3% of patients, respectively. They confirmed the benefits of incorporating a taxane (paclitaxel) in the adjuvant setting [6].
Neurotoxicity is a major concern in paclitaxel-associated treatment. As expected, we found that a significant fraction of patients had grade 3 or higher of peripheral neuropathy during treatment, but these had resolved in all patients by subsequent follow-up; and nearly a high percentage of patients experienced moderate-to-severe myalgia and arthralgia that were treated symptomatically.
Recently, in a randomized trial by Sparano, four cycles of AC were administered every 3 weeks postoperatively which was followed by one of four taxane-based treatments, specifically "paclitaxel" or "docetaxel" either weekly or every three weeks. They concluded that the group receiving weekly paclitaxel had significantly more moderate-to-severe neuropathy than the group receiving standard therapy which was consistent with our results. Moreover, the group receiving docetaxel every 3 weeks showed significantly more severe neutropenia and its associated complications which were more frequent in docetaxel group than paclitaxel [5].
Ishikawa and colleagues evaluated the feasibility of AC/EC every three weeks followed by weekly paclitaxel or Chemotherapy Research and Practice 5 four cycles of three consecutive weekly administration followed by a one-week rest (3 × 4) and reported during AC/EC, (68%) of patients developed grade 3/4 granulocytopenia, compared to (7.8%) receiving 12 PAC and (2.1%) receiving 3 × 4 PAC. Sensory neuropathy was a common adverse event during weekly paclitaxel. Although sever symptoms of grade 3 occurred in only one patient with 12 PAC, grade 1/2 neuropathy occurred in (52.7%) patients receiving 12 PAC and in (54.1%) receiving 3 × 4 PAC [16].
Among the other important findings, a high incidence of neurosensory disorders after repetitive cycles of weekly paclitaxel was also noted; however, it was not so severe for patients to withdraw from the study. This adverse effect was the major problem with long-term treatment with paclitaxel which caused substantial patients discomfort. No differences in nonhematologic toxicities such as neurosensory disorders were observed between 12 PAC and 3 × 4 PAC treatment [16].
Follow-up of patients in this study will demonstrate results for the efficacy of this treatment, which is a secondary endpoint of disease-free and overall survival.
The endpoint was the incidence of grade 4 toxic events. According to the statistical design of the trial, 9 patients (15%) experienced grade 4 toxicity with this chemotherapy regimen.
In conclusion, the present study concluded that dosedense epirubicin and cyclophosphamide plus weekly paclitaxel with G-CSF support is a feasible and tolerable regimen in node-positive breast cancer patients, particularly with regard to neurotoxicity, the major concern of paclitaxelassociated treatment. However, larger double-blind, randomized controlled trials are needed to generalize this finding. | 2016-05-12T22:15:10.714Z | 2014-09-09T00:00:00.000 | {
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5887880 | pes2o/s2orc | v3-fos-license | Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification
Abstract Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events.
INTRODUCTION
Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function (1)(2)(3) and hundreds of Cre 'deleter' lines are available to the mouse community. Cell-type specific expression of Cre allows for specific deletion of a gene of interest by the use of a 'conditional knock-out' (CKO) allele of that gene (2). Typically, for a conditional allele a critical exon(s) is flanked by two loxP sites ('floxed') and in the cells where Cre is expressed, the floxed exon(s) is removed, resulting in a deletion, or knock-out, allele. Cre deleter lines are generated either by targeted knock-in of the Cre cDNA into an endogenous locus or by pronuclear microinjection of a Cre transgene driven by a cell-type specific promoter. For the latter, the integration site is random and in most cases not known. Knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. This becomes increasingly important in complex crosses with multiple conditional alleles, as some combinations of alleles might not be possible. Importantly, integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the transgenic phenotype (4)(5)(6) or preventing the generation of homozygous transgenic animals due to embryonic lethality when the transgene is bred to homozygosity. Transgenes often integrate as a multicopy concatemer (7) and in the absence of integration site data, hemi-and homozygous animals have to be distinguished by copy number variation (CNV) analysis which involves quantitative polymerase chain reaction (PCR) and reference DNA with a known copy number. The resolution and reliability of CNV analysis for accurate genotyping decreases as copy number increases. Therefore, another important and practical use of knowing the exact transgene insertion site is that efficient, copy-number independent and locus-specific genotyping assays can be developed. For large transgenic animal facilities, where a large number of genotyping assays are PAGE 2 OF 9 performed, automated genotyping platforms and robust assays are necessary (8). CNV analysis is more labor-intensive than real-time PCR assays due to the need for an internal copy number standard and the additional requirement for replicates to ensure accurate copy-number calling (9). Alternatively, if the insertion site is known, an assay can be designed for the wild-type DNA and for the insertion, allowing for analysis without having to calculate copy number in order to determine zygosity. Thus the characterization of transgene integration sites is important both to know if any rearrangements have occurred in the endogenous sequence of the integration site and to enable automated genotyping of as many of the Cre transgenic lines as possible.
The most commonly used method for identification of transgene insertion sites is based on inverse PCR (iPCR) (10,11). This method relies on knowledge of appropriate restriction sites located in the transgene and on sequence information to design appropriate primers for the iPCR. Furthermore, iPCR works best if the number of transgene copies is low as selective amplification of the transgene concatemer reduces the chance of identifying a PCR product containing flanking genomic sequence (12). An alternative method for identification of insertion sites, 'Splinkerette', was first developed for the cloning of retroviral integration sites (13). Just as for iPCR, Splinkerette is best suited for single or low copy integrations. While iPCR and Splinkerette can be used to identify the genomic/transgene junction, both methods rely on prior knowledge of the end(s) of integrated transgene sequences and on whether the breakpoint sequence is suited for PCR amplification. In addition, neither method provides any information about structural changes at the integration site. Although both iPCR and Splinkerette methods now take advantage of next-generation sequencing (NGS) (for example, see (14)), NGS technology alternatives to iPCR and Splinkerette for transgene mapping now include whole genome sequencing (15,16) and sequence capture followed by NGS (17). Whole genome sequencing analyses are not suited for the detailed analysis of the transgene sequence since most of the generated sequencing data is uninformative. More importantly, in hemizygous mice, the generated sequencing data is not only derived from the locus of integration but also from the wild type locus, obscuring interpretation of structural changes specific to the integration site. Moreover, many transgenes contain sequences that are homologous to endogenous sequences and the presence of the endogenous homologs in whole genome sequencing data precludes sensitive and specific analyses of the transgene. Capture (or multiplex PCR based) enrichment technologies require detailed knowledge of the transgene sequence and, inherently, only provide information across sequences known to be present in the transgene and for which capture probes/PCR primers were designed. The integration site in the genome is only detected if that specific fragment was captured by the designed probes and successfully sequenced.
As an alternative to whole genome sequencing and capture based targeted re-sequencing, targeted locus amplification (TLA) enables the selective amplification and NGS sequencing of the locus of interest without the need for detailed knowledge of the region (18). The TLA technology is based on the crosslinking, fragmentation, religation and selective amplification of DNA and results in the amplification of >100 kb of sequence information at either end of a primer pair complementary to a short (trans-) gene specific sequence. As such, TLA-based targeted sequencing enables targeted complete sequencing of loci of interest and detection of all single nucleotide variations (SNVs) and structural variants. Here we describe the use of TLA to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. These seven mouse lines are all widely used by the mouse community. For example, see http://www.informatics.jax.org/home/recombinase for an up-to-date list of references for each transgenic line. With very limited sequence data available for the transgenes used to generate these lines, Cre transgenic lines provided an ideal test-case for the power of the TLA approach to transgene mapping. Because the mouse lines all have the Cre sequence in common, we designed primers on the Cre sequence and these primers could then be used in the analysis for all Cre lines. After mapping the exact integration sites and identifying structural changes, we used the information to design quantitative PCR genotyping assays that distinguish wild-type, hemizygous and homozygous animals for four of the lines. Our data illustrates the power of the TLA method to sequence transgenes and efficiently map their integration sites with very limited requirement for prior knowledge of the transgene sequence. Furthermore, our data illustrates the power of TLA to identify structural changes occurring at the site of transgene integration.
Splenocyte preparation
Mice were euthanized and the spleens dissected and stored on ice. Splenocytes were then isolated and purified through a 40 m mesh filter. The splenocytes were collected by centrifugation at 4 • C at 500 × g for 5 min. For each spleen, the supernatant was discarded and the pellet dissolved in 1 ml 1× Pharm Lyse (BD Biosciences) and the samples were incubated at room temperature for 3 min to lyse splenic erythrocytes. To terminate the lysis reaction, 0.5 ml phosphate buffered saline (PBS) was added and the splenocytes were collected by centrifugation at 4 • C, 500 × g for 5 min, the supernatant discarded and the pellet resuspended in 0.5 ml PBS. After one final centrifugation step for 2 min, the supernatant was discarded and cell pellet resuspended in 1 ml freeze medium (PBS with 10% Dimethyl Sulfoxide and 10% fetal calf serum). The samples were stored at minus 80 • C until TLA processing.
TLA
Preparation of the samples for TLA was performed as described (18). In brief, the splenocytes were crosslinked using formaldehyde and DNA was digested with NlaIII. The samples were ligated, crosslinks reversed, and the DNA purified. To obtain circular chimeric DNA molecules for PCR amplification, the DNA molecules were trimmed with NspI and ligated at a DNA concentration of 5 ng/l to promote intramolecular ligation. Importantly, NspI was chosen for its RCATGY recognition sequence that encompasses the CATG recognition sequence of NlaIII. As a consequence, only a subset of NlaIII (CATG) sites were (re-)digested, generating DNA fragments of approximately 2 kb and allowing the amplification of entire restriction fragments. Sequences of the Cre iPCR primers are (5 to 3 ): 2291 CRE1 F GGAGTTTCAATA CCGGAGAT; 2292 CRE1 R AGGGTGTTATAAGCAA TCCC; 2299 CRE2 F AGTTTCAATACCGGAGATCA; 2300 CRE2 R TTTCGGCTATACGTAACAGG.
After ligation, the DNA was purified, and eight 25l PCR reactions, each containing 100 ng template, were pooled for sequencing. Illumina NexteraXT NGS library preparations were performed according to manufacturer's protocols. We performed sequencing of TLA libraries on the Illumina MiSeq platform pooling ∼20 libraries per V2 PE150 sequencing run yielding on average 1 million reads per library.
Bioinformatics/sequence alignment
Because the TLA protocol leads to reshuffling of genomic sequences, reads were mapped using split-read aware alignment with BWA mapping software version 0.6.1-r104, settings: bwasw -b 7 (26). Although we perform paired-end sequencing, we do not use the paired-end information in the mapping owing to reshuffling of the sequence. Paired ends are therefore treated separately in the general analysis. The data were aligned to the human (hg19), mouse (mm9) and rat (rn5) genome. The resulting BAM files were analyzed using IGV software (27).
Genotyping assays
For all seven lines, the mice were first typed for presence or absense of the Cre transgene using the generic Cre Apob primers and probes listed in Supplementary Table S1. Using the same primers and probes, samples were then analyzed in triplicate or quadruplicate and C q values for the samples and the internal reference (Apob) were calculated using CFX Manager software (Bio-Rad). The means of the C q values were used to calculate C q values and these were then used to calculate transgene copy number using the 2 − C q formula (28). Genomic DNA was isolated from tail biopsies using the Agencourt DNAdvance kit, 96 well (Beckman Coulter) and tail DNA from a homozygous knock-in Cre line (two copies of Cre) was used as copy-number reference. Based on transgene integration site sequences, genotyping assays were designed using PrimerQuest software (Integrated DNA Technologies). 5 nuclease assays were created such that one primer sits 5 to the junction site, and one reverse primer sits 3 to the probe. A wild-type probe is designed to sit entirely on uninterrupted wild-type sequence 3 to the forward primer and a mutant probe is created to sit 3 of the junction site, completely in the transgene. Primer and probe sequences are provided in Supplementary Table S2. A total of 5 l reactions were constructed as follows: 1 l (∼20-100 ng) gDNA, 2.5 l of 2× Type-It Fast SNP Probe PCR Master Mix (Qiagen) and 1.5 l of primers and probes with a final reaction concentration of 500 nM each primer and 250 nM each probe. Assembled reactions were processed on a CFX384 Touch Real-Time PCR System (Bio-Rad) under the following cycling conditions: initial denaturation at 95 • C for 8 min followed by 30 cycles of 95 • C for 10 s, 60 • C for 60 s and 72 • C for 15 s. C q values were calculated using CFX Manager software (Bio-Rad). Relative fluorescence units values were averaged for duplicate samples and plotted using Prism 6 Software (GraphPad) with Cre TG probe signal on the X-axis and WT signal on the Y-axis.
Copy number analysis of seven Cre/CreERT2 lines
In the absence of detailed information on the transgene insertion site in our transgenic lines, we routinely genotype and determine zygosity for Cre transgenic mice by CNV analysis. This is done by quantitative PCR (qPCR) using Cre-specific PCR primers and a Cre probe (see Supplementary Table S1). Using a calibrator with a known number of Cre copies, the copy number value is calculated using the standard qPCR formula, 2 − C q (28) (see 'Materials and Methods' section for details). An example of CNV genotyping data for all seven Cre/CreERT2 lines used in this study is shown in Figure 1. Although CNV analysis is separate from TLA, and not necessary for accurate TLA analysis, the calculated transgene (hemizygous) copy number for each of the seven lines is listed in Table 1 for easy reference. This method is reliable but time-consuming due to the amount of manual genotype-calling needed after obtaining the qPCR data and thus we were interested in obtaining integration-site specific data for each line to be able to generate genotyping assays that can directly distinguish wild-type, hemizygous and homozygous animals, enabling full automation of the genotyping process. Added benefits to knowing the integration site would include information of proximity to nearby genes that might be affected by the transgene, and information about structural changes at the integration site.
Identification of transgene integration sites
In order to identify the exact transgene integration sites, we performed TLA on splenocytes from an individual animal from each line. An outline of the TLA process is provided in Figure 2 (please see (18) and 'Materials and Methods' section for details). We mapped the sequence data to the mouse, rat and human genomes and analyzed the results to identify integration sites in the mouse genome and to confirm, or identify, the different components of the transgenes (e.g. a rat-derived promoter or the human estrogen receptor sequences found in CreERT2 lines). TLA provides deep sequence coverage of genomic regions in close proximity to the location of the primer set and very low sequence coverage of the rest of the genome (Supplementary Figure S1). This allows for identification of the exact transgene integration site with high confidence and for detailed characterization of any structural changes at the integration site. After mapping of the transgene integration sites, the exact transition sequences sites were subsequently confirmed by PCR followed by sequencing using genomic DNA from transgenic mice and primers designed to span the identified junction. The genomic/transgene sequence for one or both of the two junctions for each transgenic line is provided in Supplementary Figure S2, and transgene integration site data for each of the seven transgenic Cre lines is summarized in Figure 3 and Table 1. The ACTB-Cre mouse line contains a transgene consisting of human ACTB (beta-actin) promoter, the Cre cDNA and human ACTB 3 UTR and polyA signal (19). TLA analysis mapped the transgene insertion site to mouse chromosome 1 in intron 4 of Tmem163. Our analysis also detected three intronic deletions of 2.2, 7.3 and 3.2 kb, respectively, surrounding the transgene integration site.
The BEST1-Cre transgene contains about 500 bp of the human BEST1 promoter, Cre cDNA, Simian Virus 40 (SV40) exons/introns and Herpes Simplex Virus Thymidine Kinase (HSV TK) polyA (20). TLA analysis mapped the transgene insertion site to mouse chromosome 9, about 58 kb upstream of Tm108. Our analysis also detected a 120 bp deletion at the transgene integration site.
The Cdh5-CreERT2 line carries a transgene consisting of a 3 kb mouse Cdh5 (VE-cadherin) promoter, a rabbit betaglobin intron, CreERT2 and SV40 intron/exon plus polyA (21). TLA analysis maps the integration site to chromosome Figure 1 and was not part of the TLA analysis.
3, about 138 kb upstream of Ankrd50. Our analysis also detected a fragment (about 40 kb) from the mouse Vat1l gene. As this sequence is not part of the transgene itself, it's possible that the fragment co-integrated with the transgene (see 'Discussion' section). In addition, our analysis indicates that a 87.6 kb genomic duplication occured at the integration site. This duplication unfortunately prevents design of a genotyping assay that can distinguish hemizygous from homozygous mice as the wild-type probe will be able to recognize both the unmodified wild-type allele and the transgene allele. In other words, true homozygous transgenic mice would be falsely typed as hemizygous due to the extra signal from the wild-type probe (see Supplementary Figure S3). The only description of the Pdx1-Cre transgene in the original publication is that Cre is driven by the mouse Pdx1 promoter (22). By mapping the sequences to both the human and mouse genome, our TLA analysis found that the Pdx1-Cre transgene in addition to the mouse Pdx1 promoter also contains human ACTB 3 UTR and polyA signal. TLA analysis mapped the transgene insertion site to mouse chromosome 8, about 48 kb upstream of Zfp423. Our analysis shows that a 1.5 kb genomic duplication occurred at the integration site. As for Cdh5-CreERT2, this duplication prevents design of a genotyping assay that can distinguish hemizygous from homozygous mice.
The Syn1-Cre transgene contains about 4 kb of the rat Syn1 promoter, 100 bp of chloramphenicol acetyltransferase vector/UTR sequence, Cre and the human growth hormone gene (exons, introns, polyA) (23). TLA analysis mapped the transgene insertion site to mouse chromosome 6. For this insertion site, the nearest gene is located more than 1 Mb away. Our analysis identified a duplication of unknown size of the genomic region at the integration site. As for Cdh5-CreERT2 and Pdx-Cre, this duplication prevents design of a genotyping assay that can distinguish hemizygous from homozygous mice. Furthermore, we also found evidence of the co-integration of a 142 Kb region from chromosome 1 along with the transgene. For this transgene we were not able to identify more than one of the two transgene/genomic transitions. A more detailed view of the TLA analysis for Syn1-Cre is provided in Supplementary Figure S1.
The Tyr-CreERT2 line carries a transgene composed of a 3.6 kb mouse Tyr enhancer element fused to a 5.5 kb mouse Tyr promoter element. The two elements map 12 kb apart on the mouse genome. Between the promoter and CreERT2 cDNA is a rabbit beta-globin intronic sequence and the CreERT2 cDNA is followed by the SV40 polyA (24). TLA analysis maps the integration site to chromosome 2, about 7.8 kb downstream of Snrpb and 9.6 kb downstream of Tgm6. Our analysis also detected a 3 kb genomic deletion at the integration site.
Finally, The Vil1-Cre line carries 12.4 kb of the mouse Vil1 promoter fused to Cre, followed by exons, introns and polyA from the mouse Mt1 gene (25). TLA analysis maps the integration site to chromosome 17, about 119 kb upstream of St6gal2. Our analysis also detected a 14.6 kb genomic deletion at the transgene integration site.
Development of real-time PCR assays based on transgene integration site data
With transgene insertion site sequences available, we next designed genotyping assays for the four lines that did not have genomic duplications surrounding the integration site (ACTB-Cre, BEST1-Cre, Tyr-CreERT2 and Vil1-Cre) to enable high-throughput and fully-automated genotyping using real-time PCR. A genomic duplication flanking the integration site does not prevent the design of a transgenespecific assay. However, the assay for the wild-type allele will also produce a signal from the transgene allele, thus preventing accurate genotyping (Supplementary Figure S3). With the integration site sequences available from this study (Supplementary Figure S2), it is also possible and easy to design three-primer assays for regular PCR. In this type of genotyping assay, a forward genomic primer is shared and reverse primers are designed specific to either the transgene or the wild-type allele, making sure the two kinds of amplicons have different sizes. All three primers are included in the PCR reaction and the three possible genotypes (wildtype, hemizygous and homozygous transgenic) can easily be identified by agarose gel separation. This type of assay, commonly used in smaller mouse facilities, is not practical for high-throughput genotyping, however. As described in the 'Materials and Methods' section and summarized in Supplementary Table S2, we therefore developed a set of primer pairs and matching probes for real-time PCR, specific for each Cre/CreERT2 transgene insertion site and their corresponding wild-type alleles. The assays were then tested using tail-derived genomic DNA. Genotyping results for the four Cre/CreERT2 lines are shown in Figure 4 says. The genotype calls were all confirmed by our standard CNV genotyping assay (data not shown).
Effect of transgene insertion on nearby genes
For the ACTB-Cre line, we mapped the insertion site to intron 4 of Tmem163 ( Figure 3 and Table 1). Our analysis also detected three intronic deletions. It is therefore possible that the normal function of Tmem163 is disrupted in this line. However, we routinely obtain homozygous transgenic mice, so Tmem163 is either not necessary for normal development or fertility, or the transgene does not significantly alter the normal regulation and expression of Tmem163. A Tmem163 knock-out mouse has to our knowledge not been reported, but a plausible role for Tmem163 in the regulation of intracellular zinc levels has been suggested (29). It can therefore not be ruled out that ACTB-Cre mice display a Zinc homeostasis phenotype, and this should be taken into account when using the ACTB-Cre line together with alleles of genes relevant for Zinc metabolism. Table 1 lists nearby genes for the other Cre transgenic lines as well. Although none of the other integration sites are located within genes, some transgenes are located in the vicinity of genes and we cannot exclude that transgene integrations influence the expression of these nearby genes. However, since we have been able to generate homozygous mice from all seven lines, it can be concluded that none of these Cre/CreERT2 transgenes disrupt genes essential for survival and development.
DISCUSSION
Compared to other methods for identifying transgene insertion sites, including other NGS methods, TLA uniquely enables targeted and complete NGS sequencing of transgenes and integration sites and the detection of single nucleotide variants and structural variants within the transgene sequence and in sequences surrounding the integration site. We have demonstrated here that even with limited knowledge of the transgene sequences (limited to the method sections of the papers where the Cre lines were first described), it is possible to generate a complete or nearcomplete picture of the transgene integration event and provide exact genomic/transgenic borders. The transgenes described in this study all contain the Cre cDNA sequence, and we could thus use the same set of Cre-specific primers to perform TLA analysis for all the seven Cre or CreERT2 lines. We are currently using this approach to map the insertion site in additional transgenic Cre lines in our facility but since only minimal information is needed about a given transgene for successful mapping, TLA is a highly efficient method that should be useful for mapping the insertion site of any transgene. Importantly, our analysis identified structural changes at the site of transgene integration in all seven lines. Three of the lines (Cdh5-CreERT2, Pdx1-Cre and Syn1-Cre) had genomic duplications surrounding the transgene concatemer. These duplications prevent the design of genotyping assays that can distinguish wild-type, hemizygous and homozygous alleles since all the samples will have a positive signal from the wild-type probe/amplicon (Supplementary Figure S3). Four lines had small or large deletions and we identified genomic DNA fragments from another chromosome integrating along with the transgene in two lines. Our observation of structural variations at the integration site is in agreement with a chromothripsis model for transgene insertion (30,31). While the mechanism for transgenesis by random insertion is still largely unknown, it is possible that the linear transgene is simply integrated at a chromosomal site already undergoing active DNA break repair. Alternatively, by an unknown mechanism, the action of injecting linear DNA into the pronucleus of a mouse zygote could itself stimulate chromothripsis followed by DNA repair and insertion of the transgene, accompanied by other structural changes (insertions, deletions, inversions). This model suggests that only when repair by the cell's DNA repair machinery is possible in a manner compatible with survival will a transgenic founder be the result. If DNA repair is not possible, due to extensive chromosomal damage, the early embryo will not survive. To our knowledge there are only a few reports in the literature (30,32) with detailed analysis of transgene integration events. Going forward, TLA should be an excellent tool for this type of analysis.
In conclusion, we have demonstrated the power of TLA not only for precisely mapping the integration site of several Cre transgenes, but also for describing the nature of the structural changes that often accompany transgene insertions. Detailed knowledge of the transgene integration event not only allows for improved genotyping assays, it also helps inform the interpretation of phenotypes obtained when using mice, or any other model organism, carrying transgenic alleles. Finally, for crosses involving conditional alleles, knowing the exact location of the Cre transgene is a clear advantage and since the use of transgenic Cre/CreERT2 lines is widespread, our results and methods should serve as a useful reference for the mouse community.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online. | 2018-04-03T01:29:20.290Z | 2017-01-04T00:00:00.000 | {
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229253834 | pes2o/s2orc | v3-fos-license | Trabajo Decente? Decent Work from a Cuban Perspective
In this article, we critically examine Cuban social and labour policies as attempts of a practical application of Decent Work principles in a state-socialist context. We start with an examination of past and present models of socio-economic development and labour relations in Cuba. This is followed by an overview of the main Cuban policy principles related to Decent Work, which we contrast with some developments in Cuban labour realities. To contextualize our analysis, we emphasize the conceptualizations of work, production and property, as well as the institutional logics that underly the Cuban labour models and policies. Finally, two policy areas are highlighted by way of example: wages and gender equality.
Introduction
In the 1944 Declaration of Philadelphia, a document that is often mentioned as a predecessor of the concept of decent work, the International Labour Organization (ILO 1944: 1) states as one of its fundamental principles that "labour is not a commodity". However, following the official proclamation of the ILO's Decent Work Agenda (DWA) in 1999, this agenda has been criticized for its narrow conception of work by focusing on core labour standards and endorsing business self-regulation and supply-side economics (for an overview of such criticism, see Hauf 2015). Subsequently, the ILO committed to extending these standards and to moving towards a broader conception of work, intending to include, for instance, informal labour and care work. This broader definition, as it is put forward by the ILO today, places slightly more emphasis on a social dimension of work. Nonetheless, the DWA still seems to deviate from its original aim 1 Dr. Osnaide Izquierdo (osnaide@ffh.uh "to reduce the commodity character of labour relationships" (Standing 2008: 258), and to lack a critical stance on the ever-increasing emphasis on the commodification and marketization of labour in the 21 st century. Drawing on former critiques of the decent work concept in this regard (e.g., Standing 2008, Izquierdo 2017, we in this article argue that work should be considered first and foremost as a social category. As a consequence, we suggest that to analyse the decency of labour policies and realities, it is important to consider the relations of labour, production and property underlying such policies. In the following, we will do so with the example of the historical development and current situation of social and labour policies and actual working conditions in Cuba. Since the 1960s, the Cuban state has promoted a wide range of principles associated with the concept of decent work. However, the reality of past and especially of present Cuban working conditions only partly reflects and realizes these principles. The first section of this article introduces and discusses the -past and present -conceptual framework of the Cuban models of socio-economic development and labour relations. This is followed by an overarching analysis of Cuban labour and employment policies in the second section, focusing on these policies' key principles that are associated with the concept of decent work. We contextualise the analysis of the Cuban models and their principles by emphasizing underlying conceptions of work, relations of production and property, as well as the (in)congruency of labour policies with institutional foundations and logics. Finally, we focus in more detail on the examples of wage policy and gender equality measures as two areas in which political claims and the principles of decent work seem to be particularly at odds with everyday realities in Cuba.
In the conclusion, we briefly summarize our analysis and reflect on the importance of a critical stance when assessing labour policies and realities in the contemporary realm of work, also beyond the Cuban case. Finally, we consider some possible future developments and approaches for solutions that would enable more decent working conditions in Cuba.
The Cuban Models of Socio-Economic Development and Labour Relations
After the seizure of political power in 1959, the Cuban revolutionary government opted for a centralist formula based on the empowerment of the state as the leading institution of economic development and guarantor of justice and social welfare. This recourse to a centralist type of governance was justified by a development strategy oriented towards accelerated industrialization, agricultural diversification, internal income redistribution and the reorientation of international economic relations. A large and dominant state sector was created, which at that point became the main actor in the Cuban economy, covering nearly all of its branches. This process (in the following referred to as stateization process) guaranteed the state monopoly on resources and their distribution. The process was based on a structure of planning, administration and distribution of power that legitimized and facilitated centralized and hierarchical decision-making on these state resources, leaving very little space for market mechanisms.
Over time, the Cuban development model was gradually adapted to the model that had been institutionalized in other socialist countries. It was based on a property system in which social property mainly took the form of state ownership. The adherence to this model during the most critical moments of Cuba's history 3 has, to a certain extent, ensured the rights of the working class, but was, at times, also accompanied by high socio-economic costs. Admittedly, the model and its institutions have since then transformed, especially regarding the institutional changes starting in 2011. With the so-called update of the Cuban socio-economic model, a far-reaching (and still ongoing) reform process was started, which seems to mark a new trend for Cuban socio-economic development. The Cuban state, as the leading institution and guarantor of people's rights and social welfare, has unquestionably begun to share responsibilities in the areas of societal production, investments, employment, marketing and the provision of certain services to the population.
However, it would be simplistic to think of these changes as a linear transformation process away from state planning and towards the liberalization of market action. According to the reformers themselves, it is better described as a slow and calculated action which first and foremost seeks greater levels of economic efficiency. Simultaneously, in the long term, the reforms should rather consolidate the state's role as the central institution that determines the rules of insertion, negotiation, and empowerment of new economic actors in the Cuban economy. Nonetheless, if we consider recent changes and their effects on the system of social relations that sustains the productive and working spaces in Cuba, we can highlight two developments in particular: Firstly, the emergence and legitimization of new forms of capital-labour relations based on the transformation of the property system, and secondly, the prevalence of bureaucratic and centralized logics of labour relations. Together with the pre-existing, long-standing systemic crisis 4 of the state sector in the Cuban economy, these factors have drastically diminished the effectiveness of a system of labour institutions designed to ensure the rights of the working class. It is important to note that, although the update officially started in 2011, this reform process cannot be understood outside the context of the crises and readjustments faced by Cuban society since the late 1980s, which widely impacted the national socio-productive sphere.
With the transformations of the past three decades, the Cuban model of labour relations, 5 developed since 1959, has also been compromised. Indeed, there had never been a transition towards truly collective ownership in Cuba, with the empowerment of the labour collective as the real manager of production and property (and without the intervention of the state apparatus). Already in the early years of the Cuban Revolution, trade unions began to take on strong state-corporatist overtones, based on the socialist state enterprise and the bipartite relationship enterprise-workers, in which unions represent both the state and the working class. In this context, Cuban trade unions became the transmission belt of the Communist Party of Cuba, i.e., a channel for ideologies and labour practices with a scope of action predominantly limited to 3 Mainly related to the crisis experienced in the 1990s due to the fall of the socialist bloc. This resulted in an almost total loss of the Cuban international market and the correspondingly large impact on production capacities and thus on the supply of goods and services within the country. 4 This crisis is now not only expressed in terms of a tendency towards technological obsolescence and low productivity, but also in a productive cycle that does not allow the use of the national economy's real assets, such as a qualified labour force, the possibilities of productive chains, and the insertion in national and international markets. 5 Understood as the system of power relations established between capital and labour, and where the state plays a mediating role in the process of building rights within this relationship. As a model, it represents the configurations of the relations between the different actors and serves as an explanatory basis for the establishment of labour agreements.
processes of unionization and membership management, the socialization of workers and the promotion of socialist principles of labour. Moreover, the very term labour relations was basically erased from Cuban scientific, political and legal discourses. Hence, the Cuban model of labour relations evolved into a system that can be summarized, in terms of participation, as highly centralized by the state regarding both resources and strategic and operational decisions. As such, the model expresses the basic contradiction of the Cuban property system, in which property was never really socialized and the expropriation of the wealth generated by the workers, i.e., the exploitation of wage-labour, has been legitimized. Moreover, this contradiction is expressed in institutions that systematically curtail active labour participation, either through normative inefficiency (as the design of the model does not ensure participation), or because participation takes place through other channels (non-labour participation structures, e.g., on the community level or in mass organizations). The equalization of social property with state ownership resulted in vertical decision-making structures and a trade union unable to represent workers in collective negotiations. Ultimately, ignoring a continued exploitative situation based on salaried work -even if in conditions of (state-)socialist management -also meant ignoring the possibility of conflict among the principal actors of labour relations, which further limited the possibilities of union action.
This historically determined reality is compounded by the socio-economic transformations that have taken place in the country since 2011. As described above, socio-economic restructuring in Cuba is an ongoing process that, although the update officially began in 2011, has been developing since before 1990 through successive reforms that had already altered the country's productive sphere. However, these reforms have not been accompanied by a well-thought-out reformulation of labour relations. These have been strongly marked by a static trend in the trade union model developed by the Workers' Central Union of Cuba (CTC) 6 , which has kept its structural and functional premises unchanged. Indeed, it is true that the update is not the first substantial restructuring of the socio-economic dynamics that have characterized the Cuban development model. 7 But with the current reform process, the state has given impetus to important development processes of non-state forms of property and labour management. This has led to the proliferation of new sectors in the economy and, consequently, to the emergence of new actors in Cuban labour relations: so-called mixed companies (mainly joint enterprises with both national and foreign capital), non-agricultural cooperatives, as well as the actors of Cuba's relatively small but growing private sector. The latter includes self-employed people and owners of small businesses (known as the economic area of cuentapropismo), as well as (very few) companies with entirely foreign capital.
These emerging economic sectors and actors also present new challenges for Cuban trade union action, which, under the aegis of the CTC, has been characterized by a practice of state-6 The Central de Trabajadores de Cuba (CTC) is the country's sole representative of the branch unions in the Cuban economy. It represents the continuity of the national trade union practice since the 1930s, which is class-based and has been moving towards a more state-corporatist projection based on the particularities of the Cuban model. At present, 16 national unions are grouped within this trade union centre by branch of the economy. The CTC is represented at the highest levels of government, participating in the main spaces of government management in the country. corporatist trade unionism under the logic of state management of the economy. It is true that, in view of the reform process and the new socio-productive situation in Cuba, there are ongoing attempts to develop trade union policies that include these new actors in Cuban labour relations. However, these policies are characterized by mechanical logics that do not favour trade union participation in the newly created labour sectors because they do not recognize the particularities of their structure and functioning that are distinct from the state sector (for example property relations, the nature of incomes and salaries, or access to and the use of technology).
To summarize, since the 1990s, Cuban labour relations have been going through a process of structural dynamization towards a multi-stakeholder and multi-sectoral society. In a bestcase scenario, the emergence of new actors and new relational structures between capital and labour could contribute to the configuration of new strategies of union action within the Cuban socialist project and allow for the construction of novel spaces of confrontation. So far, however, it seems that these changes have only made the existing Cuban trade union model less effective. What is more, the socio-productive relations of non-state forms of labour and property management are characterized by a diversity and dynamism that have still not been covered by the regulatory apparatus. This also led to the precarisation of labour in different sectors of the Cuban economy -a precarisation that is reflected, for example, in a lack of labour rights in the socalled non-state sector, or in the decreased purchasing power of wages in a large part of the state sector, as described below.
Principles and Realities of Cuban Labour Policies
The stateization process of the Cuban economy, which began with the nationalization of the main means of Cuban economic production in the early 1960s, was accompanied by an important decapitalization of the country, including both financial and so-called human capital, as a large proportion of investors and skilled workers left the country during the first years of the Revolution. New social and labour policies were developed to give support and legitimacy to the then-proposed development model, to respond to national and international political and economic dynamics, and to allow for the control of national labour resources. To this day, these policies have taken into account almost all contemporary principles of the Decent Work Agenda. However, the effectiveness of said policies, which aim to ensure the (socialist) principles of (decent) work in Cuba, is largely impaired by the socio-economic developments of the country, as well as by its framework of labour relations.
The principle of full employment, in force until the early 2000s as one of the fundamental pillars of Cuban social and labour policy, is perhaps its most controversial one. It was based, firstly, on the strong emphasis on social development, and secondly on the high degree of state control over national resources (material, financial and human). In practice, the application of this principle translated into political strategies driven by the social deficit inherited from previous governments (low and/or unequal levels of education, health coverage, territorial development, etc.). These strategies eventually generated tensions between social and economic development priorities, resulting, for instance, in the goal to strike a balance between the objective of safeguarding employment and at the same time promoting innovation, efficiency and productivity. Together with the structural crisis that began in the late 1980s/early 1990s (and the transformations carried out to overcome this crisis), this finally led to the disappearance of full employment as a principle and practice of Cuban employment policy. 8 Cuban employment policy and strategy strongly emphasized educational development. Educational policies aimed to reconfigure the qualification structure of the national labour force so that it could cope, on the one hand, with the low availability of qualified labour on the national level in the first years of the revolution, and on the other hand, with the need to develop the most socio-economically disadvantaged territories. In practice, however, the structural tensions linked to full employment also affected educational strategies and developments, generating a high supply of qualified workers not linked to installed and/or available production capacities. Nonetheless, the emphasis on education allowed for the development of so-called human capital that represents a potential asset in the contemporary Cuban development model, as well as a comparative advantage regarding the country's integration into the international economy.
The freedom to choose a job and equal opportunities to access it without discrimination of any kind are two further principles 9 that appear controversial both in the policy design and in practice. Very closely linked with the principles of full employment and educational development, they cannot be understood outside of a system of labour insertion where the educational level represents the fundamental motor of social mobility, at least until the end of the 1980s. Thus, the freedom to choose and access employment without discrimination, in a context of almost absolute state control over labour resources, was mainly equated with the freedom to choose an education and a professional career -which, in turn, was limited by the capacity of the state to offer jobs. Finally, two other principles are worth mentioning here, namely the prohibition of child labour (with an official working age of 18, and special legislation for adolescents between the ages 15 and 18 who have completed their studies in professional or vocational education), as well as health and safety in the workplace. The latter is one of the least institutionalized principles within politics, which is mainly due to a lack of control and development mechanisms.
It must be stressed that the development of each of these principles is closely related to the system of labour relations that prevailed in the country until the mid-1990s at least. The state as guarantor of each of these relations 10 has played a constant and important role for the development, the achievements, but also the deficiencies of this system. Achievements that are sustained until today are linked, first and foremost, to access to employment (both in quantitative and qualitative dimensions), which is considered a citizen's inalienable right and which is based on inter-institutional relations that follow a logic of state assurance and planning. The deficiencies of the system had already been apparent in the 1970s/80s, when the statist ownership model was enforced, but they were undoubtedly entrenched during the structural crisis of the 1990s. Over time, the lack of a de facto appropriation and socialization of both the means of production 8 Although the official disappearance of this principle from Cuban employment policy is attributed to the year 2010 (see, for example, Echevarría, Rojas and Tejuca 2019: 151), the pursuit and realization of this principle was affected much earlier in practice. 9 The principle of equal pay for equal work is also closely linked to this. 10 From the point of view of generating the necessary productive capacities and labour resources, but, more importantly, also from the perspective of generating the institutional frameworks for the functioning (and control) of this system of relations. and of the wealth created by the workers contributed to the development and institutionalization of bureaucratic mediation, which ended up undermining the sustainability of employment policies and realities, as well as economic development in general.
In addition to such policy deficiencies, a set of problematic dynamics and processes linked to the realm of work has become apparent in the past decades. In some cases, these dynamics and processes resulted from the crisis of the 1990s and the loss of trade relations with the Socialist Bloc. In other cases, they were consequences of the very strategies designed to face this reality. One of the main problems resulting from the crisis was the decline of general employment levels by up to 15% -including a significant decrease in state employment due to the decapitalization of this sector (Izquierdo 2016). We may state in this regard that although the decline in employment was already announcing itself during the so-called Process of Rectification of Mistakes and Negative Trends 11 in the last five years of the 1980s, it was certainly aggravated by the crisis of the 1990s.
One of the main strategies to face the new realities after the end of the Soviet Union was manifested by what could be called a process of de-stateization, i.e., a gradual contraction and retreat of the state in the national economy. This process began in the 1980s with the opening up to self-employment, the creation of joint ventures and the transition of most state-owned farms to the cooperative sector. This process has been ongoing ever since and has certainly been intensified with the structural readjustments of the update of the socio-economic model during the past decade. The exit strategies of the state contributed to reversing some of the impacts of the crisis in the 1990s (some of which continued into the 21 st century) and have improved employment rates, especially in the newly created (or revived) sectors of the economy. At the same time, these strategies presented new challenges for an already unstable social and labour policy. One of the main challenges was the applicability of the above-described principles in a context where the state was losing its capability to ensure productive capacities and therefore opened the economy to new actors. With this process, there emerged social and labour dynamics that were alien to many of the logics underlying Cuban employment policy and its essential principles. In its old configuration, the state remained limited in its capacity to act and to generate new institutional dynamics and regulations, especially regarding informality and informal work. This has led to high levels of labour precarisation, particularly expressed in: high unemployment rates (especially regarding youth, female and urban unemployment); the worsening of the tensions between available production capacities and the qualification and geographic location of the workers; the deregulation of labour relations; a strengthening of the informal sector and an increase in informal economic practices in the 1990s.
Today, even if certain levels of economic revival and the maintenance of the fundamental principles of social and labour policy are seen as indisputable, the precarisation of work remains visible in different areas within the state sector. This is, on the one hand, closely linked to the inability of the jobs in this sector to ensure the reproduction of workers (as human beings) in areas that are not considered to be motors of the economy. On the other hand, it is also linked to the conditions and organization of work in this sector. Precarious working conditions are also becoming increasingly evident in non-state sectors but here, they are rather linked to the normative and organizational aspects of work. What appears to be fundamental here, is the fact that the state is losing control over the real applicability of employment policy by relinquishing part of its representative role within the capital-labour relationship to the non-state sectors of the economy.
In essence, Cuban social and labour policy continues to maintain its founding principles with greater or lesser levels of institutionalization. Unfortunately, the Current Labour Code, enacted in 2014 (ANPP 2014), has even weakened many of the above-discussed principles of Cuban employment policy and has not ensured sufficient progress in labour relations. Although the code gives recognition and legitimacy to the rights of workers and trade unions under the conditions of the above-outlined contradictions, it conceives of workers as subordinate subjects and conceals their practical possibilities of action and control -to which they are constitutionally entitled. The Code is not expected to be changed until at least three years from now (at the time of writing, i.e., spring 2020) and will thus in the meantime increase the contradictions and institutional gaps Cuban labour policy is facing today.
This being said, in the context of changing socio-economic conditions and the ongoing reforms, further principles and measures have been included in Cuban employment policy over the past years. Some examples are: an increasingly territorial vision of employment policy based on the development of territorial employment programs; the strengthening of the workforce for emerging economic sectors (tourism, biotechnology and pharmaceutical industry); business resizing and rationalization of excess personnel under conditions of salary assurance (according to the time of services provided); the development of training programs regarding the re-qualification of workers who have faced processes of staff rationalization and workers who are newly looking for jobs; the assurance of job placement for higher education graduates (and protection of those who graduate from technical-vocational education); the creation of special employment programs aimed at the most vulnerable groups of the employment population (such as disabled persons, single mothers and other population segments that require it); guaranteeing the retention of qualified workers in key sectors (from a social point of view), such as education and public health (particularly in terms of rehiring regarding the former and salary regarding the latter).
However, we need to stress again that the conditions for social dialogue between the fundamental actors of the Cuban model of labour relations continue to be problematic. In view of the diversification of these actors in contemporary Cuba, this appears to be the basis of the contradictions that sustain the ineffectiveness of this social and labour policy in terms of the fight against the precarisation of labour. To further illustrate these contradictions, we will, in the following, discuss the examples of wage policies and gender equality measures -two areas in which the political claims and principles of decent work seem to be particularly at odds with Cuban everyday realities -in more detail.
Wage policy
In accordance with the second pillar of the Decent Work Agenda 12 , the principles promoted by Cuban wage policies since the early years of the revolution have formally aimed to meet decent wage criteria -within the particular framework of a national economy based mainly on state property, as explained in the previous sections. However, the reality of Cuban wages is far more complicated, especially in contemporary Cuba.
The salary system of the Cuban state sector -which, according to official statistics (ONEI 2019), still employs around two-thirds of the Cuban labour force -is based on standardized and centrally fixed wages. Historically, these can be traced back to the introduction of unified wage scales in the early 1960s. These scales, as well as the corresponding policies, have been slightly changed and adapted in the past decades. However, the system's basic principles have remained steady over time: The core of the wage system is formed by a minimum wage, a unified wage scale at the national level and two basic forms of payment: payment according to time and payment according to performance (Galtés 2017: 67). Although the attempts towards complete wage equalization and total demonetization from the late 1960s (e.g. Hernandez/Mesa-Lago 1971) were abandoned in the 1970s 13 , Cuban state wages are until today considered to be relatively low in international comparisons and to present little differentiation between different categories of occupation. However, it is often stressed that these features of Cuba's salary system have to be considered against the backdrop of the country's social policy tradition, that is, free and universal education and social protection services, subsidies (e.g., for electricity, communication and culture), and the distribution of food and basic consumer goods (e.g. Dominguez 2017).
As described previously, socio-economic developments in the past decades have presented new challenges for Cuban institutions and policies, especially in terms of social protection and decent wages. While prices increased dramatically during the crisis of the 1990s, real wages are estimated to have fallen between 70 and 90% (Mesa-Lago/Pérez-López 2014: 14; Yaffe 2012: 37). As a result, labour and income in economic spheres outside of the state sector (i.e., the private sector, mixed enterprises and the cooperative sector in the formal economy, but also informal work, underground and illegal activities) became increasingly important. The introduction of a dual currency system 14 and the boosting of the tourism industry to combat the effects of the crisis contributed to the socio-economic division in these different sectors and spheres.
In 2010, in view of the impending update of the economy, the Cuban government had announced the dismissal of a large portion of state sector employees. Although these layoffs 12 Which specifies that measures of social and labour protection should include "[p]olicies in regard to wages and earnings […] designed to ensure a just share of the fruits of progress to all and a minimum living wage to all employed and in need of such protection" (ILO 2008: vii). 13 Notably with the adoption of the System of Management and Planning of the Economy (SDPE), which was based on the Soviet model. 14 Based on the National Cuban Peso (CUP) and the US-Dollar; in 2004, the latter was replaced by a distinct convertible currency, the Convertible Peso (CUC).
eventually occurred more slowly and on a smaller scale than originally planned 15 , it is estimated that throughout the past decade, more than half a million workers were dismissed from the state sector (e.g. Bye 2020: 33). Those who have continued to be state-employed often rely simultaneously on jobs and incomes outside of the state sector (see, e.g., Echevarría /Tejuca 2017;Galtés 2015). This is what Feinberg (2016) labels the GESPI category: government employees with a significant private income. He makes a conservative estimate of this category at 10 to 20% of the public workforce (Feinberg 2016: 111). Certainly, few workers could live on their state wages without relying on other sources of income -whereby various forms of material and immaterial income supplements come into play (Ritter 2006). It is worth noting in this context that for many Cuban households, remittances have become an equally or more important source of income than work-related incomes, which contributes to poverty reduction, but also widens inequalities within society (Barberia 2017). 16 Regarding the overall situation of social protection, health care and education, it is true that the Cuban state has, until today, maintained universal access to basic goods and social services, but their availability and quality has declined (Dominguez 2017).
In the context of little available statistical data, the dual currency system, and the close entanglement of wages, social services, subsidies and food rationing, it is difficult to assess the purchasing power of state salaries. Nonetheless, until today, most Cuban incomes, and especially wages in the Cuban state sector, are considered to be below the subsistence minimum. In the past years, this has been repeatedly acknowledged even by Cuban political leaders, who, for instance, stated that "it is true that the salary does not meet all the needs of the worker and his family" (Castro 2014), and that salaries are "insufficient" to cover the necessities of the workers (e.g. EFE 2019). In the past decade, there have been wage increases in both the state enterprise system and in the public sector, and compensation schemes were further diversified (emphasizing, for example, performance-related pay). However, although nominal average salaries have grown in the past decade, this has hardly been the case for real wages, which are considered to "not cover the cost of Cuban daily life" (Echevarría/Tejuca 2017: 156). According to Togores/García (2003: 4), until the 1980s, wages in the state sector accounted on average for about three-quarters of household income, while by the 1990s, they represented only slightly more than half of it. Vidal (2016: 154) states that the purchasing power of the real average state salary in 2013 represented 25% of the 1989 salary purchasing power. In 2016, according to "personal and extra-official information provided by then Minister of Economy Murillo […] a family of four with two breadwinners would cover only 25% of the family's basic needs through their 15 In the course of the year 2010, different government officials provided varying information as to the planned number of layoffs, which ranged from 1,3 to 1,8 million employees (Bye 2020: 32). In accordance with the objectives of the reform process, the workers that were actually laid off from the state sector are supposed to be absorbed by the growing mixed, cooperative and private non-state sectors. Thus, the dismissals went hand in hand with, for example, the extension of licenses for cuentapropistas (self-employed people and owners of small businesses). For those workers who did not immediately find a new occupation, compensatory measures were provided in the form of a partial and temporary salary replacement and support through the social welfare system (Mesa-Lago/Pérez-López 2014: 124). 16 The social structure resulting from this income situation is commonly interpreted as an inverse pyramid (e.g. Galtés 2017; Luis 2017). In a nutshell, the term is used to describe disconnections (or, in normative terms, misconnections) between the three parameters qualification, work and income. The inverse pyramid is in turn seen as a cause for low productivity, working motivation and a brain drain within the Cuban economy.
average incomes" (Bye 2020: 86). 17 Anaya/Garcia (2018) estimate basic expenses for Cuban urban families of three, accounting for subsidies and food rationing. The authors conclude that in 2016, an equivalent of two to three average wages in the state sector -or nine to ten minimum wages 18 -was needed to cover the estimated amount of such basic expenses. This does not take into account additional expenditures, which are part of everyday Cuban life (household equipment, repair works, internet, etc.). Wage policy (together with the abolition of the dual currency system) has been highlighted by Cuban political leaders as one of the priorities of the current reform process. The guidelines of the Update, the Lineamientos de la Política Económica y Social, tackle this only in a very general manner, stating the aim to restore the role of work and the income obtained from it as the main way to generate quality products and services and increase production and productivity, and to achieve the satisfaction of the fundamental needs of workers and their families. (PCC 2017: 28) In the last few years, different measures and reforms have been introduced to combat some of the above-described issues related to wages and incomes in contemporary Cuba. They mostly concern regulations about employees in the state sector, such as recent increases in minimum salary (from 225 to 400 Pesos per month, the latter being equivalent to about 16 US dollars), or the wage increases for workers in the public sector in 2019. Basic operating modes of the payment and material stimulus systems within the Galtés (2017: 99) counts among the advantages of these reforms that these eliminate administrative restrictions on wages and allow for simultaneous payment systems (piece-rate and performance-based), as well as for a certain degree of autonomy for state enterprises regarding the distribution of wages. At the same time the author criticizes, among other things, that the resolutions generalize salary penalties and that the distribution of wages among the workers is deregulated and left to the managers of the firms. She also criticizes that the formal requirement of the participation of workers and unions in the annual approval of payment systems is not granted in reality.
To better understand these remarks, it is important to note that the wages of many employees in state enterprises are divided into a basic wage and a so-called stimulus (estimulación), depending on the achievement of (mainly centrally) planned and defined indicators. The fluctuations in the stimulus (which may change monthly, in a range between zero to three times the basic wage of a worker) represent a major uncertainty for workers. Díaz/Echevarría (2017: 234-235) stress that sanctions, in the case of non-achievement of the indicators (which are assessed at the company level and not individually), collide with one of the basic principles of the conception of salary payments. 19 This principle notably conceives of salaries as the expression of the quantity, the quality of, and the time dedicated to work. Referring to Resolution No 138/2017 of the MFP, these authors also criticize that the income of the workers is being sanctioned in a twofold manner (via salaries and via stimuli) in the case of non-achievement of the indicators. They further note that workers do not actively participate in the determination and approval of these indicators and critically highlight that compliance with the indicators does not always lie directly in the hands of the firms, and even less so in the workers' hands.
The previous reflections on the question of decent wages in contemporary Cuba only tackle some main issues relating to wages in the state sector. Although these are closely linked to wage and income-related issues in other sectors, and although some steps have been taken in recent years to regulate wages in the non-state sectors of the economy, many observers criticize that there are still major gaps in this area. As Galtés (2017:65-67) notes, the current labour code and corresponding legislation and resolutions provide, to some extent, norms for wage guarantees and social protection of workers in non-state areas. Nevertheless, she also stresses that the setting of salary levels in these areas does not follow the same rules as the state sector and is perceived to be much more flexible and less regulated, eventually resulting in a widening of the gap between state salaries and incomes from other sectors. This equally applies to social protection issues. Yet, it is difficult to assess the question of decent wages in some parts of the non-state sector, especially due to a lack of data. We, therefore, confine ourselves here to highlighting the gaps between the state-and non-state sectors, as well as the important loopholes in the promotion of decent wages in the informal economy.
Nonetheless, we hopefully managed to illustrate some of the contradictions of the Cuban model outlined in the previous sections. Indeed, fair wages and income from work, as well as legislation on minimum wages (as stipulated by the ILO's criteria to realize decent wage conditions) have been promoted in Cuba since the beginning of the Cuban revolution and are emphasized in the political discourse as part of the current reform process. However, as wage policy -like other areas of employment policy -faces the contradictions inherent in the conception of the Cuban production and property systems, it presents important deficiencies and does not guarantee a real appropriation of the wealth generated by the workers. Before moving on to a general conclusion on this matter, we will shortly address the topic of gender equality in the context of this contradiction.
Gender equality measures
In order to apply a "gender equality lens" (ILO 2008: 1) and to assess womens' work opportunities and working conditions in Cuban labour policies and realities, we will, in the following, first examine issues of decency (or indecency) of female work in a more narrow sense of core labour standards in the so-called productive sphere. Second, we will refer to a broader conception of the notion of decent work and take a short look at unpaid labour in the so-called reproductive sphere.
Gender equality is said to be a major objective of Cuban politics since the early years of the revolution in the 1960s. It is anchored in the Cuban Constitution (Constitución de la República de Cuba 2019), as well as in labour, social and family legislation and policies. According to the OECD's Social Institutions and Gender Index (OECD 2014a), which addresses de jure and de facto situations of discriminatory social institutions, Cuba is among the countries with very low levels of gender discrimination in social institutions. However, if we look at female labour force participation in the realm of paid work, official numbers on the employment gender gap in Cuba are very similar to the global gender gap, 20 with 49.5% of Cuban women employed, compared to 76.9% of Cuban men (ONEI 2019). 21 In the Cuban state sector, women are still relatively well represented (1.404 million women of the total 3.067 million labour force in 2018; see ONEI 2019). The proportion of highly qualified women in different areas and professions of the state sector is described as relatively high, and their representation in political and other important decision-making functions is often emphasized. 22 Nonetheless, Cuba is not completely exempt from the phenomenon of gendered occupational segregation, as patterns of educational choices seem to suggest: to date, Cuban women are over-represented in so-called social courses of study and less oriented towards technical and agricultural careers (Echevarría et al. 2019).
As described above, the current developments in Cuba come along with new tensions and problems regarding occupational security, wages, and social protection -and women are particularly affected by these developments. Taking the example of the layoffs in the state sector in the past decade, Lara Junco (2015) notes that between 2010 and 2013, the Cuban state dismissed almost 62,000 women, compared to about 4,000 men. Echevarría/Tejuca (2017: 155) mention a slightly lower number of female workers dismissed from the state sector between 2010 and 2014, namely 51,000. As recent numbers of women's share in the non-state sector show (254,700 women, compared to 1.416 million men in 2018; ONEI 2019), the female labour force coming from the state sector has been absorbed less by the non-state sector than the male labour force.
Nonetheless, especially in the often-discussed area of cuentapropismo (self-employed people and owners of small businesses), women's labour participation has been increasing (from 152,000 in 2015to 197,200 in 2018ONEI 2019). Assessing decent working conditions in this area of the Cuban economy seems particularly difficult, since it is possible to conceive of cuentapropismo as both a potential for the Cuban economy and, due to a persisting lack of regulation, a threat to basic labour standards and social protection of workers. The return of paid domestic work in Cuba (e.g. Romero 2016; 2019) may serve as an illustration for this, especially with regard to gender-related issues. Historically, after the takeover of the revolutionary government in the 1960s, paid domestic work was frowned upon and almost completely abolished, due to its entanglement with class, gender and racial discrimination. With the rise of cuentapropismo in Cuba at the beginning of the 21st century, there seems to be a resurgence of paid domestic work. As is often the case for activities in the area of self-employment and small businesses in Cuba, the sociodemographic composition of domestic workers is quite diverse and also includes highly qualified women, which is generally perceived as a major threat in terms of brain drain and occupational downgrading. Admittedly, seen from a different angle, many of today's domestic workers find themselves in a relatively good economic situation and enjoy a certain 20 The current global labour force participation is estimated to be about 49% for women and 75% for men (ILO 2018a). 21 Referring to a working age population of women between 17 and 59 years, and men between 17 and 64 years. 22 Based on national statistics (ONEI 2019; FMC 2016), Echevarría, Rojas and Tejuca (2019) highlight, for example, that more than 70% of federal prosecutors, presidents of provincial courts and professional judges are female; and that 53,3% of jobs in the areas of science, innovation and technology are occupied by women, as well as 47,2% of higher management positions. According to UN Women (2019), in 2019 the proportion of women in Cuban parliament was 53,2% -the second highest worldwide. degree of autonomy, which they might not have exercising their original profession, at least not in Cuba's current labour context. Yet, generally speaking, one of the features of cuentapropismo seems to be the reinforcement of a division into so-called typically female and typically male working activities, which often involve particularly vulnerable working conditions for occupations labelled as typically female (Echevarría et al. 2019). In addition, to date there are more women who do contract work than there are female entrepreneurs -a major explanation for this is women's limited access to seed capital, compared to men's resources of getting such capital (Díaz/Echevarría 2016). As mentioned before, regulation in the area of cuentapropismo remains at a relatively low level, while the precariousness of many activities in this area remains relatively high -with particular deficiencies regarding women's labour, such as lacking rights to paid rest or unpaid leave due to family problems; or even the denial of the right to return to work after maternity leave (Echevarría et al. 2019).
The Cuban legal framework indeed provides basic institutions that should protect working women and ensure gender equality, such as the maternity -and paternity -leaves that should be guaranteed after the birth of a child. By law (Consejo del Estado 2017), working mothers have the right to a pre-natal leave of six, and a post-natal leave of twelve weeks (with a full wage substitution, calculated from the average weekly income from wages received in the twelve months preceding the maternity leave). After that, until the child reaches the first year of life, mothers can either choose to return to work or to care for the child with a wage substitution of 60% and a job guarantee for the child's second year of life. This leave for early childcare can also be taken by the father. In reality, however, the implementation and realization of these institutions and protection measures must be regarded under the circumstances already discussed above: the regulations apply mainly to the state sector, in which wages are relatively low and are considered to be insufficient to cover costs of living. In other sectors, the regulation and implementation of these institutions seldom exist. It must also be noted in this context that despite the long tradition of promoting gender equality and women's empowerment in Cuba, machismo remains persistent in Cuban daily life. 23 The number of Cuban fathers who actually take leave for childcare is said to be quite low; some Cuban families might not even know about this possibility. 24 In Cuba (as in most parts of the world), women continue to perform a majority of the work in the so-called reproductive sphere. According to the OECD (2014b), Cuban women devote about three times as much time to unpaid care work as men. 25 Indeed, in international 23 For a recent inquiry on machismo (understood as a specific form of patriarchy, mainly in the Latin American and Caribbean context) in contemporary Cuba see, e.g., Fox & Zagumny (2017). The authors state that traditional Cuban gender roles, expectations and responsibilities continue to manifest in Cuban society, particularly in every-day social interactions and child education. 24 Unfortunately, we could not find much precise information on this matter. According to UNICEF ( comparisons, Cuba is considered to have high enrolment rates in early childhood care and education and is among the countries that have relatively low care dependency ratios (ILO 2018b), which may be interpreted as a certain easing of women's workload. However, Cuban women's double burden of paid and unpaid work also needs to be seen in a context of recurrent economic crises, environmental shocks and scarcity, in which family and domestic chores are highly labour-intensive. To give just one example of research on this matter, Davidson and Krull (2011) have observed a reinforcement of the gendered division of reproductive labour (particularly regarding chores related to food acquisition) in post-Soviet Cuba. Interestingly, the authors note that this gendered division also appears to be reflected in a reinforced and shared discourse on the supposed expertise of women in domestic work. This, in turn, seems to be backed up by the changing policy framework of the past decade, which represents new challenges regarding questions of -paid and unpaid -decent work for women, since the current reform process also seeks to reduce state subsidies and welfare services. It is worth noting that this contrasts sharply with the projected ageing of the population (comparable with demographic developments in so-called first-world countries), which raises the question of who will care for these elderly. 26 Echevarría et al. (2019) note that these demographic challenges have led to recent policies aiming to raise birth rates and reinforce women's social function in the reproductive sphere. To end this chapter with the words of these authors, in Cuba paid work is still conceived of as the only work that is valued […]. This conception explains the scarcity of measures aimed at promoting more equitable gender relations, in which the reproductive space is promoted as an important space for the development of a country. (Echevarría et al. 2019: 153)
Conclusion
The previous examination of labour policies and realities in Cuba has shown that the promotion of principles associated with the Decent Work Agenda is a necessary, but not sufficient prerequisite to realize conditions of decent work. In the analysis, our goal was to highlight the relations of labour, production and property in the Cuban development model and the resulting (im-)possibilities for workers to appropriate their work and the wealth it generates. Furthermore, we addressed some of the institutional logics and problems these relations are based on; as well as the general conception of work that underlies Cuban labour institutions and policies.
As we have attempted to show, Cuban social and labour policies are, until today, based on a socio-economic development model in which social property takes the form of state property. Historically, this model has generated a set of principles, relationships and social and labour policies that seek to propose an equitable appropriation of work (and the wealth it creates) by the workers. However, the current reality of Cuban labour relations and working conditions hardly conforms to this claim. The transformation processes related to the crisis of the 1990s (and its long-standing repercussions and follow-up crises), as well as the strategies designed to face this crisis have led to important institutional loopholes that eventually complicated the system of Cuban labour relations and contributed to the precarisation of working conditions (including the remuneration of work) in both the state and the non-state sector. In other words, the socio-economic developments of the past decades and the reforms designed to face them, especially the so-called update of the Cuban socio-economic model, have, to some extent, altered Cuban relations of production and property. The state has gradually contracted and retreated as an economic actor in areas where new actors and economic sectors have gained importance.
However, the existence and legitimization of other forms of property than state property and the emergence of new actors in Cuban labour relations have not been covered by sufficient changes in institutions and policies. Conditions for social dialogue between the fundamental actors of Cuban labour relations continue to be problematic, which, in our opinion, represents both a major source of contradictions and an obstacle in the fight against the precarisation of work. Particularly the lacking possibilities for workers' participation in execution and decisionmaking processes appear to be an important obstacle for the maintenance or creation of decent working conditions. This is also reflected in contemporary discussions on Cuban wages, as discussed in this article's section on Cuban wage policy. Moreover, the strong ideational emphasis of the Cuban development model -and its update -on economic development seems to nourish a general conception of work that understands it primarily in economic or commercial rather than in social terms. Particularly the example of women's working conditions and unpaid household and care work seems to substantiate this assumption.
In this respect, Cuba hardly differs from tendencies in other countries around the globe. It appears that the criticism pointing out the ILO's lacking critical stance on the commercialization of labour discussed in the introduction of this article is equally relevant for the Cuban context. Therefore, we would like to emphasize, once again, the importance of such a critical stance when assessing labour policies and realities (in terms of their potential to create and maintain decent working conditions) in any context, also beyond the Cuban case. In our analysis, we stressed the elements of labour and property relations, wealth appropriation, institutions and their (historically formed) logics, as well as ideational and conceptual foundations -such as the conception of work underlying development models and policies. We suggest that these elements can be used as tools for a critical analysis of social and labour policies and can thus be an important complement to other evaluation tools of the Decent Work Agenda. 27 To conclude, we briefly reflect on some general approaches that could provide a sound basis for decent working conditions in Cuba and that would respond to the challenges discussed in the previous chapters. In our opinion, there is a need for a new regulatory framework that better responds to and involves all actors of the socio-economic system. Mechanisms of economic and political management and action should be adjusted to the new reality and culture of work in contemporary Cuba, which would also require a readjustment of labour organization and execution based on decades of thinking and working within centralist budgets. Further, a revolutionary transformation of the prevailing model of labour relations is necessary, including the generation of new forms of enterprise and labour organization in the state sector that allow for a more participatory model of labour relations. This would require the adoption of a company law that recognizes the decisive authority of workers in the state sector. In the non-state sector, the problematic existence of private economic actors that distance themselves more and more from the (generic and almost euphemistic) category of self-employed workers should be addressed. Workers in the area of cuentapropismo should be made visible as legal entities and as social subjects with very particular socio-productive logics. This would also contribute to the generation of a new model of labour representation and syndicalism that surpasses the existing model of labour relations and that can represent the multiplicity of interests emerging in the consolidation process of a mixed economy. | 2020-12-17T04:11:49.248Z | 2020-12-15T00:00:00.000 | {
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239615377 | pes2o/s2orc | v3-fos-license | Grape pomace silage on growth performance , carcass , and meat quality attributes of
Grape pomace is a by-product that can be ensiled and added to animal feed for a sustainable animal production. This study evaluated the effects of grape pomace silage (GPS) on the intake, performance, and carcass and meat quality of feedlot lambs. Twenty-four male lambs (21.5 ± 3.0 kg initial body weight) were distributed into pairs to four diets levels 0, 10, 20, and 30 % of GPS. The addition of grape pomace silage influenced only ether extract (EE) intake linearly without hindering consumption. The diet did not affect performance and meat quality attributes and carcass parameters, with average daily gain (ADG) of 0.235 kg d–1, feed conversion 4.299, carcass conformation 2.7, compactness index 0.25 kg cm–1, fat thickness 1.51 mm, loin eye area 13.9 cm2, pH 5.79, natural matter moisture 74.05 g 100 g–1, and crude protein (CP) 19.94 g per 100 g–1 of dry matter (DM). Grape pomace as could be used as silage in lamb diets with up to 30 % GPS, as the chemical composition of this by-product and the results indicate that GPS did not compromise performance, carcass traits, and meat quality.
Introduction
Lamb consumption is rising and consumers are more demanding for meat quality standards, which requires production systems to evaluate sheep carcass traits quantitatively and qualitatively (Petrescu et al., 2020). In addition, systems need to be sustainable and adopt actions to preserve environment integrity.
Diets commonly used for ruminants are energy grains that are costly to produce and subject to frequent price fluctuations, besides competing with feeds used in other animal species. Recent studies have suggested partial replacement of grains by agricultural byproducts (Almeida et al., 2019;Mazza et al., 2020;Evan et al., 2020).
The processing of agricultural products generates annually thousands of tons of organic by-products that are rich in seeds, husk, pomace, and pulp with excellent nutrient composition and bioactive compounds that could be used as energy sources. Recently, agro-industrial byproducts have been used in the production of biofuels, antibiotics, biofertilizers, chemicals, enzymes, vitamins, antioxidants, and animal feed (Eleonora et al., 2014;Kodagoda and Marapana, 2017).
Grape pomace is a by-product of the wine industry. It is a low-cost ingredient with good nutritional quality and chemical composition that could be used in animal feed. In addition, grape pomace can be stored as silage (Massaro Junior et al., 2020). The use of grape pomace in different in ruminant diets showed considerable effects on animal growth performance, as well as on carcass and meat quality traits (Gómez-Cortés et al., 2018;Kafantaris et al., 2018;Chikwanha et al., 2019).
Studies have reported effects of grape pomace on animal performance; nevertheless, there is no information on the effects of adding up to 30 % of grape pomace cv. Isabel (Vitis labrusca L.) as silage and limitations of the levels added to lamb diets and the effects of adding GPS on animal performance need investigations. This study investigated the addition of 0, 10, 20, and 30 % of grape pomace silage to the diet of feedlot lambs and its effects on nutrient intake, performance, carcass, and meat quality.
Experiment and animal handling
The study was carried out in the municipality of Londrina, Panará State, Brazil (23°20'23" S, 51°12'32" W, altitude 580 m). The Ethics Committee on Animal Experiments approved the study under the identification number (Protocol n° 78/10). The experiment comprised 24 male lambs of mixed breed with approximately four months of age with an initial average live weight of 21.5 ± 3.0 kg. The animals were wormed and identified before the study. The experimental design was completely randomized with four treatments and three replications, and the pen was a repetition. The lambs were distributed in pairs and housed in pens provided with troughs and drinkers for 21 days for adaptation to the diets, to the experiment, and to the facilities, followed by a 35-day trial period. The animals were slaughtered with an average live weight of 33.81 ± 2.85 kg and average carcass weight of 15 kg, according to consumer acceptance. Grape pomace silage in diets of feedlot lambs Sci. Agric. v.79, n.5, e20200343, 2022 The animal were weighed at the beginning and end of the adaptation period and every 15 days during the experimental period. Prior to slaughter, the animals were weighed after 16 h of fasting to calculate final weight, average daily weight gain (ADG), and feed conversion. ADG was obtained by the ratio between weight gain and number of feedlot days, while feed conversion was obtained by the ratio between total feed intake and weight gain during the feedlot period.
The ration was provided in two meals with 50 % each of diet total at 7h30 a.m. and 4h30 p.m. and adjusted to provide 15 % of dry matter (DM) at ad libitum. DM and nutrient intake were calculated by subtracting from feed amount provided.
Experimental diets
Grape pomace was obtained from the juice industry in Paraná State, Brazil, shortly after processing. The byproduct evaluated came from grapes cv. Isabel (Vitis labrusca L.) and was largely composed of seeds (610 g kg -1 DM), peels, and pulp residues (390 g kg -1 DM). At the time of collection, the by-product was 11 % DM and was dehydrated outdoors and turned over three times a day, until reaching approximately 30 % DM. The by-product was then treated with 5 g kg -1 natural matter (NM) of urea and ensiled in 100-and 200-liter plastic drums stored for seven months until the beginning of the experiment. The sorghum used for silage production was cultivated under a no-tillage system at the university farm and the sorghum was stored in a bunker silo after cutting.
The ration samples were provided and 20 % of the leftovers were collected weekly directly from the trough. We analyzedthe contents of dry matter (DM), organic matter (OM), crude protein (CP), and ether extract (EE), according to the methodologies described in AOAC (1990). The neutral detergent insoluble fiber (NDF) was analyzed according to Van Soest (1963) (Table 1). Total carbohydrates (TCHO) and nonfibrous carbohydrates (NFC) were calculated according to equations proposed by Sniffen et al. (1992).
Evaluation of carcass traits
The animals were weighed and then subjected to 16 h of solid fasting and then weighed again to obtain body weight at slaughter (BWS). During this period, the animals were transported to the slaughterhouse under municipal inspection at 7h00 a.m. with a specific collective stall truck, equipped with a railing floor and a bed of rice straw, following the transportation rules proposed by the Ministry of Agriculture, Livestock and Supply (MAPA, 2017). The animals were slaughtered with stunning by the percussive method using a pneumatic pistol, followed by bleeding, skinning, and evisceration, according to the rules established in the Brazilian Regulation for Industrial and Sanitary Inspection of Products of Animal Origin (MAPA, 2017).
The carcasses were identified, weighed to determine the hot carcass weight (HCW), and cooled in cold rooms at 2 °C. Then, the carcasses were weighed and after 24 h of cooling at 2 °C, the cold carcass weight (CCW) was obtained. Hot (HC) and cold (CC) carcass yields were calculated by the percentages of hot and cold carcass weights in relation to the final weight (FW) and cooling weight loss (CWL) by the difference between the two carcass weights according to Osório and Osório (2005).
The gastrointestinal tract was collected, weighed full and empty, and the difference was used to obtain the weight of the gastrointestinal content used to determine the empty live weight (ELW) and true yield (TY), where: ELW = WG -weight of gastrointestinal content and TY = ((HCW/ELW) × 100). The carcass compactness index (CCI) was calculated according to Cezar and Souza (2007), where: CCI (kg cm -1 ) = CCW/ cold carcass internal length. After cooling, conformation (values from 1concave to 6 -convex), finishing (values from 1 -cover fat absent to 5 -abundant cover fat) and cover fat were evaluated using photographic standards (Cañeque and Sañudo, 2001). We also measured carcass length and chest depth, length, perimeter and leg and arm depths (Osório and Osório, 2005).
The left half carcasses were sectioned at the 12 th rib height to assess the loin eye area (LEA), fat thickness, depth and width of the longissimus thoracis et lumborum (LTL) muscle according to Cezar and Souza (2007). The marbling rate was subjectively evaluated using photographic standards from the American Meat Science Association -AMSA (2001), with grades ranging from 1 to 10 (1 = marbling traces and 10 = abundant marbling).
The spine was boned to obtain the LTL muscle. The muscle was divided into portions: three portions for shear force (3-cm thickness for each), one portion for color measurements, the pH, marbling and drip water loss (2-cm thickness), one portion for proximate analysis, (2-cm thick) and a portion for lipid oxidation index (2-cm thickness).
The shear force samples were obtained from two to three separate portions of the LTL muscle of each animal, depending on the piece size, according to AOAC (1990). Ten samples of each portion were taken and cooked to an internal temperature of 71 °C and seven subsamples measuring approximately 1.25 cm thick and 2.5 cm long were obtained from these samples using a cylindrical steel sampler (Whipple et al., 1990).
The shear force was measured in the seven subsamples using the 3-mm blade shear probe attached to the Brookfield® CT3 Texture Analyzer texturometer. A trained observer evaluated color of L * (brightness), a * (red-green component) and b * (yellow-blue component) components as described by Miltenburg et al. (1992). The colorimeter was set to the L*, a*, b* system and illuminant D65, observer angle of 2°, aperture size of 5.0 mm, and a closed cone. These values were used to calculate the hue angle (h*) by the equation h * = tan-1 (b*/a*) and the saturation index, or chroma (c*), from the equation c* = (2a* + 2b*) 0.5 following the methodology of MacDougall and Taylor (2007).
Drip water loss was evaluated according to the technique described by Boccard (1981) by cooling at 4 °C for 48 h. The pH was checked inside the cold chamber of the refrigerator at a temperature of 2 °C using a portable potentiometer with a Testo® 205 insertion electrode equipped with a temperature sensor, which was previously calibrated. The left palette was frozen and subsequently dissected to obtain the bone, muscle, and fat proportion, according to the methodology of Fisher and Boer (1994).
For the lipid oxidation index, the 2-thiobarbituric acid test (TBARS) was performed, weighing 5 g of homogenized sample and adding 25 mL of 7.5 % tetramethoxypropane. Subsequently, homogenization was performed for 1 min with corning tube filtration. We added 4 mL of the filtrate, 1 mL of trichloroacetic acid, and 5 mL of thiobarbituric acid to the test tube. The tubes were placed in a boiling water bath for 45 min. After cooling, a spectrophotometer reading at 538 nm was performed followed by a standard curve, according to the method described by Cherian et al. (2002).
Statistical analysis
The data were submitted to the Shapiro-Wilk and Bartlett tests to verify the assumptions of normality and homogeneity of variance, respectively. After meeting these assumptions, the data were submitted to the analysis by the mixed procedure (PROC MIXED) including the treatment as a fixed effect and the stall as a random effect. Significance was determined at the level of 0.05 according to the Tukey test of comparison of multiple means by the SAS program (v. 9.2; SAS Institute Inc., Cary, NC), following the model: where: Y ij = Variable response related to treatment i in pen j; µ = Overall average; α i = Effect of treatment i; β ij = Effect of treatment i in pen j; ε ij = Random error associated with each Y ij observation.
All data on nutrient intake and performance were analyzed by the Regression procedure (PROC REG). When applicable, the regression models were chosen by their degree of adjustment (R 2 ).
Nutrient intake and performance
GPS in the diet increased only EE daily consumption linearly (p < 0.05) ( Table 2). ADG and FC were not influenced (p > 0.05) by GPS in the diet (Table 3), with an average value ADG of 235.2 ± 13.07 g d -1 kg and FC of 4.299. The inclusion of 10 % GPS increased the final body weight lambs by 3.6 % than the control group. Lambs fed with 20 % of GPS obtained 9.4 % more ADG than the control group and the best FC among all treatments; however, difference in performance was not significant.
Carcass traits and meat quality
GPS did not influence (p > 0.05) the carcass traits of lambs (Table 4) nor carcass biometric measurements and bone, muscle, fat yield in the palette, as well as the index carcass compactness (Table 5). Grape pomace silage in diets of feedlot lambs Sci. Agric. v.79, n.5, e20200343, 2022 Muscle LTL and meat quality did not vary (p > 0.05) due to GPS in the diets (Table 6). Mean values of 1.51 ± 43.93 mm of fat thickness were observed. The compactness index of 0.25 kg cm -1 indicated good muscle proportion and an average marbling score of 1.64. The loin eye area was 13.9 cm 2 , the meat pH was 5.79, and the shear force obtained was 33 N. GPS had no influence (p > 0.05) on the centesimal composition of the LTL muscle (Table 7).
Nutrient intake and performance
The GPS levels increased; therefore, EE intake rose linearly (Table 2), which may be related to the diet chemical composition that increased EE in g kg -1 DM, as GPS alone had a higher EE content than the other ingredients (Table 1). Chikwanha et al. (2018) also observed a positive linear relationship between EE intake and inclusion level of GPS to the diets. The increased EE content in the GPS diet is due to the higher density of seeds (610 g kg -1 DM), which have a higher oil concentration (Shinagawa et al., 2015) than the peel and pulp (390 g kg -1 DM). In our work, the quality of GPS cv. Isabel (Vitis labrusca L.) showed higher EE content, due to seed density (Massaro Junior et al., 2020). Guerra-Rivaz et al. (2017) also found that the proportion of seeds in grape pomace ranged from 406 to 564 g kg -1 DM and higher EE in seeds than in pulp (31.7 vs 99.0 g kg -1 DM). Therefore, the linear increase of EE intake can be explained by the higher EE concentration in the GPS, which increased EE concentration in the diet due to the higher GPS levels added (Table 2). Values are mean ± standard error of the means. GPS = Grape pomace silage; CV = Coefficient of variation. The values for nutrient intake due to GPS addition in our study (Table 2) are in agreement with Chikwanha et al. (2019) and Zhao et al. (2018). The authors suggest that nutrient intake not was influenced by GPS inclusion. Chikwanha et al. (2019) found a DMI increase of up to 11.3 % with GPS inclusion in lamb diet and considered GPS an alternative feed to replace fiber sources rich in polyphenolic compounds to reduce feeding costs and improve performance and carcass characteristics. Although sheep have a significant selective capacity and the proportion of seeds is greater than the other GPS components, there were no leftovers from the diets with up to 30 % GPS inclusion, with GPS accounting for 165 g kg -1 DM in the total diet. The average DMI (3.5 % live weight) met the requirements of the animals and presented a value according with NRC (2007), that is, 3.5 % for the animal category analyzed with 30 kg and ADG 300 g d -1 , recommended by Mertens (1987) for ruminant animals (1.2 % of live weight); thus, the NDFI in our study is close to this recommendation. Gao et al. (2019) evaluated the inclusion of up to 15 % of GPS in lamb diets and found lower DMI and NDFI values with higher levels of GPS added. In our work, DMI and NDFI values showed no difference (Table 2) with inclusion up to 30 % of GPS in the diet, indicating that palatability was not negatively affected by adding GPS of cultivar Isabel Vitis Labrusca L. The GPS addition can be limited because due to high tannin content in seeds, which can have negative effects on nutrient digestion and palatability. However, some benefits are observed, such as alteration rumen fermentation and microbial protein synthesis, depending on the level, type, and nature of tannins (Gao et al., 2019).
The performance of feedlot lambs was not influenced by diets containing different levels of GPS. The results for ADG and FC in this study are considered satisfactory, when compared to data from other studies Values are mean ± standard error of the means. GPS = Grape pomace silage; CV = Coefficient of variation; HCY = Hot carcass yield; CCY = Cold carcass yield; TY = true yield; LC = Loss by cooling; FGT = full gastrointestinal content; EGT = empty gastrointestinal content; 1 (1 concave -6 convex); 2 (1 absent-5 abundant). Values are mean ± standard error of the means. GPS = Grape pomace silage; CV = Coefficient of variation. Grape pomace silage in diets of feedlot lambs Sci. Agric. v.79, n.5, e20200343, 2022 on GPS addition to lamb diets (Chikwanha et al., 2019;Kafantaris et al., 2018;Zhao et al., 2018;Gómez-Cortés et al., 2018). Calderón-Cortés et al. (2018) assessed the replacement of alfalfa by up to 30 % dehydrated grape by-product and did not observe variation in lamb performance, with ADG of 106.0 g. Zhao et al. (2018) found greater ADG and lower feed: gain ratio with the addition of 5 and 10 % of GPS in lamb diets than in the group control, with 177.9 and 215.3 g d -1 kg ADG and 8.1 and 6.9 of feed: gain ratio, respectively, indicating that this diet improved the feed efficiency. In our study, the average values of 235.2 g d -1 kg ADG and FC of 4.299 showed that GPS addition is a considerable ingredient for lamb diets. Kafantaris et al. (2018) also observed better performance of lambs fed with a GPS-added diet and reported that the ADG significantly increased before weaning compared to the control group. These researchers suggested that GPS may be included to the diet of growing lambs with considerable economic benefits to growers and production. GPS may also benefit performance due to its antioxidant properties that eliminate reactive oxygen species (ROS); thereby, reducing membrane damage and consequently improving intestinal functionality and reducing pathogenic microorganisms (Kafantaris et al., 2017).
Carcass traits and meat quality
The average values of final live weight and hot and cold carcass weight were similar to results obtained by Zhao et al. (2018) when supplementing lambs with up to 10 %grape marc. Gómez-Cortés et al. (2018) and Chikwanha et al. (2019) found similar average carcass yield by adding grape marc to lamb diets. Hot and cold carcass yields showed average values considered satisfactory in agreement with values found in the literature for GPS-added diets (Kafantaris et al., 2018;Gómez-Cortés et al., 2018). Chikwanha et al. (2019) also found values close to three for conformation for lamb diets with GPS addition. The average finish rate may be due to animal age and the method used for skinning. Fat coverage may have favored higher cooling loss values than the values observed by Oliveira et al. (2018) for Santa Inês lambs, since fat deposition reflects cooling loss, higher fat content leads to less water loss during cooling, due to its protective function, preventing losses and improving meat tenderness (Sañudo et al., 2000). Values are mean ± standard error of the means. GPS = Grape Pomace Silage; CV =Coefficient of variation; LWD = Loss of water by dripping; LWT = Loss of water by thawing; LWC = Loss of water by cooking; L* = lightness; a* = red intensity; b* = yellow intensity; c = croma; h = hue. The animals in our study were homogeneous, that is, with same ages, weights, and sex and were supplied with rations of similar nutritional value, which may have contributed to the similarity in biometric evaluations and bone, muscle and fat composition, and the compactness index. This similarity indicates that the animals showed the same degree of body development. The average value obtained for the compactness index of 0.25 kg cm -1 indicates good muscle proportion and is between 0.15 and 0.28 kg cm -1 for animals of different genotypes (Figueiredo et al., 2015).
The LTL muscle had average values for fat thickness of 1.51 mm, close to values found by Calderón-Cortés et al. (2018) for lambs fed with dried grape pomace. Fat thickness may be related to animal age and the skinning method. According to Ponnampalam et al. (2007), young animals have reduced fat deposition in the carcass and lean meat than old sheep. However, the average shear force values were 33 N and a slight lower value was observed for lambs fed with a GPS-added diet. Zhao et al. (2018) also observed a reduced value for lambs fed with grape pomace compared the control group. Chikwanha et al. (2019) reported shear force values below 49 N for lambs fed diets with grape pomace.
The average LEA value was 13.9 cm 2 , close to the average values of 11.40 and 13.07 cm 2 obtained by Souza Júnior et al. (2013) in lambs of large and medium frame size kept in feedlot. According to Bastos et al. (2015), the average LEA values for Santa Inês lambs are between 9.6 and 14.8 cm 2 . The addition of GPS to the diets showed no significant difference on LEA; thus, carcasses possibly showed similarity of muscularity (Table 6) due to the nonsignificance of diets on the yield and carcass, cuts, and the compactness index (Table 5). The average score of 1.64 obtained for marbling in the LTL muscle indicates little deposition of intramuscular fat in the carcass of lambs. Lonergan et al. (2019) reported that the intramuscular fat deposition occurs later than other the adipose tissue deposition in the animal and that intramuscular fat deposition occurs at a more advanced age of the growth process, justifying the low value obtained for marbling in our study.
Color is the most important characteristic to indicate freshness and quality for consumers at the time of product purchase (Van Ooijen et al., 2017). The average value 14.46 obtained for redness values in our study exceeds the threshold value of ≥ 9.5, considered acceptable by lamb meat consumers (Khliji et al., 2010). The values for color red intensity and other constituents are close to those found in the literature (Chikwanha et al., 2019;Figueiredo et al., 2015). For Zhao et al. (2017), the darker pigmentation in general in lamb masks any kind of color improvement, regardless of the diet. For the shear force, 49 N is an acceptable sensitivity in sheep meat (Hopkins et al., 2006), we found lower values in our study.
The mean pH value obtained in carcasses 24 h postmortem is within the acceptable range for sheep carcasses (Majdoub-Mathlouthi et al., 2013). Drip losses, defrosting, and meat cooking were not influenced by the GPS contents in the diets. This result was possibly influenced by the absence of dietary interference on pH, marbling, and finishing parameters. The meat centesimal composition was not influenced by diet and the values observed are close to those reported in the literature with grape pomace added to lamb diets (Chikwanha et al., 2019;Gómez-Cortés et al., 2018). GPS used in our study can be considered to be an alternative feed for lamb diets, considering the chemical composition of this by-product and results indicating that GPS did not compromise performance, carcass traits, and meat quality.
Our data suggest that the GPS levels of cultivar Isabel (Vitis Labrusca L.) increased linearly the EE intake, without affecting consumption. The DWG and FC showed were satisfactory results, with good muscle proportion and carcass conformation. The traits evaluated show that up to 30 % GPS can be added to lamb diets. In addition, this approach provides sheep meat production with sustainable resources that meet the demands of consumers. | 2021-10-21T16:20:55.798Z | 2022-01-01T00:00:00.000 | {
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19074416 | pes2o/s2orc | v3-fos-license | Recovery of a Functional Class 2 Integron from an Escherichia coli Strain Mediating a Urinary Tract Infection
ABSTRACT A class 2 integron was found in an Escherichia coli isolate mediating a urinary tract infection. Unlike other class 2 integrons from pathogens, the encoded IntI2 protein was functional. The integron possessed a dfrA14 cassette, and a second novel cassette in which a lipoprotein signal peptidase gene is predicted.
Mobilized integrons are substantial contributors to the spread of antibiotic resistance genes. The three classes of integron that mostly contribute to the problem of multidrug resistance are classes 1, 2, and 3 (1,13,14), where classes are determined based on sequence differences in the respective IntI proteins (5). Of the three, class 1 integrons are the most abundant and are found in a diverse range of other mobile elements (12,17), such as transposons and plasmids. However, class 2 integrons are also found in 4 to 20% of uropathogenic Escherichia coli strains (23,24) as well as in other human pathogens (22), other animal pathogens (16), and various commensal bacteria (2,4). In all of these cases, though, where examined, the intI2 gene is inactive by virtue of possessing a premature in-frame stop codon. Interestingly, however, in one study class 2 integrons were identified in commensal bacteria from bovine fecal material and hides. Here, the normally present stop codon is replaced with a glutamine codon and it is therefore likely that the associated IntI2 protein is functional, although this has not been demonstrated experimentally (3). Such putatively functional integrons have not been seen in either human commensals or pathogenic bacteria. Clearly, if they were to be found in such bacteria they would represent an additional mechanism by which resistance gene cassettes could be mobilized in pathogens.
In a survey of strains mediating a urinary tract infection from different individuals in Uruguay (19), 15 of 104 strains were identified that possessed a class 2 integron on the basis of generating a 789-bp PCR product with an intI2-specific primer pair (21). All 15 PCR products were sequenced, and 13 possessed the internal stop codon, confirming that the nonfunctional version of intI2 predominates in this population. However, two E. coli strains, designated 3843 and 8157, from separate individuals generated a sequence that implied a functional intI2 gene was present. Consequently, a genomic fosmid library was constructed from strain 8157 and the class 2 integron sequence was determined. The intI2 gene was different from the corresponding gene in Tn7 (accession no. AJ001816) at six positions, including in the stop codon (TAA) in Tn7, which in strain 8157 was the glutamine codon CAA. This is the first report of a potentially functional class 2 integron from a human pathogen. To confirm that the intI2 gene from E. coli isolate 8157 encoded a functional integrase, an amplicon including the gene was generated and cloned into pUC19 downstream of the P lac promoter. The recombinant plasmid was used to measure the integration frequency of the aadB cassette attC site into a target class 1 integron, as described previously (7,11). The assay used measures the ability of an integron integrase to catalyze an integrative recombination reaction between two sites: normally either attC versus attI or attC versus attC. It was found that IntI2 could efficiently catalyze an integrative recombination reaction ( Table 1). The point of insertion in the target conjugative plasmid, pMAQ495 (7), which contains a class 1 integron, was determined by PCR mapping (15). It was found that for 10/10 (two from five independent assays) cointegrate junctions, insertion had occurred at the pMAQ495 orfA attC site. This is consistent with observations that integron integrases do not efficiently recognize heterologous attI sites (8) and that orfA is the preferred attC target when attI1 is not favored (9).
To determine whether the class 2 integron was on a transferable plasmid, the strain 8157-which is sulfamethoxazole (SMX) and chloramphenicol (CHL) resistant and nalidixic acid (NAL) and rifampin (RIF) sensitive-was mated (20) with the E. coli strain Top10 (SMX and CHL sensitive and NAL and RIF resistant). Transconjugants appeared after selection on media containing SMX and NAL at a frequency of 1.0 ϫ 10 Ϫ4 /recipient. A total of 24 screened transconjugants were also resistant to CHL and RIF, 3 of which were screened by PCR and were found to be positive for the intI2 gene. Plasmid typing of the transconjugants by Inc/rep multiplex PCR (6) revealed a single amplicon consistent with the presence of an IncP plasmid.
In total, about 4,500 bp of DNA sequence was determined. The class 2 integron possessed a two-cassette array. The first of these was the dfrA14 gene cassette seen commonly in class 1 integrons (10,18). The second cassette included a novel open reading frame (ORF) not previously associated with cassettes and predicted a protein that matches a family of lipoprotein signal peptidases. The best match (4eϪ31; accession no. YP_001230301) was to a protein from Geobacter uraniireducens. Beyond this second cassette was another ORF that predicted a putative outer membrane lipoprotein (best match, 1eϪ23 to YP_411052) from Nitrosospira multiformis. This ORF was not obviously in a gene cassette in that an attC site could not be identified. Also, the putative outer membrane lipoprotein gene associated with the class 2 integron in strain 8157 is not functional as it has a stop codon at position 40 in an unprocessed predicted protein that would otherwise be 117 amino acids in length.
Class 2 integrons are found in a significant proportion of multiresistant human isolates, although the associated IntI2 protein, where examined, is nonfunctional (23,24). Here we show that a functional class 2 integron has appeared in a pathogenic E. coli strain and can autonomously acquire new cassettes and is apparently carried on an IncP plasmid that can transfer at high frequency. It will be important to determine whether this integron appears in different contexts, both geographical and genetic, beyond the single observation made here. This integron also displays evidence of acquiring new types of genes relevant to pathogens not obviously present in mobilized integrons previously. It will be interesting to investigate the context of this functional class 2 integron further to determine whether it is associated with Tn7 or some other transposon since such an association would provide another mechanism of mobilization.
b This protein is encoded within the plasmid designated pMAQ1047. c Value as measured by Collis et al. (9). 4154 NOTES ANTIMICROB. AGENTS CHEMOTHER. | 2018-04-03T01:24:22.639Z | 2008-09-15T00:00:00.000 | {
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235397617 | pes2o/s2orc | v3-fos-license | Evaluating the Chichewa version of the London Measure of Unplanned Pregnancy in Malawi: a validation update
Objective To investigate the psychometric properties of the validated Chichewa version of the London Measure of Unplanned Pregnancy in a large representative community-based sample in Malawi, a low-income country. We collected data on pregnancy intention from a cohort of 4244 pregnant women in Malawi using the validated Chichewa version of the London Measure of Unplanned Pregnancy (LMUP). We evaluated the psychometric properties of the Chichewa LMUP using classical test theory and confirmatory factor analysis to re-assess the performance of items one and six, which had weaker performance in the original smaller, facility-based validation sample. Results The Chichewa version of the LMUP met all pre-set criteria for validation. There are now nine validations of the LMUP in different low-and-middle-income countries, confirming the validity and applicability of the LMUP in these settings.
Introduction
The London Measure of Unplanned Pregnancy (LMUP) is a psychometrically validated measure of pregnancy intention that was developed in the United Kingdom in the early 2000s [1,2]. Using six questions, scored zero, one or two, it produces a score of zero-to-12, with higher scores indicating a more planned/intended pregnancy. Since its publication it has been translated and validated in diverse settings and populations around the world. As of May 2021 there are seventeen validated language versions across 14 countries, including nine low-and middle-income countries [2][3][4][5][6][7][8][9][10][11][12][13][14][15], with more in progress.
The original evaluation of the Chichewa LMUP in Malawi in 2013 found the measure to be acceptable to women and psychometrically valid [4]. The Cronbach's α (a measure of internal consistency) was 0.78 (above the standard cut point of 0.7 [16]). Item-rest correlations (which should be > 0.2 [17]) were at least 0.7 for four of the six questions, was borderline for the preconception preparation question (0.16) and was low for the contraception question (0.05). Hypothesis testing confirmed construct validity and principal component analysis confirmed unidimensionality (the Eigenvalue of the first component was 3.1), albeit with a borderline second compenent (Eigenvalue 1.0) which mostly represented the contraception question (loading 0.99). A sensitivity analysis to assess the effect of the removal of the contraceptive item (item one) showed slightly improved performance of the measure but as the LMUP was not significantly adversely affected by its inclusion, and for the purposes of international comparability, tracking of trends, and future relevance, we recommended retaining it. The LMUP includes two behavioural items: question one on contraception and question six on preconceptual preparations. There are lower levels of both contraceptive use and preconceptual preparations in Malawi, and also more generally in sub-Saharan African countries, compared with the United Kingdom and other developed countries. Despite this, the pattern of the relationship of the items of the LMUP has been remarkably stable across international contexts: items two-to-five most strongly correlated with the overall score; items one and six (the behaviour items) less strongly correlated; and all items positively correlated with each other. Given the importance of improving pregnancy intention measurement in low-and-middle-income countries, and in particular the call to provide evidence on the performance of the LMUP in such [18], we sought to re-examine the performance of the Chichewa LMUP using data from a large, representative community-based cohort study of pregnant women in Mchini District, Malawi [19]. We were particularly interested to review the performance of items one and six given their poorer performance in the original validation, which was conducted on a smaller, facility-based sample of 125 women.
Main text
Methodology 4244 pregnant women in Mchinji District, Malawi, completed the Chichewa LMUP between March 2013 and July 2014. Pregnant women were identified by a community-based surveillance system and were visited, consented and interviewed at home by one of 25 local data collectors. A full description of the recruitment and of the cohort has previously been published [19]. Mchinji District is a rural area, where most inhabitants are subsistence farmers with very low levels of education.
We evaluated the LMUP using Classical Test Theory, in keeping with its development [2] and the previous Chichewa validation [4]. Rates of missing data for each item were assessed, as high levels of missing data can indicate a problem with the understanding of acceptability of an item [20]. Item discrimination was assessed by examining the endorsement of item response options. Internal consistency was assessed using the Cronbach's α with a cut-point of 0.7 [16] and examination of each question's item-rest correlation, accepting a minimum correlation of 0.20 [17]. Inter-item correlations were assessed to check they were all positive. To assess structural (construct) validity, we conducted a principal component analysis (PCA) looking for one component with an Eigenvalue larger than one to demonstrate that all items are measuring the same construct [21]. In keeping with recent recommended standards of assessment [22], confirmatory factor analysis (CFA) was carried out to assess model fit (in this case the six items to a unidimensional model). Model fit was assessed by the comparative fit index (CFI), with > 0.95 indicating acceptable model fit, and standardised root mean squared residual (SRMR), with < 0.08 indicating acceptable model fit. We also conducted a Mokken analysis, as other validations of the LMUP have, to confirm that the items vary in 'difficulty' and that women answered the LMUP questions in keeping with how planned their pregnancy was. A Loevinger H coefficient of > 0.5 indicates a 'strong' scale [23].
Results
Missing data were very rare, with only 19 missing answers out of 25 464 questions asked (the six LMUP questions asked of 4244 women), as shown in Table 1, suggesting extremely good acceptability. No question response had more than 80% endorsement. For questions one to five the more planned option was the most frequently endorsed, whereas for question six, preconception preparations, a lack of preparation was most common.
The full range of LMUP scores from zero to twelve was captured. The median score was nine, inter-quartile range three-to-11. The Chronbach's alpha was 0.89 and, as shown in Table 2, all item-rest correlations were above 0.2. Principal components analysis confirmed that all items loaded on to one component with an Eigenvalue of 3.95. The Mokken analysis showed the LMUP was a 'strong' scale, with an overall Loevinger H coefficient of 0.733. The CFA confirmed a single factor model (CFI 0.997, SRMR 0.015).
Discussion
In this large representative community dataset, from a rural area in Malawi, a low-income country, the psychometric properties of the Chichewa LMUP met all prespecified criteria and international standards. In particular, the item-rest correlations for questions one and six, i.e. the behavioural components of contraceptive use and preconception preparations, were higher than in the initial validation and were both above the cut-point of 0.2 [4]. The pre-to-post birth stability of the Chichewa LMUP has previously been investigated in this sample [24]. This showed that the LMUP has moderate to substantial stability between pregnancy and up to 12 months postpartum (AC 0.54-0.64).
Since these data were collected there has been one other assessment of the psychometric properties of the Chichewa LMUP in Malawi by Yeatman and Greenaway [25]. In their study of 645 women, the Chichewa LMUP also demonstrated excellent psychometric properties: Hence, there have now been three analyses of the Chichewa LMUP in independent samples in Malawi, with the two more recent and larger studies demonstrating good psychometric properties. In addition to this, there are now 12 validationated language versions in a further eight low-or-middle-income countries: Uganda [14]; Sierra Leone [13]; Sri Lanka [12]; Pakistan [9]; India [6]; Iran [7]; Turkey [15] and Brazil [3]. The authors are aware of ongoing evaluations in Nepal, Botswana, Mexico, Tanzania, Mozambique, South Africa, India and elsewhere. In 2015, the Population Council's Expert meeting on 'Conceptualizing and Measuring Unintended Pregnancy and Birth' recognised that the LMUP overcame many of the limitations of previous assessments of unplanned pregnancy but were concerned that, at that time, there was limited evidence of its validity outside high-income settings [18]. This concern is no longer founded. There is also growing evidence of the limitations of other methods, such as the Demographic and Health Survey questions which have recall bias and are affected by maternal characteristics and pregnancy outcomes, which the LMUP overcomes [24,[26][27][28][29][30].
Conclusions
This large community-based dataset confirms the validity of the LMUP in a rural, low-income country setting. Furthermore, the item-rest correlations, coefficient and factor loadings for the contraception and preconception preparation questions demonstrate that these items are relevant in this context in all the analyses conducted. Given this, and the accumulating validations of the LMUP in other low-and middle-income countries, there is now convincing evidence of the validity of the LMUP, and the relevance of questions on contraception and preconception preparation, in diverse settings around the world.
Limitations
The main limitation of this study is that, as women were recruited on average in the fifth month of pregnancy, we missed abortions and early miscarriages. Despite this, we captured the full range of pregnancies intentions (scores from zero to 12). | 2021-06-11T14:04:46.835Z | 2021-06-10T00:00:00.000 | {
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59404652 | pes2o/s2orc | v3-fos-license | Effects of Irrigation Regime and Nitrogen Fertilizer Management on CH 4 , N 2 O and CO 2 Emissions from Saline – Alkaline Paddy Fields in Northeast China
Irrigation regime and fertilizer nitrogen (N) are considered as the most effective agricultural management systems to mitigate greenhouse gas (GHG) emissions from crop fields, but few studies have involved saline–alkaline paddy soil. Gas emitted from saline–alkaline paddy fields (1-year-old and 57-year-old) was collected during rice growing seasons by the closed chamber method. Compared to continuous flooding irrigation, lower average CH4 flux (by 22.81% and 23.62%), but higher CO2 flux (by 24.84% and 32.39%) was observed from intermittent irrigation fields. No significant differences of N2O flux were detected. Application rates of N fertilizer were as follows: (1) No N (N0); (2) 60 kg ha−1 (N60); (3) 150 kg ha−1 (N150); and (4) 250 kg ha−1 (N250). The cumulative emissions of GHG and N fertilizer additions have positive correlation, and the largest emission was detected at the rate of 250 kg N ha−1 (N250). Global warming potential (GWP, CH4 + N2O + CO2) of the 57-year-old field under the N250 treatment was up to 4549 ± 296 g CO2-eq m−2, approximately 1.5-fold that of N0 (no N application). In summary, the results suggest that intermittent irrigation would be a better regime to weaken the combined GWP of CH4 and N2O, but N fertilizer contributed positively to the GWP.
Introduction
The burgeoning population and increasing future rice demands have created tremendous concerns about agricultural greenhouse gas (GHG) emissions, which account for about one-tenth of total global anthropogenic GHG emissions [1].The greenhouse warming potential (GWP) of rice crop fields is approximately 4.6 times and 1.6 times that of wheat and maize, respectively [2].It is reported that paddies cover nearly 153 million ha., approximately 11% of the world's farmland and 50% of the people on the earth are supported by rice [3].China is the most important producer of rice, of which cultivated areas and rice yields both account for about 1/5 of the world's planting area [4].
CO 2 , which serves as the major greenhouse gas contributor, supplies 60% of the greenhouse effect from humans, CH 4 and N 2 O contribute 15% and 5%, respectively [5].In the global warming potential for the one hundred year horizon in terrestrial ecosystems, the contribution of CH 4 and N 2 O is 25 and 298 times larger than CO 2 respectively [6].CH 4 and N 2 O fluxes from paddies contribute approximately 30% and 11% of rural emissions, and paddies also can be either a source or sink of CO 2 [7][8][9].Soil microbial activities have an effect on the production and uptake of GHG [10] and are impacted by soil characters (e.g., soil temperature, water content and content of NH 4 + and NO 3 − ) [11][12][13][14][15]. Water regimes and N fertilizer application affect soil moisture, nutrient content and the soil's physicochemical properties and could be effective crop management tools to mitigate GHG emissions.
Single spring paddy rice is widely cultivated in northeast China, and phenological developments affected by climate change include green, tillering, booting, heading and maturity [16].CO 2 is assimilated and stored in the soil by plants and roots via rice growth.In addition, the labile carbon (C) pool with easily decomposable soil organic matter is decomposed and utilized for crop growth or emission as CH 4 or CO 2 into the air.Watanabe et al., covered that the translocation of carbon assimilated by rice into the soil organic matter (SOM) with crop growth and that the content of carbon absorbed by rice and emitted as CH 4 tended to increase by 0.003%, 0.26%, and 0.30% at the tillering, booting, and maturity stage, respectively [17].The fluxes of GHGs, including CO 2 , CH 4 and N 2 O, are highly variable during the five phenophases and are strongly affected by agricultural management.
Continuous flooding irrigation (CF) and intermittent flooding irrigation (IF) are common watering management systems during rice cultivation in northeast China.Methanogenic bacteria which decompose the dissolved organic carbon and produce CH 4 , are active in the CF pattern due to the anaerobic conditions made by a flooded water layer [18].Intermittent flooding irrigation, with drying-wetting alternation during the rice-growing period, has been recognized as a valid way of mitigating CH 4 emission in rice-production [19,20].Less CH 4 fluxes were monitored in IF pattern than CF paddies [21].However, other reports have found that the role of intermittent irrigation could be not obvious, compared to flooding irrigation [19].The effects of different irrigation regimes on crop production and gas fluxes may diverge from various practices or exhaust gas emissions for the same practice in different regions [22].
Nitrogen (N) fertilizer is an effective measure to enhance rice production which will feed more than a billion people worldwide over the next 30 years [23].Crop-growth is escalated by nitrogenous fertilizer; sufficient substrates are accommodated for methanogens and the transport of GHG emissions to the air is easier due to the larger aerenchyma [24].As reported, CH 4 emissions could be significantly induced by N fertilization.The size and structure of NH availability which stimulated methane oxidation, leading to a reduction in emissions [29].In addition, Banger et al., discovered that CH 4 emissions were stimulated at a rate lower than 140 kg N ha −1 , above which the fluxes were inhibited in rice-based cropping systems [30].These discrepancies may be related to the climate conditions and the physical and chemical properties of soil from different region [31][32][33].
Salinization is an enormous threat to environmental resources, and the area would account for more than 50% of the arable land by mid-century [34].Fields in western Jilin were barely covered with vegetation due to the land salinization before 1960s, when large-scale reclamation and rice cultivation were carried out.Nowadays, the region is a major rice-producing area and has made great contributions to grain production and food security in the Jilin province.Fields are still being reclaimed to paddies, especially after the project of irrigation from the Nen River was accomplished.Thus, newly and long-term tillage saline-alkaline paddies are chosen to estimate the impact of tillage on GHG emissions and global climate change during different reclamation years.
In the current study, we monitored the GHG fluxes from 1-year-old and 57-year-old saline-alkaline paddies in 2012 and 2013 in Qianguo.The objectives of this study were to (i) identify the difference of GHG fluxes between intermittent irrigation and continuous flooding irrigation in two tillage year paddies during rice-growing stages; (ii) ascertain the cumulative GHG emissions under different N fertilization rates during five rice-growing periods in two tillage year paddies; (iii) estimate the GWP of 1-year-old and 57-year-old paddies under different tillage treatment, comprehensively.We expected that a reasonable tillage management would be predicted for rice paddies and their reclamation which would benefit the minimization of GHG emissions.
Site Description
Field experiments were carried out during the rice-growing period of 2012 and 2013 in Qianguo town (123 1), in the west of Jilin province south of the Songnen Plain.The climate is a typical semi-arid.Monthly precipitation and air temperature during experimental period is shown in Figure 2 [35].The freezing period is between late October and mid-April (approximately 165 days) each year.Single-season rice is planted in the study area on 26 May and harvested on 2 October.The fertilizer N rate is between 60 and 250 kg N ha −1 , the mean rate is 150 kg N ha −1 , and the rice variety is Jijing88 (Super-rice I).
tillage year paddies during rice-growing stages; (ii) ascertain the cumulative GHG emissions under different N fertilization rates during five rice-growing periods in two tillage year paddies; (iii) estimate the GWP of 1-year-old and 57-year-old paddies under different tillage treatment, comprehensively.We expected that a reasonable tillage management would be predicted for rice paddies and their reclamation which would benefit the minimization of GHG emissions.
Site Description
Field experiments were carried out during the rice-growing period of 2012 and 2013 in Qianguo town (123°35′~125°18′E, 44°17′~45°28′N)(Figure 1), in the west of Jilin province south of the Songnen Plain.The climate is a typical semi-arid.Monthly precipitation and air temperature during experimental period is shown in Figure 2 [35].The freezing period is between late October and mid-April (approximately 165 days) each year.Single-season rice is planted in the study area on May 26 and harvested on October 2. The fertilizer N rate is between 60 and 250 kg N ha −1 , the mean rate is 150 kg N ha −1 , and the rice variety is Jijing88 (Super-rice I).
Experimental Design
The land (124°43′03″E, 45°00′19″N, 42.7 m × 20.4 m), barely covered with vegetation due to the soil salinization, has been reclaimed as a rice paddy since 1955, irrigated and fertilized every year.tillage year paddies during rice-growing stages; (ii) ascertain the cumulative GHG emissions under different N fertilization rates during five rice-growing periods in two tillage year paddies; (iii) estimate the GWP of 1-year-old and 57-year-old paddies under different tillage treatment, comprehensively.We expected that a reasonable tillage management would be predicted for rice paddies and their reclamation which would benefit the minimization of GHG emissions.
Site Description
Field experiments were carried out during the rice-growing period of 2012 and 2013 in Qianguo town (123°35′~125°18′E, 44°17′~45°28′N)(Figure 1), in the west of Jilin province south of the Songnen Plain.The climate is a typical semi-arid.Monthly precipitation and air temperature during experimental period is shown in Figure 2 [35].The freezing period is between late October and mid-April (approximately 165 days) each year.Single-season rice is planted in the study area on May 26 and harvested on October 2. The fertilizer N rate is between 60 and 250 kg N ha −1 , the mean rate is 150 kg N ha −1 , and the rice variety is Jijing88 (Super-rice I).
Experimental Design
The land (124 • 43 03"E, 45 • 00 19"N, 42.7 m × 20.4 m), barely covered with vegetation due to the soil salinization, has been reclaimed as a rice paddy since 1955, irrigated and fertilized every year.
The bare land (124 • 42 27"E, 45 • 00 05"N, 43.6 m × 21.8 m) without artificial disturbance before rice plantation was reclaimed in 2012.Land plots were plowed in late April before irrigation and then rice seedlings were transplanted in mid-May by machinery.
Experiment I was operated in 2012 to test the impact of water management systems continuous flooding irrigation (CF) and intermittent flooding irrigation (IF) on GHG emission.An earthen row was built into a ridge in each paddy to divide the patty into two parts (20 m × 20 m) before plowing, where CF and IF irrigations would be used.The water layer was always kept at 3 to 5 cm in CF treatment before the mature stage and drained for harvest.Under the IF irrigation mode, the water depth was 3-4 cm during the green stage, drained and rewetted, and maintained at 1 to 2 cm until the mature stage.To ensure that the nutrients were not limited, N fertilizers (150 kg N ha −1 ) were applied four times, accounting for 10%, 50%, 25% and 15% of the total amount, respectively.The fertilizer was applied before transplant seedlings (basal fertilizer), added in the third day after transplanting (green-tiller fertilizer), and broadcasted in the beginning and the end of July (booting fertilizer), respectively.The rice variety is Jijing88 (Super-rice I).Crops were planted and managed according to local farmers.
Experiment II was carried out in 2013 to find the influence of N fertilizer management on GHG.Each field was separated into four parts (10 m × 20 m) by artificial ridges before plowing.Local fertilizer application rates were ranged from 60 to 250 kg N ha −1 .Thus, four gradients were set: Control group (N0, no N fertilizer added), low N fertilizer (N60, 60 kg N ha −1 ), medium N fertilizer (N150, 150 kg N ha −1 ) and high N fertilizer (N250, 250 kg N ha −1 ).Plots were plowed and cropped (under IF) according to the methods mentioned above.
Emissions Measurements
Closed chamber technique was used to collect gas samples [36].The base (50 cm × 50 cm × 30 cm) made of acrylic sheets (6 mm) were put in the fields ahead of rice transplanting and enclosed six plants.Chambers of 50 cm (Length) × 50 cm (Width) × 50 cm (Height) were placed on the base.Grooves were designed on the top of base and middle chambers and replete with water to make the system gas-tight.Fans were fixed to homogenize the air inside [37].Holes on the chamber were used for temperature testing and gas sampling.The middle chamber was put on the pedestal and under the top box from the booting stage to maturity, when rice was growing faster and taller than 50 cm (Figure 3).Gas samples were collected every half-hour at 9:00~11:00 a.m. two times a week throughout the cropping season.Samples were drawn with an air-tight syringe and injected to pro-evacuate 50 mL vacuum bags immediately.Gas was sent to the lab of Northeast Institution of Geography and Agro-ecology, Chinese Academy of Sciences.The contents of gases were subsequently tested with a gas chromatograph (GC, Agilent 7820A, Santa Clara, CA, USA) [22].The bare land (124°42′27″E, 45°00′05″N, 43.6 m × 21.8 m) without artificial disturbance before rice plantation was reclaimed in 2012.Land plots were plowed in late April before irrigation and then rice seedlings were transplanted in mid-May by machinery.Experiment I was operated in 2012 to test the impact of water management systems continuous flooding irrigation (CF) and intermittent flooding irrigation (IF) on GHG emission.An earthen row was built into a ridge in each paddy to divide the patty into two parts (20 m × 20 m) before plowing, where CF and IF irrigations would be used.The water layer was always kept at 3 to 5 cm in CF treatment before the mature stage and drained for harvest.Under the IF irrigation mode, the water depth was 3-4 cm during the green stage, drained and rewetted, and maintained at 1 to 2 cm until the mature stage.To ensure that the nutrients were not limited, N fertilizers (150 kg N ha −1 ) were applied four times, accounting for 10%, 50%, 25% and 15% of the total amount, respectively.The fertilizer was applied before transplant seedlings (basal fertilizer), added in the third day after transplanting (green-tiller fertilizer), and broadcasted in the beginning and the end of July (booting fertilizer), respectively.The rice variety is Jijing88 (Super-rice I).Crops were planted and managed according to local farmers.
Experiment II was carried out in 2013 to find the influence of N fertilizer management on GHG.Each field was separated into four parts (10 m × 20 m) by artificial ridges before plowing.Local fertilizer application rates were ranged from 60 to 250 kg N ha −1 .Thus, four gradients were set: Control group (N0, no N fertilizer added), low N fertilizer (N60, 60 kg N ha −1 ), medium N fertilizer (N150, 150 kg N ha −1 ) and high N fertilizer (N250, 250 kg N ha −1 ).Plots were plowed and cropped (under IF) according to the methods mentioned above.
Emissions Measurements
Closed chamber technique was used to collect gas samples [36].The base (50 cm × 50 cm × 30 cm) made of acrylic sheets (6 mm) were put in the fields ahead of rice transplanting and enclosed six plants.Chambers of 50 cm (Length) × 50 cm (Width) × 50 cm (Height) were placed on the base.Grooves were designed on the top of base and middle chambers and replete with water to make the system gas-tight.Fans were fixed to homogenize the air inside [37].Holes on the chamber were used for temperature testing and gas sampling.The middle chamber was put on the pedestal and under the top box from the booting stage to maturity, when rice was growing faster and taller than 50 cm (Figure 3).Gas samples were collected every half-hour at 9:00~11:00 a.m. two times a week throughout the cropping season.Samples were drawn with an air-tight syringe and injected to proevacuate 50 mL vacuum bags immediately.Gas was sent to the lab of Northeast Institution of Geography and Agro-ecology, Chinese Academy of Sciences.The contents of gases were subsequently tested with a gas chromatograph (GC, Agilent 7820A, Santa Clara, CA, USA) [22].
Soil Properties Analysis
Fifteen samples from each paddy were collected in metal cylinders (100 cm 3 ) before the field experiment, and dried at 105 • C for 48 h to calculate water content and report soil bulk density on a dry basis.Twenty-five samples taken with soil auger (7 cm) from each field were mixed and air dried at room temperature, then passed sequentially through a sieve (2 or 1 mm) and stored for physical-chemical analysis (Table 1).Soil organic carbon (SOC) was tested using a total organic carbon (TOC) analyzer (Shimadzu TOC-V, Japan).Total nitrogen was determined using Kjeldahl method.Total phosphorus was measured according the method of Andetsen (1975) [38].PH and EC were measured using PHS-3C (08) pH meter and DDS-307 conductivity meter (Rex China) at a ratio of 5:1 (water to soil).CEC (cation exchange capacity) was tested using an EDTA-ammonium acetate exchange method, and a flame emission photometric method was used for exchangeable sodium.ESP is the percentage of exchangeable sodium to CEC.Experiments were performed in triplicate.
Calculation of CH 4 , N 2 O and CO 2 Emission
Fluxes were computed from the following formula [39,40].
where F is the flux of CH 4 , N 2 O or CO 2 (mg m −2 h −1 ), ρ is the density of gas (mg cm −3 ), V and A are the volume (m 3 ) and surface area (m 2 ), ∆c/∆t is the rate of gas increase inside of chamber (mg m −2 h −1 ), and T is the absolute temperature in-house.(F cal ) is the calibration of the average emission of CH 4 , N 2 O or CO 2 , F is the mean flux of a certain gas (mg m −2 h −1 ), and C is the calibration coefficient (1.24) [41].
Seasonal GHG emission was computed as follows: where F C is the accumulative fluxes in every rice-growing season (g m −2 ) and D i is the days of the ith period.
Global warming potential of main GHG was calculated using the following equation [6]: where F c is the accumulative flux (g CO 2 -eqv m −2 ),
Statistical Analysis
SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analyses.One-way and two-way analyses of variance (ANOVA) were performed to analyze the differences of average and cumulative CH 4 , N 2 O and CO 2 flows in different irrigation regimes and N fertilization managements (LSD; p < 0.05).Microsoft Excel 2003 software was used to cypher the standard deviation of means.SigmaPlot 12.5 (Systat Software, Inc., San Jose, CA, USA) was applied for plots.
GHG Emissions under IF and CF Regime
CH 4 showed single-peak emission during the rice growing period.The peak values under intermittent irrigation were lower but earlier (4 days) than in the continuous flooding condition, with crest values of 1.98 mg m −2 h −1 and 24.02 mg m −2 h −1 from 1-year-old and 57-year-old paddies, respectively (Figure 4A,B).The largest average flux appeared in the booting stage, i.e., 1.70 ± 0.16 mg m −2 h −1 (1-year-old, CF) and 1.25 ± 0.25 mg m −2 h −1 (1-year-old, IF), but the lowest mean value was observed in the green period.Comparatively, CH 4 from long-term tillage paddy had higher emission flux, with cumulative emissions of approximately 9.21-(CF) and 8.36-(IF) fold higher than that of new fields.CH 4 was sensitive to irrigation regimes during booting, heading and mature stages, which was susceptible to tillage year (Table 2).
In the time of the rice-growing season, N 2 O flux from CF was low, with a mean rate of 0.08 mg m −2 h −1 (1-year-old) and 0.07 mg m −2 h −1 (57-year-old).Three peaks appeared in the green, booting and heading stages and then dropped speedily to low levels (0.02~0.09 mg m −2 h −1 ).N 2 O flux from IF showed similar emission trends, although the peak appeared in the tillering stage, earlier than booting, and the maximum rate in heading was much higher (0.22 mg m −2 h −1 , 1-year-old; and 0.19 mg m −2 h −1 , 57-year-old) (Figure 4C,D).No significant differences of N 2 O fluxes between the two treatments (IF and CF) were observed except for the mature stage, and the total cumulative emissions were 0.23 mg m −2 (1-year-old, CF), 0.29 mg m −2 (1-year-old, IF) and 0.20 mg m −2 (57-year-old, CF), 0.24 mg m −2 (57-year-old, IF) (Table 2).
CO 2 emissions in CF condition were lower than that from IF fields.During the green stage, the emission flux was lowest, with an average value of 169.03 to 435.65 mg m −2 h −1 .The maximum was observed in tillering stage for the 57-year-old field, 818.75 mg m −2 h −1 (CF) and 1053.95mg m −2 h −1 (IF), and maturity for the 1-year-old paddy, 852.78 mg m −2 h −1 (CF) and 893.70 mg m −2 h −1 (IF) (Figure 4E,F).There were significant differences in soil carbon dioxide during growing periods between water regimes, except for green and mature period, while cumulative emissions from the 57-year-old paddies were significantly higher than from the 1-year-old paddy (Table 2).
GHG Emissions under Fertilizer N Addition
Total cumulative CH4 fluxes ranged from 19.32-36.88g m −2 and were positive correlated with N addition rates; the maximum was observed in the N250 treatment of 57-year-old paddy.Emissions in the booting stage were the highest, accounting for 41.25-52.67%,and in the green stage, the lowest, the emissions accounted for 0.48-1.27%.The cumulative fluxes were also positively correlated to N fertilization at different stages, but exceptions appeared in booting when the cumulative emissions in the N60 and N150 treatments were lower than the control (N0).More emissions were detected from long-term tillage paddy, approximately 1.56-to 1.75-fold higher than newly developed field in booting period (Figure 5A,B).
N2O fluxes were promoted by the application of nitrogen fertilizers, increasing with seasonal cumulative emissions of 90.34 mg m −2 -174.42 mg m −2 .Cumulative N2O fluxes reached a peak in the booting stage in all fields and were significantly higher in N250 treatment than N0 from long-term tillage fields during growing periods (Figure 5C,D).N2O emissions were lower in 1-year-old paddy soil than in the long-term tillage fields.
GHG Emissions under Fertilizer N Addition
Total cumulative CH 4 fluxes ranged from 19.32-36.88g m −2 and were positive correlated with N addition rates; the maximum was observed in the N250 treatment of 57-year-old paddy.Emissions in the booting stage were the highest, accounting for 41.25-52.67%,and in the green stage, the lowest, the emissions accounted for 0.48-1.27%.The cumulative fluxes were also positively correlated to N fertilization at different stages, but exceptions appeared in booting when the cumulative emissions in the N60 and N150 treatments were lower than the control (N0).More emissions were detected from long-term tillage paddy, approximately 1.56-to 1.75-fold higher than newly developed field in booting period (Figure 5A,B).
N fertilizer stimulated CO2 emissions, and the responses of seasonal cumulative CO2 emissions ranged from 1584.72 to 3575.02 g m −2 (Figure 5E,F).The contributions of CO2 emissions in the green stage and in the heading stage were less than others, accounting for 2.05-5.55%and 9.80-14.83% of total, respectively.The cumulative emissions from 57-year-old soils were always more than of 1-yearold fields, at a maximum of 1.5-fold higher in the booting period.
Area-Scaled Global Warming Potential
GWPs of CH4 and N2O from CF were much taller than that from IF: 1112 ± 18 g CO2-eq m −2 for 57-year paddy throughout rice growth (Table 3).In contrast, the total GWPs of IF were higher than that of CF when CO2 was considered, at 2813 ± 23 g CO2-eq m −2 (1-year-old) and 4271 ± 241 g CO2-eq m −2 (57-year-old).GWPs were larger in the long-term paddy than the new paddy.
The GWPs of paddy soils increased with N application rate in all treatments (N0, N60, N150, N250).Moreover, the greatest values were reported among 57-year-old paddies.Annual GWP of CH4 and N2O was highest in N250 treatments from long-term tillage fields (974 ± 132 g CO2-eq m −2 ), but the value was more than four-fold higher than the former if CO2 was taken into account.N 2 O fluxes were promoted by the application of nitrogen fertilizers, increasing with seasonal cumulative emissions of 90.34 mg m −2 -174.42 mg m −2 .Cumulative N 2 O fluxes reached a peak in the booting stage in all fields and were significantly higher in N250 treatment than N0 from long-term tillage fields during growing periods (Figure 5C,D).N 2 O emissions were lower in 1-year-old paddy soil than in the long-term tillage fields.
N fertilizer stimulated CO 2 emissions, and the responses of seasonal cumulative CO 2 emissions ranged from 1584.72 to 3575.02 g m −2 (Figure 5E,F).The contributions of CO 2 emissions in the green stage and in the heading stage were less than others, accounting for 2.05-5.55%and 9.80-14.83% of total, respectively.The cumulative emissions from 57-year-old soils were always more than of 1-year-old fields, at a maximum of 1.5-fold higher in the booting period.
Area-Scaled Global Warming Potential
GWPs of CH 4 and N 2 O from CF were much taller than that from IF: 1112 ± 18 g CO 2 -eq m −2 for 57-year paddy throughout rice growth (Table 3).In contrast, the total GWPs of IF were higher than that of CF when CO 2 was considered, at 2813 ± 23 g CO 2 -eq m −2 (1-year-old) and 4271 ± 241 g CO 2 -eq m −2 (57-year-old).GWPs were larger in the long-term paddy than the new paddy.
The GWPs of paddy soils increased with N application rate in all treatments (N0, N60, N150, N250).Moreover, the greatest values were reported among 57-year-old paddies.Annual GWP of CH 4 and N 2 O was highest in N250 treatments from long-term tillage fields (974 ± 132 g CO 2 -eq m −2 ), but the value was more than four-fold higher than the former if CO 2 was taken into account.CH 4 emissions fluctuated with rice-growth.The flux was high from middle tillering to the end of the heading stage, observing a peak value in the booting stage in which the cumulative CH 4 emissions accounted for approximate half of the flux.The lowest cumulative gas flux was in the green and mature stages, with just 6% of the total proportion.CH 4 production can be influenced by many factors, like rainfall, temperature and crop growth [33,42].In July and August, the temperature is high, rice plants grow fast and roots develop rapidly, producing more H 2 with the help of fermentative bacteria and producing acetic acid bacteria.More root exudates accelerate the decomposition of SOC and provide adequate substrate for microbes.The relatively high soil temperature increases the methanogenic activity, and the flooding layer creates a strict anaerobic condition for soil, promoting the production of CH 4 [5].However, during the mature stage the field drained and soil permeability was better, increasing methanotroph activities and inhibiting methanogens, and thus less CH 4 emission flux was observed.
Throughout the entire rice-growing season, N 2 O emission flux showed minor fluctuations except for three drastic changes.In the green stage, the cumulative N 2 O emissions were lowest, but they were highest in the booting period.N 2 O gas produced from nitrification and denitrification, susceptible to temperature, soil moisture and fertilizer [43].Rapid growth was primarily stimulated by the application of urea, increasing the concentration of the reaction substrate and promoting the formation and emission of gas.
The emission curves of CO 2 changed with rice growing, increasing gradually from transplanting and reaching a maximum in tillering, decreasing slightly in booting, and recovering to a high level in the late heading period.The highest emissions were detected in tillering and late-heading stages, and the highest cumulative emissions of CO 2 were detected in tillering, booting and maturity periods.CO 2 in paddy fields mainly comes from plant and soil respiration [8].In the green period, plant respiration is weak due to the limitation of biological factors, such as plant height and leaf area.In the tillering stage, the air temperature gradually increased, plants grew quickly, soil microbial activities were enhanced and root exudates production increased, providing suitable conditions for soil respiration.The rate of respiration could be the reason for decrease in booting, when the photosynthetic rate is low.During the mature period, litter provides favorable conditions for the transformation of soil organic matter, promoting soil respiration and gas emissions [44].
Effects of Irrigation Regimes on GHG Emissions
Soil moisture is one of the great significance indicators of soil properties during rice-growing seasons and has impacts on greenhouse gas emissions by changing soil permeability, microbial activity and Eh (soil redox potential) [45].Water content did not change significantly in continuous flooding conditions; the water layer had a constant height of 3-5 cm.This level changed drastically in certain periods during the entire season in IF treatments.
CH 4 emissions, showing a single peak pattern in all fields, were sensitive to irrigation regimes, and the annual cumulative CH 4 fluxes in IF were approximately 23% lower than that of CF (Table 2).These results are consistent with records showing that intermittent irrigation management can reduce CH 4 emissions [46,47].Diffusion rates of O 2 and CH 4 and activities of methane-oxidizing bacteria are impressed by soil water content [47].Intermittent irrigation provides better habitat for the growth of methanotroph (obligate aerobic bacteria), beneficial to bacterial reproduction [48], increasing CH 4 oxidation capability relative to CF (Figure 4).The impact of water regime on CH 4 emissions differs with rice-growing stages, and gas fluxes were more sensitive in booting, heading and mature stages, when soil water content changed considerably in the intermittent irrigation mode (Table 2).
N 2 O emissions under two water regimes were the same as in previous studies, except for the rapidly increased peaks [36,46].The lowest peak value was noted in the heading stages under CF conditions, with no significance differences between CF and IF treatments (Table 2).Over rice-growing periods, three peaks came out instantly following nitrogen application, compliance with the fact that pulses of N 2 O emission are commonly induced by N fertilization addition [24].With the application of booting fertilizer, the peaks in intermittent irrigation appeared much earlier than in CF conditions, mainly due to the gradual drainage of paddy fields at the late tillering period, promoting the synergistic effects of nitrification and denitrification.N fertilizer addition increased the concentration of substrate, thereby promoting the formation and discharge of gas.During heading stages denitrification (N 2 to N 2 O) was impeded because of the relatively high floodwater depth (3-5 cm) under CF conditions, with high water content and strictly anaerobic conditions, lowering the release of N 2 O [49].However, no significant differences between two water regimes were noticed over the entire rice-growing period (Table 2).
CO 2 emissions were more likely influenced by water management, especially in the tillering, booting and heading stages (Table 2).CO 2 emissions from flooding treatment are always lower than in intermittent irrigation treatment.During rice-growth stages, the paddy field is drained on occasion, supplying rice roots with sufficient oxygen and strengthening root respiration which can possibly promote the activity of aerobic microorganisms in the soil, decomposing soil organic matter [49].CO 2 from soil and plant root respiration can be more easily released into the atmosphere due to good soil permeability under IF treatments.In contrast, soil water content in long-term submerged paddy soil is higher, and the relatively anaerobic environment suppresses the aerobic activity of aerobic soil, decreasing soil respiration intensity and the production of CO 2 gas.The flooded layer promotes the dissolution of CO 2 hinders its diffusion into the atmosphere.
Impacts of N Fertilization Rate on Regulating GHG Emissions
Application of N fertilizer can promote crop growth and increase food production while affecting CH 4 emissions.Three processes are involved in CH 4 emissions from paddy soil, which can be influenced by nitrogen fertilizer addition, increase or decrease production, and consumption, and release gas at various ecosystems [42].Application of N fertilizer could increase crop size and provide rich organic matter for methanogens, utilizing roots and root exudates as a carbon source [50].In addition, researchers reported that the use of N fertilizer in paddy fields promoted the growth and activity of methanogens, promoting CH 4 emissions [25,51,52].In the present study, the total cumulative CH 4 fluxes were encouraged by fertilizer additions; in contrast CH 4 emissions were inhibited in N60 and N150 treatments during the booting stage, in contrast to some reports showing that lower N rates tend to increase CH 4 emissions but higher N rates could potentially inhibit CH 4 emissions [25].The reason for the difference may be severe soil salinization, lower SOM content and high pH value [53].
Due to nitrification and denitrification processes, the emission of N 2 O from cropland was caused predominantly by nitrogenous fertilizers put on fields [54].In the current research, the reaction substrate concentration was increased through urea application, promoting the emission of N 2 O [55,56].As more N fertilizer was used, more cumulative N 2 O emissions were detected at each rice-growing stage.The positive effect is consistent with other reports [4,57,58].
It is reported by Burton et al., that CO 2 was inhibited by N fertilizer application as the decreased activities of extracellular enzyme and depletion in fungal populations [59][60][61].In contrast, the increased emissions of CO 2 attributed to N fertilizer were caught by Iqbal et al. [62].However, in the current study, the cumulative CO 2 emissions had a positive correlation with N fertilizer rate; as more urea was added, more CO 2 gas emitted.Nitrogen fertilizer increased the activity of roots, improved soil nutrient content, enhanced the biological activity in paddy soil, and promoted the mineralization of organic matter which increased soil respiration [63].In addition, the carbon source and available nitrogen increased.The appropriate C/N ratio not only ensures soil microbial activities but also promotes the vigorous growth of rice plants, thereby enhancing the respiration of soil and plant populations, promoting CO 2 emissions from paddy fields.
Relationships between GHG Emissions and Tillage Year
GHG emissions from two paddy fields showed similar trends, and the gas emissions from 57-year-old paddies were much higher; GWPs are always higher (1.4-1.8 times) than for the 1-year-old paddy field (Figure 4A,B, Tables 2 and 3).In the current study, CH 4 and CO 2 emissions were significantly influenced by tillage year, and N 2 O by N fertilizer (Table 2, Figure 4).
Previous studies have reported that GHG can be easily affected by soil chemical-physical properties such as pH value, soil organic matter and salinity [14,38].Microbial growth and activities are strongly influenced by pH.The optimum pH-value for methanogens is more than four and less than seven [64].The pH was as high as 9.72 in the 1-year-old paddy, reducing methanogenic archaea and methanotroph bacteria, thus lowering CH 4 and CO 2 production.However, no significant differences in total N 2 O emissions from the paddy fields were caught, mainly being ascribable to the weak influences of pH on the nitrification and dentrification process [65].
It has reported that CH 4 and CO 2 were influenced by soil organic matters [66].SOC also could be the reason why there were more greenhouse gas emissions in longtime tillage paddies [8].The 57-year-old paddy had a long tillage history, planted rice for many years and accumulated more organic matter [18].The soil was fertile and SOC content was five-fold higher than in the new tillage field, providing a superior growth environment for rice plants, root and soil microorganisms and more available organic carbon substrate for microorganism by root exudation and microbial decomposition (Table 1).Rice plants were tall and sturdy, and photosynthesis and respiration processes were stronger, transforming more oxide into soil for N 2 O and CO 2 production [67].Moreover, more gases escaped via rice tissue from paddies to atmosphere with the help of well-developed aerenchyma in roots, rhizomes and stems [62].In addition, plant respiration may increase GHG emissions from 57-year-old paddies, where above-ground biomass was much higher.
Conclusions
We measured CH 4 , N 2 O and CO 2 fluxes in saline-alkaline paddy fields with typical Chinese irrigation regimes, continuous flooding irrigation and intermittent flooding irrigation, and nitrogen fertilizer managements (N0, N60, N150 and N250).Relative to continuous flooding, intermittent irrigation reduced CH 4 but promoted CO 2 , and had little effect on N 2 O emissions from rice paddy soils.The N fertilizer showed a positive effect on greenhouse gases compared to no N addition.The cumulative emissions of CH 4 , N 2 O and CO 2 all increased with the supply of urea, with a maximum at the rate of 250 kg N ha −1 .The fluxes in the 57-year-old paddy were higher than in the 1-year-old soil, with higher pH and lower SOC.Intermittent irrigation versus continuous flooding irrigation would mitigate the GWP of CH 4 and N 2 O, and N fertilizer contributes to the GWP of net CH 4 , N 2 O and CO 2 fluxes.
Figure 1 .
Figure 1.Location of Jilin province in China and Qianguo in Jilin province.
Figure 2 .
Figure 2. Monthly mean precipitation and air temperature during the rice-growing period from 2012 to 2013.
Figure 1 .
Figure 1.Location of Jilin province in China and Qianguo in Jilin province.
Figure 1 .
Figure 1.Location of Jilin province in China and Qianguo in Jilin province.
Figure 2 .
Figure 2. Monthly mean precipitation and air temperature during the rice-growing period from 2012 to 2013.
Figure 2 .
Figure 2. Monthly mean precipitation and air temperature during the rice-growing period from 2012 to 2013.
Figure 3 .
Figure 3. Structure of static chamber monitored greenhouse gas (GHG) fluxes in paddy fields.
Figure 3 .
Figure 3. Structure of static chamber monitored greenhouse gas (GHG) fluxes in paddy fields.
Figure 4 .
Figure 4. Seasonal variations in GHG (CH4, N2O and CO2) fluxes from two paddies, i.e., 1st tillage (1year-old) and 57th tillage (57-year-old), with different irrigation treatments: Continuous flooding irrigation and intermittent irrigation.Error bars denote standard deviation.The vertical arrows indicate N fertilizer application except for the base fertilization before transplant of seedlings.
Figure 4 .
Figure 4. Seasonal variations in GHG (CH 4 , N 2 O and CO 2 ) fluxes from two paddies, i.e., 1st tillage (1-year-old) and 57th tillage (57-year-old), with different irrigation treatments: Continuous flooding irrigation and intermittent irrigation.Error bars denote standard deviation.The vertical arrows indicate N fertilizer application except for the base fertilization before transplant of seedlings.
Figure 5 .
Figure 5. Cumulative GHG (CH4, N2O and CO2) emissions from two paddies affected by different N fertilizer treatments over five growing stages.Capital letters over the bars showed significant differences among different N fertilizer rates (kg N ha −1 ), whereas lowercase letters indicate differences between 1-year-old and 57-year-old paddies.
Figure 5 .
Figure 5. Cumulative GHG (CH 4 , N 2 O and CO 2 ) emissions from two paddies affected by different N fertilizer treatments over five growing stages.Capital letters over the bars showed significant differences among different N fertilizer rates (kg N ha −1 ), whereas lowercase letters indicate differences between 1-year-old and 57-year-old paddies.
Table 2 .
Cumulative emission of GHG from continuous flooding irrigation (CF) and intermittent flooding irrigation (IF) of two tillage paddies during rice-growing seasons (n = 3).
Table 3 .
Area-scaled GWP (global warming potential, g CO 2 -eqm −2 ) under different water regimes, continuous flooding (CF) and intermittent irrigation (IF), and N fertilization application rates over entire rice-growing season.Mean Value ± Standard Error.Different lowercase letters indicate significantly different at significance level of p < 0.05 among four N fertilization application rates. 1 | 2018-12-24T03:17:35.751Z | 2018-02-11T00:00:00.000 | {
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2035923 | pes2o/s2orc | v3-fos-license | Radio emission of Shakhbazian Compact Galaxy Groups
Three hundred fifty three radio sources from the NRAO VLA Sky Survey (NVSS) (Condon et al. 1998) and the FIRST Survey (White et al. 1997}, are detected in the areas of 179 Shakhbazian Compact Groups (ShCGs) of galaxies. Ninety three of them are identified with galaxies in 74 ShCGs. Six radio sources have complex structure. The radio spectra of 22 sources are determined. Radio luminosities of galaxies in ShCGs are in general higher than that of galaxies in Hickson Compact Groups (HCGs). The comparison of radio (at 1.4 GHz) and FIR (at 60 $\mu$m) fluxes of ShCG galaxies with that of HCG galaxies shows that galaxies in ShCGs are relatively stronger emitters at radio wavelengths, while galaxies in HCGs have relatively stronger FIR emission. The reasons of such difference is discussed.
Introduction
A couple of decades ago it was found that radio emission is observed by about three times more often from double galaxies and members of groups of galaxies than from single, isolated galaxies (Tovmassian 1969;Sulentic 1976). It is widely accepted that galactic nuclear activity, one of the manifestations of which is a radio emission, is a result of interaction between galaxies. Events of interaction should take place much more often in dense environments of Compact Groups (CGs) of galaxies. Therefore it was expected that some of member galaxies in CGs should be relatively strong radio emitters. The first systematic search for radio emission from Hickson Compact Groups (HCGs), revealed a radio emission at 18 cm above a flux limit of 1.5 mJy from 41 out of 88 observed member galaxies (Menon & Hickson 1985). It was found (Tovmassian & Shahbazian 1981) that in groups of galaxies the radio emission is most often observed from the optically first-ranked galaxies. Menon (1992) showed that the radio emission in HCGs is almost always associated with the first-ranked elliptical galaxies, while the radio detected spirals are uniformly distributed among the three brightest members of a group.
Prior to HCGs the lists of the so called Shakhbasian Compact Groups of Compact Galaxies (SCGCGs) were published (Shakhbazian 1973, Baier &Tiersch 1979, andreferences therein). It is the largest and a relatively homogeneous sample of galaxy groups known up to now. Shakhbazian's CGs were found on the POSS prints by an eye search. They were identified according to the following criteria: -the groups consist of 5 -15 members; -the apparent magnitude of the individual galaxies is between 14 m and 19 m ; -the groups are compact, i.e. the distances between galaxies are only 3 -5 times the diameters of galaxies; -nearly all member galaxies are extremly red, at most 1-2 blue galaxies in a group is an exception; -members of groups are compact (relatively high surface brightness, borders not diffuse); -the groups are isolated.
ShCGs originally were called compact groups of compact galaxies, because the images of most of the constituent galaxies in these groups seemed very compact. Later observations of these groups with high angular resolution revealed that member galaxies mostly are of E and S0 types. The fraction of early type galaxies is 77% in SCGs compared to 51% in HCGs and 40% in field galaxies (Tiersch et al. 1996a). Member galaxies in these groups are significantly redder than the field galaxies of the same morphological type. The difference in B-V and V-R is by ∼ 0.2. m redder compared to galaxies of the RC3. Anyhow, there were found emission-line, and even Seyfert galaxies among Shakhbazian groups members.
Since, as it was found out, members of Shakhbazian groups turned to be not "compact", these groups in recent papers were called Shakhbazian Compact Groups of Galaxies (SCGGs) (Tovmassian et al. 1998a, hereafter TMTST, 1998b, or in analogy with HCGs, -Shakhbazian Compact Groups (ShCGs) (Tovmassian et al. 1998c). Galaxies in ShCGs are relatively weak, because they are at least by three times further than HCGs (TMTST). Mainly for this reason, and probably for very unusual supposed composition the ShCGs attracted little attention until recently. The redshifts of only about 70 relatively bright and nearby ShCGs have been measured so far (Robinson & Wampler 1973;Arp et al. 1973;Mirzoyan et al. 1975;Amirkhanian & Egikian 1987;Amirkhanian 1989;Kodaira et al. 1988Kodaira et al. , 1990Kodaira & Sekiguchi 1991;Lynds et al. 1990;del Olmo & Moles 1991;Tiersch et al. 1997).
The densities of galaxies in ShCGs approaches to 10 3 − 10 4 Mpc −3 . Hence the interaction and merging processes should be frequent in them. As a consequence some ShCGs could be a radio and FIR emitters. However due to larger distances and different morphological content (Tiersh et al. 1996a) we could expect smaller rate of radio and FIR detections from ShCGs in comparison with HCGs. The results of the search for FIR emission of ShCGs were published recently by TMTST. In this paper we present the results of a search for radio emission from ShCGs.
Observational data
Three hundred seventy seven groups were included in the original lists of ShCGs. The positions of the group centers were given with accuracies of about 1 ′ . For identification of radio sources with ShCGs we used accurate coordinates of member galaxies measured afterwards from the Digitized Sky Survey (DSS) by Stoll et al. (1997, and references therein). Positions of four groups, ShCG 206, 241, 301 and 353, were largely incorrect in original lists, and they were not found during accurate position measurements. It was revealed also that the group ShCG 214 coincides with ShCG 252, and the group ShCG 340 consists of two groups, ShCG 340a and ShCG 340b. Spectral observations showed that five groups, ShCG 12, 13, 78, 146, and 180, consist mainly of stars. Thus we searched for a radio emission at the positions of 367 ShCGs.
For looking for a radio emission from ShCGs we first examined the NRAO VLA Sky Survey (NVSS) (Condon et al. 1998) at 1.4 GHz at the positions of all 367 ShCGs. Generally an area ±3 ′ centered on each group was investigated. The FIRST Survey (White et al. 1997), made at the same frequency, was used in addition for those 175 ShCGs that are located in the area of the sky, covered by the Survey until 1st November 1998.
The errors of the radio position measurements in the used Surveys are small enough. For the NVSS sources they vary from ≤ 1 ′′ for sources stronger than 15 mJy, to 7 ′′ at the Survey flux limit at F ∼ 2.5 mJy . For the FIRST radio sources the errors are smaller: ≤ 0.5 ′′ for sources with flux densities > 3 mJy, and 1 ′′ at the survey treshold of 1 mJy (White et al. 1997).
Identifications
Three hundred fifty three NVSS radio sources were found within boundaries of 179 ShCGs. Fifty three of these sources were registered also in the FIRST. Seven more sources were found only in the FIRST. The more correct positions and fluxes on the latter radio sources from the FIRST were used in the farther discussion.
High positional accuracies of the used radio surveys allowed to identify with high confidence some of the found radio sources with certain galaxies in dense environments of ShCGs. The positions of these radio sources coincide appreciably well, generally within 2 ′′ − 3 ′′ , with corresponding objects in ShCGs.
The probability of the reality of radio identifications was estimated by the "Likelihood Ratio" (LR) (de Ruiter et al. 1977). It was assumed that the density of background radio sources is ρ = 5.16 × 10 −4 per arcsec 2 for high galactic latitudes (Cohen et al. 1977) The identification was considered as reliable if LR > 2. This may not be very correct in this case, since we identify radio sources in dense groups of galaxies. For this reason we inspected each identification on the DSS images.
Ninety three of the found 353 radio sources were identified with certain objects in 74 ShCGs. The list of identifications is presented in Table 1. The Shakhbazian designation of the group and the number of the galaxy (as labelled in the original ShCG finding charts), considered to be the radio emitter, is given in the first row of column 1. The RA and Dec (J2000.0) of the galaxy are given in columns 2 and 3 of the first row. In the second row the designation of the identified radio source (column 1), its coordinates (column 2 and 3), and the flux density (column 4) are given.
In five cases (ShCG 054,163,298,347,and 352) the NVSS radio source is located between two nearby galaxies, and it was not possible to determine which of them is the radio emitter. It could not be excluded, however, that both galaxies are radio emitters.
Two or more radio emitting galaxies were found in thirteen ShCGs.
Radio sources found within boundaries of nine ShCGs: 051,057,120,203,248,250,330,346,and 352, coincide appreciably well with the positions of relatively weak objects, not considered as member galaxies of corresponding groups in the original ShCG lists. These identifications should be considered as tentative. Though the mentioned objects could, however, be members of corresponding groups. Future spectral observations of these weak objects may clarify whether they are really members of the corresponding groups.
Two hundred sixty radio sources detected within the boundaries of 99 ShCGs were not identified with any visible object. They may be just background radio sources.
Remarks on identifications
ShCG 001.01. The central brightest galaxy of S0 type. There is an emission line at 3727Å in the spectra of this galaxy (Robinson, & Wampler 1973).
ShCG 003.01. The central brightest galaxy. The group is known also as VV 153.
ShCG 009.07. The galaxy is located at the edge of the group.
ShCG 011.04. One of the brightest galaxies.
ShCG 016.01. The brightest galaxy, spiral. As a typical radio galaxy it has two radio emitting lobes on two sides of the galaxy, and a weaker component coinciding with the galaxy itself (Fig. 1a). Radio lobes are at a projected distance of about 10 kpc from the galaxy. The redshift of the group, known also as I Zw 167 and Arp 330, is 0.02913 (Amirkhanian 1989).
The FIR source in TMTST was tentatively identified with galaxy No. 3, which according to Stoll et al. (1996) is an HII region. The consideration of Fig. 2 in TMTST shows that both galaxies, No. 3 and also No. 1 with radio emission, may be FIR emitters. FIR observations with better angular resolution may clarify the situation.
ShCG 018.02. One of the bright galaxies.
ShCG 023.02. One of the bright galaxies. ShCG 041.01. The brightest galaxy of the group.
ShCG 042.11. A weak galaxy, membership to the group should be proved spectroscopically. The brightest galaxy No. 2, of possibly S0 type, is located nearby.
ShCG 051.01. The brightest galaxy, located at the periphery of the group.
ShCG 051.Anon. A weak object that could possibly be a member of the group. It is not excluded, however, that the radio source may be just a projected one.
ShCG 053.01. The brightest galaxy of the group, that is known also as Abell cluster A1050.
ShCG 053.04. One of the bright galaxies.
ShCG 054.06. One of galaxies of the group. Its membership to the group should be proved by spectral observations. The group is known also as Abell cluster A1067.
ShCG 062.01. The brightest galaxy, located at the periphery of the group. More stronger radio source FIRST J112552.1+382153 is located very close to the first one. It may, probably be ejection from the galaxy ShCG 062.01, or a projected source.
ShCG 065.07. A galaxy located at the periphery of the group, known also as Abell 1284. The FIR source found here is the galaxy No. 27 in the southern part of the group (TMTST).
ShCG 074.08. Relatively weak galaxy, that is very close to the brighter galaxy No. 3.
The FIR source was identified with galaxy No. 9 (TMTST). The center of the FIR source is located almost between galaxies No. 8 and 9. Hence both galaxies may be FIR emitters.
ShCG 104.03. Galaxy is located at about 10 ′′ to the south from the brightest galaxy No. 2. The latter was identified as the FIR source (TMTST). It is not excluded, however, that just galaxy No. 3 may really be a FIR emitter.
ShCG 120.Anon. The central brightest radio source FIRST J110431.2+355157 coincides with a weak object between galaxies No. 4 and 6, located at the periphery of the group. Two weaker radio sources FIRST J110432.6+355212 and FIRST J110433.1+355222 seem to be radio lobes ejected from the central object (Fig. 1c). The group is known also as a cluster A1151.
The belonging of the object to the group should be checked by spectral observations. The FIR emitter, located just here, was considered as an uncertain identification in MTST. Apparently the same object may really be a FIR emitter.
ShCG 149.01. One of the brightest galaxies in the central region of the group.
ShCG 163.01/03. Brightest galaxies (probably interacting) in the center of the group.
ShCG 166.02. One of the bright galaxies of the group, known also as a cluster A2247.
ShCG 168.06. One of the bright galaxies of the group. The FIR source was identified with the brightest galaxy No. 1 (TMTST). ShCG 203.Anon. A pair of relatively strong radio sources is identified with very weak object in the north part of the group (Fig. 1d). The pair was not considered as a member of the group in the original ShCG lists. Spectral observations are needed to clarify the case.
ShCG 205.03. One of the brightest galaxies.
ShCG 209.01. The brightest galaxy located in the center of the group.
ShCG 219.01. Radio source FIRST J145233.5+275751 is identified with the brightest galaxy. It seem to be in interaction with galaxy No. 2. Galaxy No. 1 has halo or spiral arms. The group is known also as a cluster A1984.
Two other radio sources, FIRST J145231.6+275807 and FIRST J145234.2+275751, seem to be ejected from galaxy No. 1 (Fig. 1e). This galaxy and the galaxy No. 1 are in interaction.
ShCG 234.04. Relatively weak galaxy, located in the central part of the group. Its membership to the group should be proved by spectral observations. ShCG 248.04. The the brightest galaxy of the group. In TMTST the FIR source was identified with the same galaxy.
ShCG 248.Anon. Relatively bright galaxy located at about 1 ′ to the west from the group. It may be a member of the same group. The consideration of the isophotes of the FIR source (TMTST) shows that both this galaxy and the galaxy No. 4 of the group may be FIR emitters.
ShCG 250.Anon. Weak object that could possibly be a member of the group. It is not excluded, however, that the radio source may be just a projected one to this group. ShCG 320.09. One the bright galaxies, 17. m 88.
ShCG 330.Anon. A weak object that could possibly be a member of the group. It is not excluded, however, that the radio source may be just a projected one.
ShCG 339.01. One of the three brightest galaxies.
ShCG 344.07. Galaxy of Sab type. Seems to be in interaction with another, relatively weak galaxy.
The FIR source was identified in TMTST with the brightest galaxy No. 1 of S0 type. The reconsideration of the FIR isophotes here with taking into account the errors of the IRAS positional measurements in this case (40 ′′ × 23 ′′ ) allows to suggest that galaxy No. 7 may also be a FIR emitter.
ShCG 346.Anon. A weak object, that could possibly be a member of the group. It is not excluded, however, that the radio source may be just a projected one.
ShCG 347.01. The brightest galaxy, located at the end of the elongated group.
ShCG 347.03/04. The radio source is located between galaxies No. 3 and 4, and could be identified with one of them.
ShCG 348.02. One of two brightest galaxies in the group.
ShCG 351.06. The dominant spiral galaxy (UGC 06212). It is located at the periphery of the group. In TMTST the FIR source was identified with the same galaxy.
ShCG 352.Anon. The position of the radio source coincides well with the weak object at about 17 ′′ to the S-W from the brightest galaxy No. 1.
ShCG 360.01. The brightest galaxy of S0 type. This very compact group, is known also as the cluster A2113.
ShCG 362.01/04. The radio source is located at about 12 ′′ south of galaxy No.4, and could be identified with it, or with the brightest galaxy No. 1, located nearby. These two galaxies seems to be interacting. The group is known also as III Zw 108.
ShCG 371.02. The group is very dense. The radio source is identified with one of the three central bright galaxies of the group, Nos. 2, 3 or 4. The latter is the brightest in the group. In TMTST the FIR source was tentatively identified with galaxy No. 2. However, the optical spectrum of these galaxy is a normal one with absorption lines, while the spectrum of galaxy No. 4 has emission lines. (The results of the spectral observations of this group will be published elsewhere). On the basis of spectral data we assume that galaxy No. 4 may also be a FIR emitter.
ShCG 372.03. One of the bright galaxies.
ShCG 376.04. The dominant galaxy. The FIR source was identified with the same galaxy (TMTST). Spectral observations of this group (the results of which will be published elsewhere) show that this galaxy, and also some others in the group, have emission lines.
3.3. The optical rank of the radio emitting galaxy A radio emission, as we already mentioned, is most often observed from the optically first-ranked galaxies or other brightest members of groups of galaxies (Tovmassian & Shakhbazian 1981, Menon 1992. We identified most of radio sources detected in ShCGs (64) with the brightest or one of the bright galaxies in corresponding groups. Only in nineteen cases the identified objects are not the brightest members. If the found regularity (Tovmassian & Shakhbazian 1981, Menon 1992 holds in the case of ShCGs as well, then some of the latter nineteen identifications with weaker members of groups may not be correct. The membership of these objects to the corresponding groups should be checked by spectral observations.
The structure of radio sources
Due to large distances of ShCGs from us the angular sizes of member galaxies are generally small enough. For this reason the angular resolution of the FIRST (5 ′′ ) and, especially of the NVSS (45 ′′ ) does not allow in most cases to distinguish whether the observed radio radiation is emitted from the nuclear region of the galaxy, or from its disk. Hence we assumed that the measured flux refers to the disc.
However, in some cases the high angular resolution of the FIRST Survey allowed, to resolve detected radio sources. Radio sources, identified with ShCG 016.01, 021.01, 219.02, and 317.01 have composite structure (Fig. 1). Some of them consist of two lobes located diametrically in two sides of the parent galaxy, that is characteristic to classical radio galaxies. In the case of ShCG 016.01 and 219.01 the radio sources are of FR II type. Radio sources identified with ShCG 120.Anon and 203.Anon also seem to consist of two components. In ShCG 016.01, 120.Anon, and 317.01 the radio emission of the central galaxy itself is also observed. The radio source in ShCG 317 is a very complex one (Fig. 1).
Radio spectra
Radio sources detected within ShCG areas were cross-identified with sources of the CATS (astrophysical CATalogs Support system) database (Verkhodanov et al. 1997), that unifies 200 radio astronomical catalogs, including the Texas catalog at 365 MHz (Douglas et al. 1996), 6C at 151 MHz (Baldwin et el. 1985), high sensitivity WENS at 327 MHz (Rengelink et al., 1997), and others. We used the task match in the identification circle of a 90 ′′ radius. We found that twenty two of the detected radio sources have been observed at least at two frequencies. The spectra of these radio sources are presented in Fig. 2. The spectral indices of these sources were determined. For determination of spectral indices we used the least square methods to fit the obtained data sample for construction of the spectra.
The spectral indices are presented in Tab. 2. Most of the spectra have normal slopes.
The radio source, identified with ShCG 248.04 have very unusual, too steep spectrum. It is possible that the flux density of this source at 365 MHz is overestimated. The spectra of three sources, ShCG 041.01, 051.04 and 163.01/03 are inverted.
Radio luminosities
Redshifts of only a handful ShCGs have been measured until recently (Robinson & Wampler 1973;Arp et al. 1973;Mirzoyan et al. 1975;Amirkhanian & Egikian 1987;Amirkhanian 1989;Kodaira et al. 1988Kodaira et al. , 1990Kodaira & Sekiguchi 1991;Lynds et al. 1990;del Olmo & Moles 1991). The redshifts of only 37 ShCGs with detected radio sources are known. Most of these redshifts are yet unpublished (Tiersch et al., 1999). We derived the radio luminosities of these sources at 1.4 GHz by assuming H = 50 km s −1 Mpc −1 and q 0 = 0.5. The mean redshift of the group members, if available, was used in calculations. The derived radio luminosities are presented in Table 3.
We compared radio luminosities of galaxies in ShCGs with that of in HCGs (Fig. 3). For drawing Fig. 3 we used total fluxes of 56 HCG spiral galaxies (Menon 1995) at 1415 MHz, and also 34 more HCG galaxies identified with the NVSS and FIRST radio sources by us. The list of the latter galaxies is presented in Table 4.
The consideration of Fig. 3 shows that radio sources in ShCGs are more powerful. Indeed, the radio luminosities of more than half of HCGs located at redshifts < 0.05 are less than 22.0 W Hz −1 , while only one out of eleven ShCGs at the same distances has such low radio luminosity. It is seen also that most of powerful ShCG radio sources are located at larger distances, where no HCGs were found.
The comparison of the radio and FIR emitting properties of ShCGs and HCGs
Since redshifts are known for only a limited sample of ShCGs, it is not yet possible to study the radio luminosity function of ShCG galaxies. Such study may be done after compilition of the program initieted by Tiersch et al. (1999) on the spectral study of ShCGs. The available data allow, anyhow, to compare the radio and FIR emission of HCGs and ShCGs.
For comparison of the radio and FIR emission abilities of ShCGs and HCGs we draw the graph log F 60 -log F 1.4 (Fig. 4). The 60 µm IRAS band was by far the most sensitive for the detection of extragalactic objects (see, for example, 1989, 1993).
For star forming galaxies a very close connection between two apparently unrelated physical mechanisms, the thermal emission from a dust and the synchrotron radio emission from relitivistic electrons, was found (Dickey & Salpeter 1984, Helou, Sofier & Rowan-Robinson 1985, Hickson et al. 1989, Helou & Bicay (1993. It was shown that the ratio of the FIR and radio fluxes of starburst galaxies is almost constant.
If the ratio of the FIR and radio fluxes of galaxies in the considered CGs is also constant, as it is in the star forming galaxies, then due to different distances from us, and also due to differences in the emitted fluxes, the CG galaxies should be distributed on Fig. 4 along a diagonal line.
For drawing Fig. 4 we used: -thirty four spiral HCG galaxies from Menon's list (1995), and also twelve HCG galaxies from Tab. 4 (this paper), the FIR emission of which at 60 µm was mesured by Allam et al. (1996). Nine galaxies from Table 4 also are spirals.
The high accuracies of radio positional measurements allowed to identify with high confidence the detected radio source with a certain galaxy in the corresponding CG. The situation is not the same in the case of the IRAS FIR observations. Absolute positions provided by the IRAS are accurate up to 6 ′′ (within ±3σ) in the in-scan and ∼ 25 ′′ in the cross-scan directions respectively. In the case of HCGs, that are nearer to us and have relatively larger angular dimensions in comparison with ShCGs, in most cases (37 out of 47) it was possible to identify the detected FIR source with a certain galaxy in the corresponding group (Allam et al. 1996). The FIR sources detected in ShCGs (TMTST) are in general weaker than those in HCGs. As a result their positional measurement accuracies reach usually values of about 10 ′′ to 30 ′′ in the in-scan and the cross-scan directions respectively. For this reason, and also for the relatively smaller angular sizes of ShCGs it was not possible to determine with certainty which galaxy in a dense group is the FIR emitter (TMTST). As a probable one the brightest member of the group was usually mentioned. For construction of Fig. 4 we attributed the measured FIR flux eather to a single galaxy or to two nearby galaxies (see Subsection 3.2). This uncertainty does not however influence the made conclusions.
The consideration of Fig. 4 shows that most of HCG galaxies (30 out 47, i.e. ∼ 64%) are located along and somewhat lower of the arbitrarily drawn diagonal dashed line. These galaxies thus obey the correlation between the thermal emission from a dust and the synchrotron radio emission from relitivistic electrons found for star forming galaxies, and hence they also are SB galaxies. Galaxies that are lower of the dashed line apparently have relatively stronger FIR emission. Along the same dashed line are distributed, as it was expected, also most of Markarian SB galaxies.
Seventeen HCG galaxies (∼ 36%) are higher of the dashed line. It means that they have stronger radio emission than SB galaxies. It is remarkable that two of these galaxies, HCG 92c and HCG 96a, are Seyferts, and three others, HCG 56b, HCG 68a and HCG 68b, are E and S0 type galaxies.
The situation is different for ShCG galaxies. Nine of them (82%) are located above the arbitrarily drawn diagonal line. Above this line are located also twelve out of thirteen (∼ 92%) Markarian-Seyfert galaxies. Hence most of ShCG galaxies are not SB galaxies. If the identification of the FIR source with a radio emitting galaxy is not correct then the galaxy with detected radio emission and thus with smaller FIR emission would move on Fig. 4 to the left and hence would be located even higher of the dashed line. The blending of a few probable FIR sources in dense groups would have the same effect.
It is worth to note that those HCG and ShCG galaxies for which either FIR or radio emission was detected would also have different locations on the logF 60 -logF 1.4 graph.
There are 73 ShCG galaxies, fluxes of which at 1.4 GHz exceed 1 Jy, but only upper limits of fluxes at 60 µm were determined (TMTST). These galaxies would apparently be located on Fig. 4 higher of the dashed line. At the same time there are only fifteen ShCG galaxies with determined FIR fluxes (TMTST), and radio fluxes lower than 1 Jy detection limit (this paper). The latter would be located lower of the dashed line. In the case of HCG galaxies the situation is vice versa. There are 34 HCG galaxies with 1.4 GHz fluxes exceeding the 1 mJy limit (Menon 1995, and present paper), and the FIR fluxes lower than the limiting value (Allam et al. 1996). These galaxies would be located on Fig. 4 upper of the dashed line. Much more HCG galaxies, 65, with measured FIR fluxes and upper limits of radio fluxes, would be located below the dashed line, in the lower part of Fig. 4. Thus in the case of galaxies with eather one of the fluxes (eather 60 µm or 1.4 GHz) measured, we see the same trend: most ShCG galaxies would be located upper of the dashed line on Fig. 4. Meanwhile HCG galaxies would be located mainly below this line. Sulentic & De Mello Rabaca (1993) claimed that the FIR sources in HCGs are likely the combined contribution of two or more members. If to assume that in all Hickson's groups with detected FIR emission, the FIR emitters are in reality two galaxies with equal fluxes, then the corresponding points on Fig. 4 should move to the left by 0.3, and still they would be located lower than ShCG galaxies. Moreover, if the same is valid also for the more dense ShCGs, then the corresponding positions of ShCG galaxies on Fig. 4 should also move to the left, and the found trend would certainly not be altered.
Hence, one may conclude that galaxies in ShCGs are relatively stronger radio emitters, while HCG galaxies are stronger FIR emitters. It means that physical conditions in ShCGs are somehow favorable for triggering the AGNs with relatively strong synchrotron emission of relativistic electrons, while in HCGs the conditions are favorable for formation of SB galaxies with relatively strong thermal dust emission. What could be the reason for such difference?
CGs are generally very dense formations. According to N-body simulations (Carnevali et al. 1981, Barnes 1985, 1989, Mamon 1990, Zheng et al. 1993) the member galaxies at high density environments of CGs should interact and merge in one large galaxy in about 10 8 years. For this reason the very existence of CGs has been questioned by some authors (Walke & Mamon 1989, Mamon 1986, Hernquist et al. 1995. Meanwhile Hickson & Rood (1988), Mendes de Oliveira & Giraud (1994), Mendes de Oliveira (1995), Oleak et al. (1995) and recently Tovmassian et al. (1998c) presented firm evidences on the reality of CGs. For explaining the existence of CGs Governato et al. (1996) proposed a second generation merger scenario, according to which CGs permanently aggregate new members from their surrounding areas. Such scenario could be valid since, according to Rood & Williams (1989), Vennik et al. (1993 and Ramella et al. (1994), most of HCGs are associated with loose groups of galaxies.
ShCGs and HCGs differ from each other by the number of members and by morphological content. If HCGs contain generally four-five members, ShCGs have up to fifteen members. The relative number of E or S0 type galaxies in HCGs compose only ∼ 50% of all members (Hickson et al. 1989), while ShGGs are more rich in early type galaxies. About 77% member galaxies in the latter are of E and S0 type (Tiersch et al. 1996a). For this reason ShCGs are considered as more evolved systems in comparison with HCGs.
Since almost half of HCG galaxies are spirals with sufficient amount of gas and dust, the gravitational interaction between them and with a suggested newcomer galaxy may trigger starburst processes in interacting galaxies. The FIR emission is characteristic to such events. Meanwhile in the more evolved ShCGs most galaxies are of E and S0 type, and the number of spirals is relatively small. The gas was pushed out from them during previous interaction processes between galaxies, and filled the intergalactic space. The spirals here should have shed also their dust content. The presence of intergalactic gas in CGs was proved by the detection of diffuse X-ray emission (Ebeling et al. 1994, Pildis et al. 1995, Saracco & Ciliegi 1995, Tiersch et al. 1996b) from some of the groups. Due to the interaction with a newcomer galaxy to the group this intergalactic gas may be falling in the form of cooling flows directly into the center of the preferentially dominant early type galaxy in the group. In the result of this an active nucleus of the galaxy with sufficiently strong radio emission may be formed. Due to small amount or even absence of dust in such galaxies their FIR emission would be very weak or even completely absent.
CONCLUSIONS
Three hundred fifty three NVSS ) radio sources are found within boundaries of 179 ShCGs. Sixty sources were registered also in the FIRST (White et al. 1997) survey, of which seven sources -only in the FIRST.
Ninety three of the found radio sources are identified with corresponding galaxies in 74 out of 366 ShCGs.
Radio spectra of 22 radio sources are constructed.
It is shown that ShCG galaxies have in general higher radio luminosities than galaxies in HCGs.
The comparison of the radio and FIR fluxes of galaxies in HCGs and ShCGs showed that the latter are more stronger radio emitters, while HCG galaxies are generally more stronger FIR emitters. It is suggested that the reason for this may be that ShCGs are more evolved in comparison with HCGs, and galaxies in them do not have enough dust for attributing the FIR emission. On the other hand the conditions in ShCGs are more favorable for formation of AGN with relatively stronger radio emission. , & Longo, G. 1993, Astron. Nachr., 314, 393 Verkhodanov, O.V., Trushkin S.A., Andernach H., Chernenkov, V.N. 1997The CATS database to operate with astrophysical catalogs", in "Astronomical Data with astrophysical catalogs. In "Astronomical Data Analysis Software and Systems VI", Editors: Gareth Hunt and H. E. Payne, ASP Conference Series, V. 125, P.322-325. | 2014-10-01T00:00:00.000Z | 1999-05-01T00:00:00.000 | {
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221112873 | pes2o/s2orc | v3-fos-license | Effect of Mach number on droplet aerobreakup in shear stripping regime
Abstract The present experimental study investigates the shear stripping breakup of single droplets in subsonic and supersonic gaseous flows. In contrast to most research that places emphasis on the Weber number (We), we focus on the individual effects exerted by flow Mach (M∞) and Reynolds numbers (Re). Millimeter-sized droplets made of either ethylene glycol or water are exposed to shock-induced flows. Shadowgraph and schlieren images of the breakup process are recorded by an ultra-high-speed camera. The experimental We is constrained at 1100, while M∞ is varied from 0.3 to 1.19 and Re from 2600 to 24,000. A systematic analysis of the experiment series reveals that the breakup pattern alters with M∞ although a constant We is maintained. The classical stripping behavior with fine mist shed from the peripheral sheet changes to rupture of multiple bags along the periphery at M∞ = 0.63, and further to stretching of ligament structures from the leeward surface at M∞ = 1.19. The corresponding breakup initiation is delayed and the resultant fragments are sized less uniformly and distributed over a narrower spread. In terms of the early-stage deformation, droplets experience less intense flattening and slower sheet growth at higher M∞. The change of Re introduces additional variations, but only to a minor extent. Graphical abstract
Introduction
Droplet breakup, also termed secondary atomization, refers to the fragmentation of a droplet subjected to aerodynamic forces. This phenomenon is relevant in diverse applications, such as fuel injection (Reitz and Diwakar 1986), spray coatings (Mostaghimi et al. 2002) and metal powder production (Lagutkin et al. 2004). It has been widely recognized that the breakup morphology is primarily determined by the Weber number (We) and the Ohnesorge number (Oh) (Lane 1951;Hinze 1955): where ρ g and u g are the density and the velocity of the gas flow, and d 0 , σ, μ d and ρ d are the initial diameter, the surface tension, the dynamic viscosity and the density of the liquid droplet, respectively. The Weber number represents the ratio between the disruptive aerodynamic force and the restorative surface tension, and the Ohnesorge number compares (1) We = g u 2 g d 0 ∕ (2) the viscous force to the surface tension. According to Guildenbecher et al. (2009), the influence of liquid viscosity on the breakup regime diminishes when Oh drops below 0.1, leaving We as the dominant factor. Several breakup mechanisms have been identified in the literature and are classified as bag breakup at 11 < We < 35 and stripping breakup at We > 80, with so-called multimode breakup in the intermediate range (Hsiang and Faeth 1995;Schmehl 2003). The bag breakup is conventionally understood as a result of the Rayleigh-Taylor instability developed at the droplet front (Joseph et al. 1999). However, some studies suggest different physical mechanisms, including the pressure imbalance between the front and rear side (Opfer et al. 2014), the stress repartition around the surface (Villermaux and Bossa 2009) and the structure of flow vortices in the wake (Inamura et al. 2009). The cause of the stripping breakup is also under debate. No agreement has been achieved on whether the viscous shear or the aerodynamic drag is the driving force. Correspondingly, the name of this regime varies among shear stripping (Ranger and Nicholls 1969), sheet thinning (Liu and Reitz 1997) and shear-induced entrainment (Theofanous and Li 2008). For the current work, we focus on the stripping breakup and adopt the concept proposed by Theofanous and Li (2008).
Although the breakup mechanism is mainly governed by the Weber number, other non-dimensional parameters influence the breakup behavior as well. Chou et al. (1997) conduct experiments with Ohnesorge numbers below 0.1, and observe larger micro-drops generated at higher Oh. Pilch and Erdman (1987) analyze the effect of Oh on the breakup time and conclude a consistent postponement of the breakup initiation as Oh increases. Lee and Reitz (2000) experimentally show that the liquid-gas density ratio (ε = ρ d /ρ g ) exerts negligible effects on the breakup process at values higher than 100. In numerical simulations by Kékesi et al. (2014); however, new breakup patterns appear for density ratios below 100. In terms of the flow Reynolds number (Re = ρ g u g d 0 /µ g ), the work of Liu and Reitz (1997) indicates that the breakup behavior is independent of Re when Re > 500. The dependence becomes important only in the Stokes flow (Aalburg et al. 2003) and in liquid-liquid breakup systems (Hsiang and Faeth 1995).
Another non-dimensional parameter, which is of significance but not fully explored, is the flow Mach number M ∞ . Most of preceding experiments are conducted at subsonic conditions, where the effect of the flow compressibility is marginal. However, with the recent development of supersonic combustion systems including pulse detonation engines (Kailasanath 2003), scramjet engines (Curran 2001) and supersonic gas atomizers (Anderson et al. 1991), droplet breakup in high-speed flows becomes of increasing importance. Dinh et al. (2003) and Theofanous et al. (2004) investigate various breakup regimes in a highly rarified flow at M ∞ = 3. They find that the morphologies differ significantly from those categorized in subsonic flows and attribute the differences to changes in pressure fields. Ortiz et al. (2004) measure the drag coefficients of droplets in different airstreams and observe a rapid increase as the flow Mach number approaches supersonic conditions. Xiao et al. (2017) numerically simulate the deformation of droplets exposed to supersonic flows and conclude that the onset of breakup is postponed compared to subsonic cases. Igra and Takayama (2003) and Meng and Colonius (2015) conduct experimental and numerical research on water column breakup in highspeed flows, respectively. Both works quantitatively show a slower temporal increase of the cross-stream diameter at higher M ∞ .
Although the abovementioned research reveals distinct breakup features in supersonic flows, the experimental database addressing the effect of M ∞ is rather limited. Moreover, a change of M ∞ in experiments is commonly accompanied with a change of Re, which renders the independent investigation of M ∞ difficult. In the present work, we constrain the Weber number at 1100 and decouple the correlation between M ∞ and Re by applying different liquid-gas combinations and carefully choosing operating conditions. This creates a test matrix which allows us to study the effects of M ∞ and Re individually.
Experimental setup
The layout of the shock tube used for the current experiments is depicted in Fig. 1. The tube, which has an overall length of 24 m and an inner diameter of 290 mm, consists of three segments: the driver, the driven and the test sections. A cookie cutter is installed in front of the test section to remove the boundary flows and to contract the cross section to a 190 × 190 mm 2 square. In the experiments, a 0.15 mm-thick Mylar diaphragm is placed between the driver and driven sections and in contact with a pair of 0.1 mm-thick crossed NiCr heating wires. Each section of the shock tube is first filled to pressure levels corresponding to desired flow conditions. Then, a single droplet is produced in the test section by expelling liquids through a hypodermic needle with an outer diameter of 0.9 mm. The droplet falls through a pair of aligned laser emitter and receiver. This triggers the rupture of the Mylar diaphragm by supplying an electric current of 3 A to the heating wires. Subsequently, a planar shock wave The pressure variation along the shock tube is measured by flush-mounted PCB Piezotronics ICP ® fast-response pressure sensors. The measured signals are acquired by a LTT transient data recorder at a sampling frequency of 1 MHz. We calculate the shock velocity based on the time lag between moments when the incident shock passes two pressure sensors ahead of the test section. The combination of the shock velocity and initial pre-shock conditions yields post-shock flow properties based on moving shock relations. Another sensor in the test section measures the pressure rise across the incident shock. A representative pressure signal normalized against the theoretical post-shock pressure is provided in Fig. 2. The shock-induced freestream remains steady over 1.6 ms which well covers the investigated period of droplet breakup (maximally 1.4 ms). The slight decline in the pressure signal is attributed to the growth of the boundary layer as well as the nature of the piezoelectric sensors. This pressure signal also serves as a trigger for the image recording.
As for the flow visualization, a Shimadzu HyperVision HPV-X ultra-high-speed camera is integrated in a Z-type shadowgraph/schlieren photography system. The camera records 128 consecutive images with a resolution of 0.087 mm/pixel at a framing rate of 100 kfps.
For the present experiments, the combinations of two liquids (ethylene glycol and water) and two gases (air and CO 2 ) are exploited to analyze the effects of M ∞ and Re independently at a constant We. The Weber number, which is constrained at 1100 with a standard deviation of 50, lies within the stripping breakup regime and allows a wide variation of M ∞ and Re. Figure 3 shows the inversely proportional correlation between M ∞ and Re for different liquid-gas combinations. The eight labelled points represent the operating conditions selected for current experiments, and the associated error bars (magnified twice in the plot) stand for the ranges of M ∞ and Re from repeated experiments. The eight operating conditions are numbered as i·j, where the values of i and j correspond to the relative magnitudes of M ∞ and Re, respectively. Detailed parameters averaged from repeated experiments at each operating condition are summarized in Table 1.
For water (ρ d = 998 kg/m 3 , σ = 7.28e-2 N/m, μ d = 8.9e-4 kg/m s), the average droplet diameter is 3.1 mm, resulting in an Oh of 0.002. For ethylene glycol (ρ d = 1113 kg/m 3 , σ = 4.73e-2 N/m, μ d = 1.61e-2 kg/m s), the lower surface tension leads to smaller droplets with an average diameter of 2.6 mm and the considerably higher viscosity yields an Oh of 0.043. Concerning all the experiments, the freestream Mach number M ∞ varies from subsonic (0.3) to supersonic (1.19) levels and the droplet diameter-based flow Reynolds number Re ranges over an order of magnitude (from 2.6e3 to 2.4e4). The liquid-gas density and viscosity ratios are also provided in Table 1
Results and discussion
A brief overview of the typical stripping breakup process in subsonic flows (Case 1.4, M ∞ = 0.3, liquid: ethylene glycol, gas: air) is provided in Fig. 4. Here, the experimental time t is regarded as zero at the moment when the incident shock impacts on the droplet. Furthermore, t is normalized against the characteristic transport time derived by Ranger and Nicholls (1969) based on droplet deformation in incompressible flows, yielding the dimensionless time T as Fig. 2 Step-wise pressure rise across the incident shock measured in the test section The time scaling in Eq. (3) does not account for the compressibility effects, which govern the droplet breakup in the current study. Nevertheless, this scaling is used to present the results in a consistent and comparable way with previous literature. Presented images are processed with subtraction of the background noise, contrast stretching and super resolution using MATLAB's Very Deep Super-Resolution convolutional neural network (Kim et al. 2016).
The entire breakup process is divided into four stages. It starts with the shock-droplet interaction which establishes a flow field resembling that around a solid sphere (T < 0.1). Then, the droplet is flattened along the streamwise direction (T = 0.2) and a liquid sheet emerges at the equator (T = 0.3). The rupture of the sheet indicates the breakup initiation (T = 0.4) and the coherent body is continuously eroded at the periphery afterwards. The last stage is achieved when the whole droplet disintegrates into fragments distributed widely in the flow field (out of the time window shown in Fig. 4). Figure 4 also presents the quantitative change of the droplet cross-stream diameter d c . The error bar represents the uncertainty (90% confidence level based on the Student's t-distribution) calculated from four repeated experiments. Before the onset of breakup (T < 0.37), the increase of d c indicates the flattening of the intact body and the associated uncertainty is low. Once the breakup starts, the intact body is shadowed by the fine mist. The interpretation of d c changes to a description of the spatial distribution of liquid fragments. The corresponding uncertainty significantly increases as micro-drops are more sensitive to local flow disturbance than the coherent body. Considering the uncertainty levels are similar for all cases, the error bars are omitted in the following plots for the sake of brevity.
In following sections, we compare cases with variations in flow Mach and Reynolds numbers with respect to (2001) and Meng and Colonius (2015). The main change at M ∞ = 0.63 is that the separation zone behind the droplet extends over a wider region. As the freestream speed enters the transonic range (M ∞ = 0.83), a normal shock (NS) appears behind the droplet and fluctuates slightly around the displayed location. This suggests that the surrounding flow accelerates to supersonic conditions as it bypasses the droplet. For the case at M ∞ = 1.19, an oblique shock cone (OS) appears, stretching downstream over a broad region. Meanwhile, the reflected shock ahead of the droplet settles as a detached bow shock.
Wave dynamics
Considering the droplet does not undergo noticeable deformation during the short period of shock-droplet interaction, the surrounding pressure field is expected to resemble that around a solid sphere, which is also implied by the wave patterns presented in Fig. 5. As M ∞ changes, the pressure imposed on the spherical surface differs significantly (Charters and Thomas 1945;Bailey and Haitt 1972). These differences are held accountable for the more pronounced distinctions in succeeding deformation and breakup processes (Hanson et al. 1963).
The travelling velocity of the reflected shock is plotted in Fig. 6 with respect to the position relative to the droplet front. The distance s between the reflected shock and the droplet leading edge is normalized by the droplet diameter d 0 . The Mach number of the reflected shock relative to the freestream flow is calculated as M r = (ds/dt + u g )/a, where a is the speed of sound in the freestream. For cases with M ∞ < 1, the reflected shock decays to sonic waves, and the corresponding M r falls towards 1. For the supersonic case with M ∞ = 1.19, however, the decreasing M r settles at M ∞ and the normalized distance s/d 0 is stabilized around 0.65 which matches the value measured for a solid sphere at similar conditions (Liepmann and Roshko 2001).
Early-stage deformation
The early-stage droplet deformation for subsonic and supersonic cases is compared in Fig. 7. As described in Fig. 4, the droplet at M ∞ = 0.3 exhibits typical deformation features such as flattening at frontal and rear surfaces and stretching of the liquid sheet around the equator. Generally, droplets at higher-M ∞ follow similar deformation patterns, but a thorough examination reveals noticeable distinctions that are summarized as follows: • the liquid sheet emerges further downstream in the supersonic flow (T = 0.20); • the flattening of the leeward surface becomes weaker as M ∞ increases (T = 0.30); • the liquid sheet grows more rapidly at lower M ∞ (T = 0.48).
As the windward surface of the droplets gets flattened, kinks form around the droplet equator at T = 0.2. These kinks are the origins of the subsequent development of liquid sheets. In subsonic flows, the kink is located ahead of the equatorial plane and the angle of inclination of the line connecting the kink to the droplet center is approximately 80° with respect to the flow direction. However, the position of the kink is shifted considerably downstream in the supersonic flow and the corresponding angle of inclination is 92°. The kink locations are in good accordance with the trajectory of the separation point at the surface of a solid sphere measured by Charters and Thomas (1945). In their work, the separation point stays with an angle of inclination between 70° and 80° at subsonic conditions and drifts downstream beyond 90° at M ∞ = 1.2.
During the period shown in Fig. 7, the leeward surface of the droplet is continuously flattened. The extent of the flattened area at T = 0.3 is estimated relative to the initial droplet diameter. As M ∞ increases from 0.3 to 1.19, the corresponding flattened area shrinks from 0.84d 0 to 0.5d 0 . This could be associated to the change of the pressure imposed on the droplet rear at different flow conditions. Karyagin et al. (1991) experimentally measure the pressure distribution over the surface of a sphere, and observe that the pressure at the rear surface drops consistently as M ∞ increases. The same trend is also reported in the numerical work by Nagata et al. (2016).
By T = 0.48, the liquid sheets for all cases have grown to considerable sizes and stretched out radially from the main body. Similarly to the experimental observation by Theofanous et al. (2012) and the numerical analyses by Jalaal and Mehravaran (2014), the growth of the liquid sheets is enhanced by the emergence of propagative waves at the droplet surface, as shown in Fig. 8. These surface waves are induced by Kelvin-Helmholtz instabilities at the windward surface, where the liquid-gas interface suffers strong shear. The waves are transported towards the equator under drag forces and then merge with the preceding waves. Papamoschou and Roshko (1988) point out that increasing the flow Mach number reduces the growth rate of the shear layer between two streams rapidly, because the associated compressibility effect tends to stabilize the flow disturbance. Moreover, according to Liepmann and Roshko (2001) and Nayfeh and Saric (1971), the pressure distribution around small-scale waves is out of phase with the wave profile in supersonic flows and thus suppresses the development of instabilities. Consequently, the growth rate of the liquid sheet is lower at higher M ∞ . This explains the observation at T = 0.48 in Fig. 7 that the liquid sheet becomes smaller as M ∞ increases. Figure 9 compares the droplet contours at T = 0.4 between Case 3.2, 5.2 and 5.1. The dimensions are normalized against the initial droplet diameter, and the origin represents the position of the initial droplet center. Case 5.2 has the same M ∞ as Case 5.1, and shares a comparable Re with Case 3.2. The resemblance of the droplet contours between Case 5.2 and Case 5.1 indicates that Re exerts negligible effects on the early-stage deformation, while M ∞ plays a critical role in determining the flattening intensity and the sheet development.
To further quantify the droplet deformation, streamwise displacements of the leading edge, the mass center and the trailing edge are measured. These parameters are of particular interest for numerical validations.
The position of the mass center, which is calculated with the assumption that the droplet cross section normal to the flow direction is axisymmetric, is plotted in Fig. 10. For all cases, the trajectory of the droplet mass center approximates to be parabolic over the shown period. The drag coefficient CD is estimated by fitting the data into the relation xmc/d 0 = 3/8CDT2 derived by Ranger and Nicholls (1969). The subsonic case at M ∞ = 0.3 experiences a drastic acceleration around T = 0.25. This results from the rapid growth of the cross-stream diameter, as shown in Fig. 7, and yields a relatively high CD of 1.4. For the other three cases that share comparable cross-stream diameters before T = 0.4, the streamwise drift of the droplet mass center is faster at higher M ∞ . The corresponding drag coefficients are calculated to be 0.9 for M ∞ = 0.63, 1.0 for M ∞ = 0.83 and 1.2 for M ∞ = 1.19. This trend agrees with the drag coefficients for a solid sphere measured by Bailey and Haitt (1972) and Charters and Thomas (1945), but the values are much higher due to the droplet flattening. Figure 11 compares the streamwise displacements of the leading edge and the trailing edge at different flow conditions. It is noteworthy that the difference of the leading edge shift is negligible among all present cases. Therefore, as also stated by Theofanous (2011), the displacement of the leading edge is not a proper parameter to represent the drag. In terms of the trailing edge, the displacement shows certain degrees of variations between cases and becomes smaller at higher M ∞ . This tendency is consistent with the observation
Breakup patterns
Breakup patterns of droplets at different flow conditions are displayed in Fig. 12. The first row represents the individual breakup initiation which is defined as the onset of the formation of liquid fragments. Each of the remaining rows corresponds to a specific time moment. Although the breakup process shows certain degrees of chaotic behavior (Hardalupas and Whitelaw 1994;Engelbert et al. 1995), the features discussed in the following are consistently observed in repeated experiments. Generally speaking, three types of breakup patterns are categorized: The droplet at M ∞ = 0.3 experiences a typical stripping breakup initiated with the fracture of the liquid sheet. Afterwards, the windward surface is continuously flattened and expands over an increasingly broad region. At regions encircling the smooth front, small-scale waves generated by Kelvin-Helmholtz instabilities appear. These waves propagate towards the periphery peeling micro-drops off from the droplet. The micro-drops are entrained in the flow and distributed widely in the cross-stream direction.
The breakup pattern is altered as the flow Mach number increases. At M ∞ = 0.63, the breakup onset is not indicated by entrainment of micro-drops, but by the formation of multiple bags along the periphery. Figure 13 shows the evolution from the bending of the liquid sheet to the inflation of the multiple bags. As the sheet extends downstream, the peripheral region is straightened to directly face the freestream flow (T = 0.54). Then, multiple bags form along the rim and inflate rapidly (T = 0.59). These bags rupture into fine mist and the rings that the bags are attached to disintegrate into larger interconnected fragments as indicated at T = 0.8 in Fig. 12. The remaining coherent body of the droplet deforms into a crescent shape and the succeeding breakup process resembles the typical stripping pattern.
According to Theofanous et al. (2004) and Guildenbecher et al. (2009), the wave number n of the fastest-growing wave induced by Rayleigh-Taylor instabilities at the droplet front can be calculated with the following relation: For Case 2.3 at T = 0.56, d c is 1.53 times of the initial diameter d 0 (see Fig. 15) and the average value of C D is 0.9 (see Fig. 10), which yields n = 11.7. The actual value of n should be higher considering the real-time C D is growing over time. Nevertheless, the calculated wave number agrees well with the observation in Fig. 13 that the straightened edge, where bags develop occupies 1/12.5 of the entire cross-stream diameter. This implies that the Rayleigh-Taylor instability is the underlying reason for the local bag formation. Similar bag structures are also observed in the numerical simulation of diesel jet breakup at We = 1270 by Shinjo and Umemura (2010). For Case 5.1 at M ∞ = 1.19, the droplet breakup is characterized by the generation of long and thin streamwise ligaments in the wake, similarly to the observation by Liu and Reitz (1997). Figure 14 presents the deformation of the liquid sheet which induces the succeeding generation of ligaments. After emerging from the main body, the liquid sheet bends along the flow direction and folds to wrap the droplet rear before the breakup is initiated. The sheet becomes thinner as it stretches and thus is increasingly sensitive to instabilities. According to the study of sheet breakup in coflow by Stapper and Samuelsen (1990), surface instability is mainly caused by the growth of the streamwise vortical waves when the flow velocity is high. As a result, streamwise ligaments connected by thin films are formed (T = 0.69 in Fig. 12). After the films burst into micro-drops, the ligaments are stretched under the viscous shear of the flow and break into larger fragments in the presence of Rayleigh-Plateau instabilities. The subsequent breakup of the droplet continues in the pattern that new ligaments form and fragment. It is occasionally observed that large liquid clumps (T = 1.3 in Fig. 12) detach from the intact body and disintegrate separately. It is noteworthy that recent studies (Jalaal and Mehravaran 2014;Meng and Colonius 2018;Biasiori-Poulanges and El-Rabii 2019;Dorschner et al. 2020) propose the mechanism of transverse azimuthal modulation as an alternative explanation for the generation of streamwise ligaments. It is stated that the growth of Kelvin-Helmholtz instabilities near the droplet periphery triggers Rayleigh-Taylor instabilities in the transverse plane which further lead to the formation of ligaments.
The breakup process of Case 5.2 is also presented in Fig. 12. The droplet exhibits few breakup features in common with the identical-Re Case 3.2, but resembles approximately the same characteristics as the identical-M ∞ Case 5.1. A minor difference between Case 5.2 and Case 5.1 is that the former generates slightly thinner ligaments than the latter. This suggests that M ∞ plays a dominant role in determining the breakup patterns, while Re only affects detailed structures. Figure 15 compares the change of the droplet cross-stream diameter d c between subsonic and supersonic conditions. Before T = 0.25, the droplet deformation is characterized by the flattening of the main body. During this stage only marginal differences are identified among all cases. Afterwards the liquid sheet starts to develop, resulting in a rapid growth of d c . The growth rate is significantly higher for M ∞ = 0.3 than the others, which is in accordance with the observation in Fig. 7. Although the differences between high-M ∞ cases (Case 2.3, 3.2 and 5.1) are comparatively small, there exists a consistent tendency that the cross-stream diameter grows more slowly as M ∞ increases. Once the breakup is initiated (marked by the red points in Fig. 15), d c represents the cross-stream spread of the liquid fragments instead. With the breakup onset as a separating point, the overall d c profile is divided into two power-law stages. The short-duration plateaus ahead of the breakup initiation correspond to the periods when the liquid sheet is bent along the flow direction rather than stretched out radially.
For all cases, the breakup begins when the normalized d c reaches approximately 1.5. The subsonic Case 1.4 at M ∞ = 0.3 experiences the earliest breakup, and the corresponding initiation time T = 0.39 matches the empirical correlation proposed by Pilch and Erdman (1987). The postponement of the breakup initiation as M ∞ increases is a consequence of the change in the breakup pattern as shown earlier. The fragmentation of multiple bags and the disintegration of streamwise ligaments at high M ∞ need significantly longer time than the direct sheet rupture at low M ∞ .
Fragment sizes and spatial distributions
In industrial applications that involve atomization processes, the fragment sizes are of particular significance. For instance, small fragments are desired in fuel injections to achieve efficient evaporation and combustion. Figure 16 compares the droplet fragmentation at T = 2.0 for Case 1.4, 2.3, 3.2 and 5.1. At this stage, the coherent body is difficult to identify as the windward surface is severely eroded. Droplets break into uniformly fine mist at subsonic conditions, while the supersonic flow leads to large discrete particles that are scattered among tiny micro-drops. These large particles are mainly caused by the disintegration of ligament structures.
Apart from the size distribution, the spatial spread of the fragments is also of practical importance, since it determines the likelihood of micro-drops coalescing into larger particles. Figures 17, 18 present the outlines of dispersed fragments for various cases at T = 2.0. The outlines are extracted with MATLAB ® based on the modified Moore-Neighbor tracing algorithm (Gonzalez et al. 2004). The differences among Case 2.3, 4.3 and 5.3 (Fig. 17), which share the same Re, indicate that higher M ∞ leads to a narrower cross-stream spread of the fragments. Such an effect could be related to the fact that the windward surface of the droplet is less flattened but more curved at higher M ∞ (see Figs. 7,12). Correspondingly, the fragments gain lower cross-stream momentum from the gas flow when detaching from the droplet periphery and are hence distributed less widely in the crossstream direction. The comparison between Case 5.1, 5.2 and 5.3 (Fig. 18), which share an identical M ∞ , shows that lowering Re also tends to constrain the spatial distribution of the fragments, but much less effectively than increasing M ∞ . at M ∞ = 1.19). This is consistent with the observation by Stapper et al. (1992) that less viscous liquids sustain shorter ligaments.
Conclusions
The present experimental work compares the stripping breakup of liquid droplets in subsonic and supersonic flows. Millimeter-sized droplets are impacted by a planar shock wave generated in a shock tube. The process of deformation and fragmentation is visualized in a shadow/schlieren system and recorded by an ultra-high-speed camera. Through controlling the shock strength and employing different liquids and gases, the Weber number is maintained around 1100, while the flow Mach number M ∞ varies from 0.3 to 1.19 and the Reynolds number Re from 2600 to 24,000. The effects of changing flow conditions on the breakup process are summarized as follows.
The droplet deformation prior to the breakup, including flattening of windward and leeward surfaces and growth of the liquid sheet at the equator, is weakened by increasing M ∞ . The sheet is initiated further downstream along the droplet surface at higher M ∞ .
In subsonic flows, the stripping breakup starts with the fragmentation of the liquid sheet. As M ∞ increases, distinct breakup features emerge at the breakup initiation, such as multiple bags formed along the periphery and ligament structures stretching in the wake. Correspondingly, the breakup initiation is significantly postponed. In subsonic flows, the breakup generates uniformly fine fragments spreading widely in the cross-stream direction. At higher M ∞ , the fragments are of less uniform sizes and constrained within a narrower region behind the main body. Although decreasing Re tends to have a complementary role to increasing M ∞ , the effect is marginal on main breakup behaviors. Lowering Oh reduces the size of liquid sheets and ligaments and results in earlier breakup initiation.
Although many features discussed in the present study focus on detailed breakup structures, they play crucial roles in determining the final fragmentation pattern. The dependency of fragment sizes and spatial distributions on the flow Mach number is especially important for industrial applications, where the atomization process is constantly optimized to generate widely-spread uniformlysized fragments. | 2020-08-13T10:07:18.944Z | 2020-08-09T00:00:00.000 | {
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2673133 | pes2o/s2orc | v3-fos-license | Commutativity and ideals in algebraic crossed products
We investigate properties of commutative subrings and ideals in non-commutative algebraic crossed products for actions by arbitrary groups. A description of the commutant of the base coefficient subring in the crossed product ring is given. Conditions for commutativity and maximal commutativity of the commutant of the base subring are provided in terms of the action as well as in terms of the intersection of ideals in the crossed product ring with the base subring, specially taking into account both the case of base rings without non-trivial zero-divisors and the case of base rings with non-trivial zero-divisors.
Introduction
The description of commutative subrings and commutative subalgebras and of the ideals in non-commutative rings and algebras are important directions of investigation for any class of non-commutative algebras or rings, because it allows one to relate representation theory, non-commutative properties, graded structures, ideals and subalgebras, homological and other properties of non-commutative algebras to spectral theory, duality, algebraic geometry and topology naturally associated with the commutative subalgebras. In representation theory, for example, one of the keys to the construction and classification of representations is the method of induced representations. The underlying structures behind this method are the semi-direct products or crossed products of rings and algebras by various actions. When a non-commutative ring or algebra is given, one looks for a subring or a subalgebra such that its representations can be studied and classified more easily, and such that the whole ring or algebra can be decomposed as a crossed product of this subring or subalgebra by a suitable action. Then the representations for the subring or subalgebra are extended to representations of the whole ring or algebra using the action and its properties. A description of representations is most tractable for commutative subrings or subalgebras as being, via the spectral theory and duality, directly connected to algebraic geometry, topology or measure theory.
If one has found a way to present a non-commutative ring or algebra as a crossed product of a commutative subring or subalgebra by some action on it of the elements from outside the subring or subalgebra, then it is important to know whether this subring or subalgebra is maximal abelian or, if not, to find a maximal abelian subring or subalgebra containing the given subalgebra, since if the selected subring or subalgebra is not maximal abelian, then the action will not be entirely responsible for the non-commutative part as one would hope, but will also have the commutative trivial part taking care of the elements commuting with everything in the selected commutative subring or subalgebra. This maximality of a commutative subring or subalgebra and associated properties of the action are intimately related to the description and classifications of representations of the non-commutative ring or algebra.
Little is known in general about connections between properties of the commutative subalgebras of crossed product rings and algebras and properties of the action. A remarkable result in this direction is known, however, in the context of crossed product C * -algebras. When the algebra is described as the crossed product C *algebra C(X) ⋊ α Z of the algebra of continuous functions on a compact Hausdorff space X by an action of Z via the composition automorphism associated with a homeomorphism σ : X → X, it is known that C(X) sits inside the C * -crossed product as a maximal abelian subalgebra if and only if for every positive integer n, the set of points in X having period n under iterations of σ has no interior points [25,Theorem 5.4], [24,Corollary 3.3.3], [26,Theorems 2.8,5.2], [27,Proposition 4.14], [10,Lemma 7.3.11]. This condition is equivalent to the action of Z on X being topologically free in the sense that the non-periodic points of σ are dense in X. In [23], a purely algebraic variant of the crossed product allowing for more general classes of algebras than merely continuous functions on compact Hausdorff spaces serving as "base coefficient algebras" in the crossed products was considered. In the general set theoretical framework of a crossed product algebra A ⋊ α Z of an arbitrary subalgebra A of the algebra C X of complex-valued functions on a set X (under the usual pointwise operations) by Z acting on A via a composition automorphism defined by a bijection of X, the essence of the matter is revealed. Topological notions are not available here and thus the condition of freeness of the dynamics as described above is not applicable, so that it has to be generalized in a proper way in order to be equivalent to the maximal commutativity of A. In [23] such a generalization was provided by involving separation properties of A with respect to the space X and the action for significantly more arbitrary classes of base coefficient algebras and associated spaces and actions. The (unique) maximal abelian subalgebra containing A was described as well as general results and examples and counterexamples on equivalence of maximal commutativity of A in the crossed product and the generalization of topological freeness of the action.
In this article, we bring these results and interplay into a more general algebraic context of crossed product rings (or algebras) for crossed systems with arbitrary group actions and twisting cocycle maps [17]. We investigate the connections with the ideal structure of a general crossed product ring, describe the center of crossed product rings, describe the commutant of the base coefficient subring in a crossed product ring of a general crossed system, and obtain conditions for maximal commutativity of the commutant of the base subring in terms of the action as well as in terms of intersection of ideals in the crossed product ring with the base subring, specially taking into account both the case of base rings without non-trivial zero-divisors and the case of base rings with non-trivial zero-divisors.
Preliminaries
In this section, we recall the basic objects and the notation, from [17], which are necessary for the understanding of the rest of this article.
Gradings. Let G be a group with unit element e. The ring R is G-graded if there is a family {R σ } σ∈G of additive subgroups R σ of R such that R = σ∈G R σ and R σ R τ ⊆ R στ (strongly G-graded if, in addition, ⊇ also holds) for every σ, τ ∈ G.
Crossed products. If R is a unital ring, then U (R) denotes the group of multiplication invertible elements of R. A unital G-graded ring R is called a G-crossed product if U (R) ∩ R σ = ∅ for every σ ∈ G. Note that every G-crossed product is strongly G-graded, as explained in [17, p.2].
2.1. Crossed systems. Definition 2.1. A G-crossed system is a quadruple {A, G, σ, α}, consisting of a unital associative ring A, a group G (with unit element e), a map σ : G → Aut(A) and a σ-cocycle map α : G × G → U (A) such that for any x, y, z ∈ G and a ∈ A the following conditions hold: Remark 2.2. Note that, by combining conditions (i) and (iii), we get σ e (σ e (a)) = σ e (a) for all a ∈ A. Furthermore, σ e : A → A is an automorphism and hence σ e = id A . Also note that, from the definition of Aut(A), we have σ g (0 A ) = 0 A and σ g (1 A ) = 1 A for any g ∈ G.
Remark 2.3. From condition (i) it immediately follows that σ is a group homomorphism if A is commutative or if α is trivial.
Definition 2.4. Let G be a copy (as a set) of G. Given a G-crossed system {A, G, σ, α}, we denote by A ⋊ σ α G the free left A-module having G as its basis and in which the multiplication is defined by for all a 1 , a 2 ∈ A and x, y ∈ G. Elements of A ⋊ σ α G may be expressed as formal sums g∈G a g g where a g ∈ A and a g = 0 A for all but a finite number of g ∈ G.
Explicitly, this means that the addition and multiplication of two arbitrary elements Remark 2.5. The ring A is unital, with unit element 1 A , and it is easy to see that (1 A e) is the multiplicative identity in A ⋊ σ α G.
By abuse of notation, we shall sometimes let 0 denote the zero element in A⋊ σ α G. The proofs of the two following propositions can be found in [ Proposition 2.6. Let {A, G, σ, α} be a G-crossed system. Then A ⋊ σ α G is an associative ring (with the multiplication defined in (1)). Moreover, this ring is G-graded, A ⋊ σ α G = g∈G Ag, and it is a G-crossed product.
Proposition 2.7. Every G-crossed product R is of the form A ⋊ σ α G for some ring A and some maps σ, α.
Remark 2.8. If k is a field and A is a k-algebra, then so is A ⋊ σ α G.
The base coefficient ring A is naturally embedded as a subring into A⋊ σ α G. Consider the canonical isomorphism We denote byà the image of A under ι and by A G = {a ∈ A | σ s (a) = a, ∀s ∈ G} the fixed ring of A. which is a re-ordering formula frequently appearing in physical applications.
3. Commutativity in A ⋊ σ α G From the definition of the product in A ⋊ σ α G, given by (2), we see that two elements s∈G a s s and t∈G b t t commute if and only if for each g ∈ G. The crossed product A ⋊ σ α G is in general non-commutative and in the following proposition we give a description of its center.
Proposition 3.1. The center of A ⋊ σ α G is as follows r s σ s (a) = a r s , ∀a ∈ A, (s, t) ∈ G × G .
Proof. Let g∈G r g g ∈ A ⋊ σ α G be an element which commutes with every element of A ⋊ σ α G. Then, in particular g∈G r g g must commute with a e for every a ∈ A. From (3) we immediately see that this implies r s σ s (a) = a r s for every a ∈ A and s ∈ G. Furthermore, g∈G r g g must commute with 1 A s for any s ∈ G. This yields and hence g∈G r g g commutes with every element of A ⋊ σ α G. A few corollaries follow from Proposition 3.1, showing how a successive addition of restrictions on the corresponding G-crossed system, leads to a simplified description of Z(A ⋊ σ α G). Corollary 3.2 (Center of a twisted group ring). Let σ ≡ id A . Then, the center of Corollary 3.3. Let G be abelian and α symmetric 1 . Then, the center of A ⋊ σ α G is as follows Corollary 3.4. Let A be commutative, G abelian and α ≡ 1 A . Then, the center of A ⋊ σ α G is as follows Remark 3.5. Note that in the proof of Theorem 3.1, the property that the image of α is contained in U (A) is not used and therefore the theorem is true in greater generality. Consider the case when A is an integral domain and let α take its After a change of variable via x = s −1 t the first condition in the description of the center may be written as From this relation we conclude that r x = 0 if and only if r sxs −1 = 0, and hence it is trivially satisfied if we put r x = 0 whenever x ∈ σ −1 (id A ). This case has been presented in [18, Proposition 2.2] with a more elaborate proof.
The final corollary describes the exceptional situation when and hence (i) follows by Remark 2.9. By the assumption, 1 A s ∈ Z(A ⋊ σ α G) for any s ∈ G and by Proposition 3.1 we see that σ s = id A for every s ∈ G, and hence (ii).
but α(x, y) = 0 A which implies xy = yx and also α(x, y) = α(y, x), which shows (iii) and (iv). The converse statement of the corollary is easily verified. 4. The commutant ofà in A ⋊ σ α G From now on we shall assume that G = {e}. As we have seen,à is a subring of A ⋊ σ α G and we define its commutant by Comm(Ã) = {b ∈ A ⋊ σ α G | ab = ba, ∀a ∈Ã}. Theorem 4.1 tells us exactly when an element of A ⋊ σ α G lies in Comm(Ã). Theorem 4.1. The commutant ofà in A ⋊ σ α G is as follows Proof. The proof is established through the following sequence of equivalences: Here we have used the fact that α(s, e) = α(e, s) = 1 A for all s ∈ G. The above equivalence can also be deduced directly from (3).
In the case when A is commutative we get the following description of the commutant as a special case of Theorem 4.1.
When A is commutative it is clear thatà ⊆ Comm(Ã). Using the explicit description of Comm(Ã) in Corollary 4.2, we are now able to state exactly whenà is maximal commutative, i.e. Comm(Ã) =Ã.
Example 4.4. In this example we follow the notation of [23]. Let σ : X → X be a bijection on a non-empty set X, and A ⊆ C X an algebra of functions, such We now have a Z-crossed system (with trivial σ-cocycle) and we may form the crossed product A ⋊σ Z. Recall the definition of the set Sep n In particular, this means that f n is identically zero on Sep n A (X). However, f n ∈ A \ {0} is not identically zero on X and hence Sep n A (X) is not a domain of uniqueness (as defined in [23,Definition 3.2]). The converse can be proved similarly.
Corollary 4.5. Let A be commutative. If for each s ∈ G \ {e} it is always possible to find some a ∈ A such that σ s (a)−a is not a zero-divisor in A, thenà is maximal commutative in A ⋊ σ α G. The next corollary is a consequence of Corollary 4.3 and shows how maximal commutativity of the base coefficient ring in the crossed product has an impact on the non-triviality of the action σ.
Corollary 4.6. Let the subringà be maximal commutative in
The description of the commutant Comm(Ã) from Corollary 4.2 can be further refined in the case when A is an integral domain.
2 By an integral domain we shall mean a commutative ring with an additive identity 0 A and a multiplicative identity 1 A such that 0 A = 1 A , in which the product of any two non-zero elements is always non-zero.
The following corollary can be derived directly from Corollary 4.6 together with either Corollary 4.5 or Corollary 4.7.
Corollary 4.8. Let A be an integral domain. Then σ g = id A for all g ∈ G \ {e} if and only ifà is maximal commutative in A ⋊ σ α G. Remark 4.9. Recall that when A is commutative, σ is a group homomorphism. Thus, to say that σ g = id A for all g ∈ G \ {e} is just another way of saying that ker(σ) = {e} or equivalently, that σ is injective.
. , x n ] be the polynomial ring in n commuting variables x 1 , . . . , x n and G = S n the symmetric group of n elements. An element τ ∈ S n is a permutation which maps the sequence (1, . . . , n) into (τ (1), . . . , τ (n)). The group S n acts on C[x 1 , . . . , x n ] in a natural way. To each τ ∈ S n we may associate a map A → A, which sends any polynomial f (x 1 , ..., x n ) ∈ C[x 1 , . . . , x n ] into a new polynomial g, defined by g(x 1 , . . . , x n ) = f (x τ (1) , . . . , x τ (n) ). It is clear that each such mapping is a ring automorphism on A. Let σ be the embedding S n ֒→ Aut(A) and α ≡ 1 A . Note that C[x 1 , . . . , x n ] is an integral domain and that σ is injective. Hence, by Corollary 4.8 and Remark 4.9 it is clear that the embedding of C[x 1 , . . . , x n ] is maximal commutative in C[x 1 , . . . , x n ] ⋊ σ S n .
One might want to describe properties of the σ-cocycle in the case whenà is maximal commutative, but unfortunately this will lead to a dead end. The explaination for this is revealed by condition (iii) in the definition of a G-crossed system, where we see that α(e, g) = α(g, e) = 1 A for all g ∈ G and hence we are not able to extract any interesting information about α by assuming thatà is maximal commutative. At the end of [17, Remark 1.4.3, part 2] it is explained that in a twisted group ring A ⋊ α G, i.e. with σ ≡ id A ,à can never be maximal commutative (for G = {e}). When A is commutative, this follows immediately from Corollary 4.6.
We will now give a sufficient condition for Comm(Ã) to be commutative.
This shows that Comm(Ã) is commutative.
This proposition is a generalization of [23, Proposition 2.1] from a function algebra to an arbitrary unital associative commutative ring A, from Z to an arbitrary abelian group G and from a trivial to a possibly non-trivial symmetric σ-cocycle α. Proof. Let A be commutative. Thenà is also commutative. Let I ⊆ A ⋊ σ α G be an arbitrary non-zero two-sided ideal in A ⋊ σ α G.
Part 1:
For each g ∈ G we may define a translation-deformation operator Note that, for any g ∈ G, I is invariant 3 under T g . We have This resulting element will then have the following properties:
Part 2:
Next we define a kill operator for each a ∈ A. Note that, for each a ∈ A, I is invariant under D a . By assumption A is commutative and hence the above expression can be simplified The operators {D a } a∈A all share the property that they kill the coefficient in front e. Hence, if a e = 0 A , then the number of non-zero coefficients of the resulting element will always be reduced by at least one. Note that Comm(Ã) = a∈A ker(D a ). This means that for each non-zero s∈G a s s in A ⋊ σ α G \ Comm(Ã) we may always choose some a ∈ A such that s∈G a s s ∈ ker(D a ). By choosing such an a we note that, using the same notation as above, we get
Part 3:
The ideal I is assumed to be non-zero, which means that we can pick some non-zero element s∈G r s s ∈ I. If s∈G r s s ∈ Comm(Ã), then we are finished, so assume that this is not the case. Note that r s = 0 A for finitely many s ∈ G. Recall that the ideal I is invariant under T g and D a for all g ∈ G and a ∈ A. We may now use the operators {T g } g∈G and {D a } a∈A to generate new elements of I. More specifically, we may use the T g :s to translate our element s∈G r s s into a new element which has a non-zero coefficient in front of e (if needed) after which we use the D a operator to kill this coefficient and end up with yet another new element of I which is non-zero but has a smaller number of non-zero coefficients. We may repeat this procedure and in a finite number of iterations arrive at an element of I which lies in Comm(Ã)\à and if not we continue the above procedure until we reach an element which is of the form b e with some non-zero b ∈ A. In particularà ⊆ Comm(Ã) and hence I ∩ Comm(Ã) = {0}.
The embedded base ringà is maximal commutative if and only ifà = Comm(Ã) and hence we have the following corollary. Then the following assertions hold: Proof. If I is a (possibly one sided) ideal in A, it is clear that J is an additive for arbitrary s∈G a s s ∈ J and t∈G b t t ∈ A ⋊ σ α G and hence J is a right ideal. (ii). Let I be a two-sided ideal in A such that I ⊆ A G . By (i) it is clear that J is a right ideal. Let s∈G a s s ∈ J and t∈G b t t ∈ A ⋊ σ α G be arbitrary. Then which shows that J is also a left ideal.
Theorem 5.4. Let σ : G → Aut(A) be a group homomorphism and N be a normal subgroup of G, contained in σ −1 (id A ) = {g ∈ G | σ g = id A }. Let ϕ : G → G/N be the quotient group homomorphism and suppose that α is such that α(s, t) = 1 A whenever s ∈ N or t ∈ N . Furthermore, suppose that there exists a map β : Let I be the ideal in A ⋊ σ α G generated by an element s∈N a s s for which the coefficients (of which all but finitely many are zero) satisfy s∈N a s = 0 A . Then, Proof. Let I ⊆ A ⋊ σ α G be the ideal generated by an element s∈N a s s, which satisfies s∈N a s = 0 A . The quotient homomorphism ϕ : G → G/N, s → sN satisfies ker(ϕ) = N . By assumption, the map σ is a group homomorphism and σ(N ) = id A . Hence by the universal property, see for example [7, p.16], there exists a unique group homomorphism ρ making the following diagram commute: By assumption there exists β such that β(ϕ(s), ϕ(t)) = α(s, t) for each (s, t) ∈ G × G. One may verify that β is a ρ-cocycle and hence we can define a new crossed product A ⋊ ρ β G/N . We now define Γ to be the map and show that it is a ring homomorphism. For any two elements s∈G a s s and and due to the assumptions, the multiplicativity follows by and hence Γ defines a ring homomorphism. We shall note that the generator of I is mapped onto zero, i.e. When A is commutaive, σ is automatically a group homomorphism and we get the following corollary.
Let ϕ : G → G/N be the quotient group homomorphism and suppose that α is such that α(s, t) = 1 A whenever s ∈ N or t ∈ N . Furthermore, suppose that there exists a map β : G/N × G/N → U (A) such that β(ϕ(s), ϕ(t)) = α(s, t) for each (s, t) ∈ G × G. Let I be the ideal in A ⋊ σ α G generated by an element s∈N a s s for which the coefficients (of which all but finitely many are zero) satisfy s∈N a s = 0 A . Then, When α ≡ 1 A there is no need to assume that A is commutative, in order to make σ a group homomorphism. In this case we may choose β ≡ 1 A . Thus, by Theorem 5.4 we have the following corollaries.
Corollary 5.6. Let α ≡ 1 A and N ⊆ σ −1 (id A ) = {g ∈ G | σ g = id A } be a normal subgroup of G. Let I be the ideal in A ⋊ σ α G generated by an element s∈N a s s for which the coefficients (of which all but finitely many are zero) satisfy s∈N a s = 0 A . Then, Corollary 5.7. Let α ≡ 1 A . Then the following implication holds: , the ideal I g generated by the element n∈Z a n g n for which n∈Z a n = 0 A has the property I g ∩à = {0}. Proof. Suppose that there exists a g ∈ (Z(G)∩σ −1 (id A ))\ {e}. Let I g ⊆ A⋊ σ α G be the ideal generated by n∈Z a n g n , where n∈Z a n = 0 A . The element g commutes with each element of G and hence the cyclic subgroup N = g generated by g is normal in G and since σ is a group homomorphism N ⊆ σ −1 (id A ). Hence I g ∩à = {0} by Corollary 5.6.
Corollary 5.8. Let α ≡ 1 A and G be abelian. Then the following implication holds: Proof by contrapositivity. Since G is abelian, G = Z(G). Suppose that (ii) is false, i.e. there exists g ∈ G \ {e} such that σ g = id A . Pick such a g and let I g ⊆ A ⋊ σ α G be the ideal generated by 1 A e − 1 A g. Then obviously I g = {0} and by Corollary 5.7 we get I g ∩à = {0} and hence (i) is false. This concludes the proof.
Example 5.9. We should note that in the proof of Corollary 5.8 one could have chosen the ideal in many different ways. The ideal generated by 1 A e − 1 A g + 1 A g 2 − 1 A g 3 + . . . + 1 A g 2n − 1 A g 2n+1 = (1 A e − 1 A g) n k=0 1 A g 2k is contained in the ideal I g , generated by 1 A e − 1 A g, and therefore it has zero intersection withà if I g ∩à = {0}. Also note that for α ≡ 1 A we may always write and hence 1 A e − 1 A g is a zero-divisor in the crossed product A ⋊ σ α G whenever g is a torsion element.
Example 5.10. We now give an example of how one may choose β as in Theorem 5.4. Let N ⊆ σ −1 (id A ) be a normal subgroup of G such that for g ∈ N , α(s, g) = 1 A for all s ∈ G and let α be symmetric. Since α is the σ-cocycle map of a G-system, we get α(g, s) α(gs, t) = σ g (α(s, t)) α(g, st) ⇐⇒ α(g, s) α(gs, t) = α(s, t) α(g, st) ⇐⇒ α(gs, t) = α(s, t) for all (s, t) ∈ G×G. Using the last equality and the symmetry of α we immediately see that α(gs, ht) = α(s, t) ∀s, t ∈ G for all g, h ∈ N . The last equality means that α is constant on the pairs of right cosets which coincide with the left cosets by normality of N . It is therefore clear that we can define β : G/N × G/N → Aut(A) by β(ϕ(s), ϕ(t)) := α(s, t) for any s, t ∈ G.
Theorem 5.11. Let A be an integral domain, G an abelian group and α ≡ 1 A . Then the following implication holds: Proof. This follows from Corollary 4.8 and Corollary 5.8.
The ring C q [x, x −1 , y, y −1 ] is known as the quantum torus. Now let • α(s, t) = 1 A for all s, t ∈ G. It is easily verified that σ and α together satisfy conditions (i)-(iii) of G-systems and it is not hard to see that A ⋊ σ α G ∼ = C q [x, x −1 , y, y −1 ]. In the current example, A is an integral domain, G is abelian, α ≡ 1 A and hence all the conditions of Theorem 5.11 are satisfied. Note that the commutation relation (4) implies y n x m = q mn x m y n , ∀n, m ∈ Z.
It is important to distinguish between two different cases: Case 1 (q is a root of unity). Suppose that q n = 1 for some n = 0. From equality (5) we note that y n ∈ Z(C q [x, x −1 , y, y −1 ]) and hence C[x, x −1 ] is not maximal commutative in C q [x, x −1 , y, y −1 ]. Thus, according to Theorem 5.11, there must exist some non-zero ideal I which has zero intersection with C[x, x −1 ].
Case 2 (q is not a root of unity). Suppose that q n = 1 for all n ∈ Z \ {0}. One can show that this implies that C q [x, x −1 , y, y −1 ] is simple. This means that the only non-zero ideal is C q [x, x −1 , y, y −1 ] itself and this ideal obviously intersects C[x, x −1 ] non-trivially. Hence, by Theorem 5.11, we conclude that
Ideals, intersections and zero-divisors
Let D denote the subset of zero-divisors in A and note that D is always nonempty since 0 A ∈ D. ByD we denote the image of D under the embedding ι. for all a+ Ann(c) ∈ A/ Ann(c). Note that Ann(c) is invariant under σ s for all s ∈ G and thus it is easily verified that ρ is a well-defined automorphism on A/ Ann(c). By introducing the function it is easy to verify that {A/ Ann(c), G, ρ, β} is in fact a G-crossed system. Now consider the map For any two elements s∈G a s s, t∈G b t t ∈ A ⋊ σ α G the additivity of Γ follows by where have used that β = θ • α and θ(σ s (b t )) = ρ s (θ(b t )) for all b t ∈ A and s ∈ G. This shows that Γ is a ring homomorphism. Now, pick some g = e and let I be the ideal generated by d g. Clearly I = {0} and we see that Γ| I ≡ 0. Note that ker(θ) = Ann(c) and in particular we have Γ(ae) = 0 =⇒ a ∈ Ann(c).
Take m e ∈ I ∩ Ã \D . Then Γ(m e) = 0 and hence m ∈ Ann(c) ⊆ D, which is a contradiction. This shows that and by contrapositivity this concludes the proof.
Example 6.2 (The truncated quantum torus). Let q ∈ C \ {0, 1}, m ∈ N and consider the ring which is commonly referred to as the truncated quantum torus. It is easily verified that this ring is isomorphic to A ⋊ σ α G with One should note that in this case A is commutative, but not an integral domain. In fact, the zero-divisors in C[x]/(x m ) are precisely those polynomials where the constant term is zero, i.e. p(x) = m−1 i=0 a i x i , with a i ∈ C, such that a 0 = 0. It is also important to remark that, unlike the quantum torus, A ⋊ σ α G is never simple (for m > 1). In fact we always have a chain of two-sided ideals independent of the value of q. Moreover, the two-sided ideal J = x m−1 is contained in Comm(C[x]/(x m )) and contains elements outside of C[x]/(x m ). Hence we conclude that C[x]/(x m ) is not maximal commutative in C[x,y,y −1 ] (y x−q x y , x m ) . When q is a root of unity, with q n = 1 for some n < m, we are able to say more. Consider the polynomial p(x) = x n , which is a non-trival zero-divisor in C[x]/(x m ). For every s ∈ Z we see that p(x) = x n is fixed under the automorphism σ s and therefore, by Theorem 6.1, we conclude that there exists a non-zero two-sided ideal in C[x,y,y −1 ] (y x−q x y , x m ) such that its intersection withà \D is empty.
Comments to the literature
The literature contains several different types of intersection theorems for group rings, Ore extensions and crossed products. Typically these theorems rely on heavy restrictions on the coefficient rings and the groups involved. We shall now give references to some interesting results in the literature.
It was proven in [22, Theorem 1, Theorem 2] that the center of a semiprimitive (semisimple in the sense of Jacobson [6]) P.I. ring respectively semiprime P.I. ring has a non-zero intersection with every non-zero ideal in such a ring. For crossed products satisfying the conditions in [22,Theorem 2], it offers a more precise result than Theorem 5.1 since Z(A ⋊ σ α G) ⊆ Comm(Ã). However, every crossed product need not be semiprime nor a P.I. ring and this justifies the need for Theorem 5.1.
In [12,Lemma 2.6] it was proven that if the coefficient ring A of a crossed product A ⋊ σ α G is prime, P is a prime ideal in A ⋊ σ α G such that P ∩à = 0 and I is an ideal in A ⋊ σ α G properly containing P , then I ∩à = 0. Furthermore, in [12,Proposition 5.4] it was proven that the crossed product A ⋊ σ α G with G abelian and A a G-prime ring has the property that, if G inn = {e}, then every non-zero ideal in A ⋊ σ α G has a non-zero intersection withÃ. It was shown in [2, Corollary 3] that if A is semiprime and G inn = {e}, then every non-zero ideal in A ⋊ σ α G has a non-zero intersection withÃ. In [13,Lemma 3.8] it was shown that if A is a G-prime ring, P a prime ideal in A ⋊ σ α G with P ∩à = 0 and if I is an ideal in A ⋊ σ α G properly containing P , then I ∩à = 0. In [16,Proposition 2.6] it was shown that if A is a prime ring and I is a non-zero ideal in A ⋊ σ α G, then I ∩ (A ⋊ σ α G inn ) = 0. In [16,Proposition 2.11] it was shown that for a crossed product A ⋊ σ α G with prime ring A, every non-zero ideal in A ⋊ σ α G has a non-zero intersection withà if and only if C t [G inn ] is G-simple and in particular if |G inn | < ∞, then every non-zero ideal in A ⋊ σ α G has a non-zero intersection withà if and only if A ⋊ σ α G is prime. Corollary 5.2 shows that ifà is maximal commutative in A ⋊ σ α G, without any further conditions on the coefficient ring or the group, we are able to conclude that every non-zero ideal in A ⋊ σ α G has a non-zero intersection withÃ. In the theory of group rings (crossed products with no action or twisting) the intersection properties of ideals with certain subrings have played an important role and are studied in depth in for example [3], [11] and [21]. Some further properties of intersections of ideals and homogeneous components in graded rings have been studied in for example [1], [14].
For ideals in Ore extensions there are interesting results in [4, Theorem 4.1] and [8, Lemma 2.2, Theorem 2.3, Corollary 2.4], explaining a correspondence between certain ideals in the Ore extension and certain ideals in its coefficient ring. Given a domain A of characteristic 0 and a non-zero derivation δ it is shown in [5, Proposition 2.6] that every non-zero ideal in the Ore extension R = A[x; δ] intersects A in a non-zero δ-invariant ideal. Similar types of intersection results for ideals in Ore extension rings can be found in for example [9] and [15].
The results in this article appeared initially in the preprint [19]. | 2007-09-29T11:30:47.000Z | 2007-09-29T00:00:00.000 | {
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236425805 | pes2o/s2orc | v3-fos-license | Pandemic Pivots: The Impact of a Global Health Crisis on the Dissertation in Practice
Five scholarly practitioners in an educational leadership for social justice doctoral program share their intentional, community-minded pivots during a global pandemic that disrupted their Dissertations in Practice (DiP). Embodying their Ed.D. program’s CPED framework (Carnegie Project on the Education Doctorate, 2019), the authors, at varying stages in the dissertation process, sought inventive solutions to COVID-19-related challenges that included the development of a new topic and research questions, adjusting study settings and participant pools, and embracing new methodologies to account for virtual-only approaches. Although uncertain how the global health crises would impact their DiP, by fostering a shared sense of community, the authors became critical friends, supporting each other in their personal, professional, and academic lives. Each narrative highlights the potential of oppositional praxis of threading identities of practice, reflection, and research–to respond creatively to the needs of their diverse research communities with compassion, vision, and
INTRODUCTION
As scholarly practitioners in the fifteenth cohort of a doctoral program in educational leadership for social justice, we began the spring semester of 2020 preparing for our proposal defenses. With a curriculum grounded in the CPED framework, faculty continued to push us to "develop a critical and professional stance with a moral and ethical imperative for equity and social justice" (Carnegie Project on the Education Doctorate, 2019). Drawing from our own extensive experience as full-time educators, we developed dissertation topics centered on social justice issues, incorporating both theoretical and applied, field-based research methods-a cornerstone of the professional doctorate in education programs. Although our problems of practice are unique to our individual professional contexts (our dissertations include domestic and international studies across K-12 and higher education settings), we became critical friends, supporting each other's work (Carnegie Project on the Education Doctorate, 2019). However, in March 2020, COVID-19 forced us to pivot-from radical topic and setting changes, to new methodologies and data collection approaches. Storey and Maughan (2016) note that "the research process should help the practitioner scholars combine research techniques with experiential knowledge and deepen their ability to think about problems and search for practical solutions simultaneously" (p. 225). The pandemic impacted our research at varying stages of the dissertation process, but through a shared sense of community and support for one another's scholarly and professional journeys, we responded by implementing practical solutions to complex problems that strengthened our capabilities. The following five examples highlight how we, as scholarly practitioners, responded creatively to the needs of our diverse communities through our Dissertations in Practice (DiP). Each narrative highlights the potential of oppositional praxis (Sandoval, 2000)-of threading identities of practice, reflection, and research-to respond creatively to the needs of diverse communities with compassion, vision, and agility.
COVID Community
During the spring semester of my second year in the doctoral program, and after several false starts, my dissertation was finally falling into place. I would investigate how parental participation was impacted by a school district's transition to digital communication. At the same time, COVID-19 began sweeping the world. When schools began to close, my chair and I realized that this topic would need to shift.
In the days following the closure, I was overwhelmed by my sense of loneliness due to the lack of interactions with students and other staff members. It was this loneliness and a desire to investigate how others were attempting to recreate the supportive physical school community that propelled me to a new topic. Additionally, I was noticing that even with teachers scrambling to create online educational experiences, some of our students, especially those already disengaged from school, were disappearing; I wanted to know why. This is when I pivoted to my new topic of building an eschool community during the emergency COVID-19 closure.
A student's sense of belonging at school has been positively linked to higher attendance and I pondered whether this would carry over into the digital realm (Frazier et al., 2015;Osterman, 2000). Since a student's sense of belonging to a school community can provide a safety net for early detection of mental health issues, and due to the sudden disruption of my students' lives, I was concerned about how this would be affected by emergency distance teaching (Frazier et al., 2015;Levitt et al., 2007). These concerns, along with my own desire to be a part of a community, drove my research as I raced to complete my proposal for the review committee. I successfully defended my proposal and started data collection in the summer.
I reached out to the school community with interview requests and was amazed at how quickly, even eagerly, people responded. I was concerned that conducting interviews with physical distance via the Zoom platform, the interviews might be less honest, less illuminating, and less connected. I have been gratefully surprised by the careful consideration given to my questions and the openness of their responses. Their willingness to engage with me in this research can be contributed to the community and relationships formed before the closure and maintained once separated. I have been struck by the deep sense of loss the participants express. Using a three-part interview protocol, participants discuss the history of what our school community was before the closures, what it was like during the closure, and then reflect on the alterations since the closure. Preliminary findings suggest school staff is mourning the loss of student connections built from the daily physical interactions. They discuss handshakes, class discussions, and small moments of comfort that they miss. The teachers are lamenting their lack of skills in the digital classroom; their reduction in becoming a first-year teacher again coupled with the anxiety of the unknown. The technology coach has been alarmed by her loss of boundaries between work and family life. Thinking of the poverty present at the school site pre-COVID, the school psychologist worried about all the students who had no quiet, safe place to work, especially those in group home placement.
Parents too expressed their grief. Parents of eighth-grade students mourned the end-of-year events, the virtual promotion, and the lack of real goodbyes. Several parents cried recounting the loss of the school community and its effects on their children. After many interviews were concluded, the participants desired a debrief conversation to express the emotions unearthed by the interview.
Overall, the final question of the interview investigates views, thoughts, and concerns about the 2020-2021 school year. Every participant shared unique fears and concerns, but each response contained an aspect of uncertainty, a fear of the unknown, and unknowable. These fears, their sadness, and anxiety could be alleviated somewhat by a strong, supportive school community, but unfortunately, that community is still under construction.
Student Voice and Agency
School communities are microcosms of the larger society. In the previous narrative, the focus was on the impact of the pandemic on an entire school community. The relationships forged within that community were strained by the school's closure. Research, in the form of a case study, opened the door to spaces that helped capture that story while offering a way to reconnect. That is the power of research that answers to communities and thus leverages the production of knowledge to uplift (Patel, 2016). My own direct work with immigrant-origin youth has greatly informed how much respect and admiration I have for young people who leave home in search of a better life, in fulfillment of their dreams. The identities of these youth often defy the school system's narrow definitions of who they are. Labels such as English Learners or newcomers fail to capture their rich and complex identities, their political stances, and their influence on the spaces that seek to educate them. For my study of unaccompanied youth, I wanted to center their epistemology to inform emancipatory educational practices at school sites. I realized that the only way to honor the youth's complex identities was to include youth as co-researchers. My aim was to engage in research that answered to immigrant youth and was not simply about them.
Using CRT (Solorzano & Yosso, 2002) and Youth Participatory Action Research (YPAR, Cammarota & Fine, 2008) methodologies, the project sought to produce actionable items that could positively impact the school site and other educational agencies. However, by mid-March 2020, the pandemic meant that schools would not reopen for months.
This complicated the research project in ways that neither I nor the youth co-researchers could have anticipated. Before the school closure, four youth co-researchers were recruited to interview each other, interview other unaccompanied youth, hold a focus group, and review school documents as part of the data collection.
Due to COVID-19, the research project lost a common shared space: the school. The co-researchers were all enrolled in a research course and their class discussions and learning often spilled into our after-school meetings over pizza or chicken wings. The first draft of the interview protocol was created over breakfast in a small cafe where we discussed at length what would help us learn more about the unaccompanied youth identity and their experiences 7 in school. The conversations we had were often grounded in a theoretical article on CRT (Solorzano & Yosso, 2002). Taking those organic and rich discussions to a virtual space appeared to be close to impossible.
Secondly, student lives were greatly impacted by the pandemic. One of the youth co-researchers became the only source of income for her family. She continued cleaning houses for a few clients that felt safe enough to allow her into their homes. Another student traveled to San Jose to be with his older brothers. A third student became the babysitter for her nieces as her older brothers looked to save money when their own work hours were reduced. The fourth student had also lost his part-time employment at a car wash.
In late spring, I called a virtual meeting just to check in with the group. I was also feeling disconcerted and exhausted from working non-stop. We scheduled a second meeting and decided to rethink our design. As the principal investigator, I designed a deck of Google slides to help us organize our Zoom meetings and capture our learning. During those first few awkward meetings, the researchers and I realized that the community we had built in a physical space could continue in our virtual space. We agreed to continue our important work through weekly Zoom meetings. We opened every meeting with a short meditation to center ourselves and to focus our collective energy back on our project. The study was scaled back to include fewer interviews, no focus group due to scheduling difficulties, and I completed the document analysis.
In order to mitigate the difficulty of having virtual interviews, the team decided that we would always start with a check-in before the interview, to re-establish a sense of connection. This part was not recorded. The recording itself required additional creative steps. Often, the interview was conducted via a video call. The audio was recorded on the computer to protect the anonymity of the participant. The IRB did not include a video recording of the interview. This population of students is highly vulnerable and the team wanted to protect all the participants. For the data analysis, we had to create a shared Google drive that contained all the transcripts. I would often share my screen as a way for the team to conduct the first round of analysis. Padlets were used to do the initial coding of the data. This allowed the team to collaborate in real-time when needed. Virtual, collaborative tools allowed us to continue working together, without having to be in the same physical space. As the co-researchers' work schedules became more complicated, the team's ability to meet on a weekly basis dwindled. As the new school year neared I was even busier preparing for a new virtual school year.
In spite of all the challenges, the findings of the study yielded rich descriptions of the complicated identities of these immigrantorigin youth. The youth seamlessly navigate across all their identities through what Chela Sandoval (2000) refers to as differential consciousness, "a cyberspace, where the transcultural, transgendered, transsexual, transnational leaps necessary to the play of effective stratagems of oppositional praxis can begin" (p. 68.8).
The essential question for public schools that seek to educate immigrant-origin youth is to consider how their current spaces can be more open and receptive to the voices of these youth. In the midst of a pandemic, scholarly practitioners with a social justice orientation must center their research in a way that is (a) responsive to the contexts under study and (b) leverages the knowledge production of marginalized voices, to foreground research as a liberatory force in education.
Onward through Online Interviews
Similar to my co-authors above, the challenge of building student trust and community in a virtual setting was not limited to K-12 spaces. In response to the growing COVID-19 crisis, the university where I work moved to online instruction and I began working remotely from home. At this same time, I successfully passed my proposal defense and received IRB approval for my exploratory mixed methods study that seeks to understand community college transfer student awareness of nationally competitive awards (NCAs), like the Benjamin A. Gilman Scholarship and Fulbright U.S. Student Program. My plan had been to conduct in-person interviews with transfer students from one university. Before proceeding with my qualitative interviews, I needed to make some quick-thinking adjustments to account for the abrupt shift to a virtual modality.
Having confirmed a university site for my interview participant pool, I was eager to begin my data collection. Yet, I was concerned that students would be unavailable or unwilling to participate in a 45minute interview, considering they were experiencing abrupt life challenges such as moving back home and transitioning unexpectedly to online instruction. Moreover, I worried students would not be as open or honest in an online setting. To my surprise, over thirty transfer students expressed an interest in participating. This led to my first major pivot, conducting ten student interviews instead of my initial plan of five. This proved propitious given that after my interviews, when I was ready to disseminate my newly developed survey, two of my three confirmed sites apologetically withdrew their support, citing COVID-19 as the primary factor.
However, I knew that increasing the number of participants would not mitigate my concerns about authentic dialogue in an online space. This led to my second pivot in which I reexamined my interviewing approach to account for the new setting. Drawing from Noddings' (2013) ethics of care, I sought to "work cooperatively with the student in his struggle toward competence in that world" (p.177), which now included postponed graduations, job losses, and canceled summer internships. I began every interview by thanking them for their participation (Galletta & Cross, 2013), followed by a question about their well-being. I also wanted to ensure participants felt comfortable with me even though we were communicating through a screen. Mann and Stewart (2000) expound on the importance of developing an enthusiastic rapport with online interview participants and recommend building trust by being open about the structure and purpose of the research. After my initial wellness check, I provided participants a clear outline of the topics we would discuss and paused for questions throughout. Students demonstrated a genuine interest in the study's focus and not one of the ten participants appeared reserved or reticent during the 45minute interviews. Online environments may in fact encourage shy individuals to speak more openly and freely because it's a "culturally neutral" space in which participants have chosen their physical location to engage in the research (Mann & Stewart, 2000, p. 201). Thus, the virtual format may have actually elicited more thoughtful responses which often included COVID-19 as a topic of concern.
This led to a third pivot during my qualitative interviews, taking into account the impact of the pandemic on participant responses. Given that several of my exemplar NCAs, like Gilman and Fulbright, provide funding for study abroad or international research, it was likely that a student's interest in these opportunities may be influenced by the global health crisis. Because I employed a semi-structured phenomenological interview protocol, I was able to "rephrase the questions, and make changes according to the interview situation" (Galletta & Cross, 2013, p. 75). I phrased questions in a way that allowed participants to consider their pre-COVID-19 worldviews, such as when one participant stated that he no longer thought it was possible to study abroad due to the virus. I responded with "Yeah, we're in a very unique situation right now. When would you have liked to have gone abroad if everything had worked out?" I leaned into these responses, encouraging participants to explain how their situations may have changed, but also kept the focus on my research questions. Ultimately, several participants noted that COVID-19 had changed their plans to study or travel abroad. Yet, by creating a space that encouraged participants to express these changes, they were able to look beyond the pandemic and most expressed interest in future international opportunities.
Conducting qualitative interviews during a pandemic led to quick-thinking shifts in both the modality of the interview setting, as well as my questioning method and participant interactions. This led to intense self-reflection about the kind of researcher I want to be. Noddings (2013) notes that "the primary aim …of every educational effort must be the maintenance and enhancement of caring" (p.175). I believe this extends to educational research and I have strived to situate caring as a primary characteristic of my DiP-especially after COVID-19's unpredictable impact.
Research Left Behind
I was preparing to defend my dissertation proposal on the experience of Muslim international students in the United States when COVID-19 hit the United States, turning my plans for defense and research upside down. As I navigated the tremendous disruptions in my personal and professional life, I realized that my research participants were having the same experience and I needed to approach my study with an even greater element of care for them.
After two years in my doctoral program, I can confirm the impact of doing a Dissertation in Practice on social justice in my professional endeavors. I provide leadership to our university's office supporting international students, and my dissertation work thus far had led me to engage in robust research on student support and rich conversation with students around their need for support and formation of a strong sense of community.
In 2019, international students comprised 5.5% of the total US higher education population (Institute for International Education [IIE], 2019); they are among the most heavily monitored populations in the immigration system as their visas require constant reporting of enrollment information. Offices that support international students on campus play a critical role in helping students maintain their student visa status, adjust to, and thrive in the American university environment. These offices should be staffed with colleagues who have adequate intercultural and international experiences to be able to understand and relate to the challenges of international students (Rao, 2017).
As our campus closed, our own international students faced travel restrictions, navigated online courses across time zones, experienced financial challenges, and struggled to stay current on regulations concerning their student visas. Public discourse around the relationship between many of their countries and the US added additional layers of tension. Past studies have identified international student concerns about the historical and political relationship between their countries and the US, leading to feelings of prejudice against them (Sherry et al., 2010). These feelings were heightened by the July 6, 2020 announcement from the US Government barring international students from returning to the US if their courses were fully on-line (US Immigration and Customs Enforcement, 2020).
Through the spring and summer, I provided leadership to my unit in responding to and supporting students and collaborated with senior administrators to join the subsequent and ultimately successful lawsuit filed by several other institutions to rescind the July 6 announcement. I composed communication with students to affirm their value to our institution, working in partnership with senior leaders and colleagues to make sure our international students' education would not be disrupted as the university decided on a fully online format for Fall 2020. Faculty were encouraged to develop asynchronous course content, we formed a partnership with the library to assist students overseas in accessing course materials, encouraged our academic resource center to hired tutors to support students from different time zones, and briefed the entire community on how best to support our international students. I worked personally with countless students and families at all hours of the day and evening, and my children's' frequent disruption of my video calls seemed to provide them with a measure of comfort, knowing we were all experiencing a major upheaval and were doing the best we could.
As most universities in our region began their fall semester entirely online, I realized the need to pivot and change my methodology and plans for data collection. No longer would I have the opportunity to invite participants to join in-person focus groups, nor would I be able to engage in the participant observation my ethnographic methodology required. I created a new plan for a phenomenological study, utilizing individual interviews with our Muslim international students, broadening my research questions to capture their individual lived experiences navigating this time of COVID-19. In particular, I included questions to explore how they navigated the demands of their courses across time zones in the online environment, their views on the changes to international student visa regulations by the U.S. government, and how they felt the university supported them through these challenging times.
As I write this article, I am still working remotely, taking doctoral courses online, and continuing to homeschool my children. I believe that my own students are in a place where they know they are supported and continue to feel a sense of community despite our physical distance. I look forward to moving forward with my research and hope that my findings will include all participants feeling supported, valued, and knowing their institution navigated this time with their best interest in mind.
Virtual Village
Similar to the study described above, my work also focused on international education. I first became acquainted with Senegal High School (a pseudonym) in 2019 when I visited Senegal as a Fulbright Fellow. During my fellowship, I had many opportunities to learn about Senegal's languages and culture and teach lessons with my host teacher. We were invited to many homes for meals and ataya-the traditional tea ceremony of sorts, that usually occurs after lunch or dinner. During my second visit to Senegal, I lived in the home of my host teacher and participated in Senegalese life as a member of the family. I was thrilled to experience and learn more about Senegalese culture and traditions. We traveled to historic sites and important cities around the country and I taught at the village high school. Not only that, but I attended a wedding and a baby naming ceremony in a village situated just off the government road. Reconnecting with teachers, administrators, and community members not only reaffirmed my desire to study the school's learning community but moved me closer to becoming a member of their extended family. I was eager to return a third time to officially begin gathering data.
My proposed research design was a case study. I initially wanted to know more about Senegal High School, the students, and the people who support them. Specifically, I wanted to understand how parents, families, and community members support their children's education and the factors that empower the parents, families, and communities to act as advocates on the students' behalf. Using a conceptual framework that includes Ubuntu (Metz, 2007), my research would consist mainly of interviews, observations, and document and artifact analysis. In addition to interviewing parents, family members, and community members, I would also interview teachers and administrators of Senegal High School, a total of approximately 20 individuals.
When COVID-19 began sweeping across the globe, forcing countries to close their borders, I felt that my study was in danger. I was no longer sure that the study would be feasible for my dissertation, but I had come too far to give up. The basis of the research would remain the same, but the methodology would require several pivots, some creativity, collaboration, and trust.
The major pivot in my study related to the methodology. First, because I could no longer travel to Senegal to physically collect data, I needed the support of a research assistant-someone in Senegal who could conduct the interviews, collect documents and artifacts, share the data and debrief with me. I also knew that this task was going to be demanding, and the assistant had to be willing to devote the time to complete the assignment, which would include at least 40 hours. My former host teacher graciously offered to support the study; this researcher assistant completed the necessary training and passed the necessary assessments to participate in research with human subjects. Additionally, I would have to incorporate the use of technology wherever possible, to be "present" in the interviews with the participants (Matthews & Cramer, 2008), and to debrief with my research assistant. As a result, I included a prerecorded video in the interview procedure where I introduced myself, described the nature and purpose of the study, and thanked them for considering participating in the study. This way, the participants would not only have a name but a face associated with the questions that my research assistant would ask. Furthermore, because we had time, thanks to the quarantine, my dissertation committee encouraged me to increase the robustness of the study by including student voices. I decided to add student interviews to the data collection to hear students' perspectives about how their education is supported. This decision, however, added an additional layer of approval and level of scrutiny to my already complicated study. My research protocol would need a full IRB review, which meant more waiting. In this time of waiting, however, I had opportunities to consult with my new extended family in Senegal to clarify concepts, constructs, and cultural expressions when revising the interview protocol. I had time to work with my colleagues more than 6,000 miles away and rely on others to secure the appropriate permissions to conduct the study. I had time to think and be explicitly clear about the research protocol because my research assistant would be gathering data. I knew that it would be critically important that the interview protocol not only address the research questions but connect to the conceptual framework, without exerting additional effort.
Ultimately, research in the time of COVID-19 has taught me a wealth of lessons that have impacted not only my practice as a researcher but my practice as a classroom teacher as well. This process has taught me the art of practicing patience. I frequently had to revise, rethink, or reconsider methodological approaches, and instead of being stymied by the roadblocks, I learned that there is always a way to get something accomplished. This process has also taught me to look for and celebrate the capital in others (Coleman, 1988). I often sought the wisdom and expertise of others so that my study could be approved. And, as much as I cling to the paper and pencil, I have learned that technology can provide practical solutions to challenges that seem daunting. Research in the time of COVID-19 has become more than just an attempt to conduct an international case study about one school surrounded by several villages and has challenged me to recognize and rely on others' wisdom, expertise, and various forms of capital every day.
IMPLICATIONS
Navigating the dissertation in practice process during a global pandemic led to shifts in research topics and methodologies, as well as to our needs as doctoral students. We relied heavily on our dissertation chairs and program faculty to guide us towards alternative research approaches that would maintain our prepandemic plans as much as possible. For example, when in-person interactions were no longer feasible, faculty recommended replacing observations with document analysis. Similarly, when one researcher's inability to travel to Senegal threatened to upend her study, the chair suggested hiring a Senegalese research assistant who would conduct the interviews for her. These troubleshooting conversations mostly took place via Zoom and email, adding another layer of change during the dissertation process. Ultimately, both students and faculty needed to be nimble and adaptable, working together to find quick, creative solutions to seemingly insurmountable challenges.
Scholarly practitioners are strategically positioned to respond to their contexts in ways that are informed by their experiences, knowledge of their communities of practice, and scholarly work. Furthermore, we all possess elements of what Collins (1991) presents as the ethic of caring, an epistemological dimension used by African American women. Elements of this ethic include an emphasis on individual uniqueness which respects human beings as unique beings. All researchers demonstrated a high respect for our participants and the context of our research. The second element of this ethic involves emotions in dialogue, meaning the understanding that emotions and thoughts are inextricably connected and cannot be separated. Each researcher created space to hold the emotional toll of the pandemic on our participants and devised ways to include this aspect of the current conditions in pre-interviews or during check-in sessions. The third element of this ethic is empathy. We were empathetic to the difficulties faced by our participants and communities. Their high level of empathy is what motivated us to pivot during the pandemic as a means to acknowledge the impact of the pandemic on our scholarly work. Finally, there is an element of personal accountability for the knowledge that is being created that is deeply rooted in values and ethics. "Neither emotion nor ethics is subordinated to reason. Indeed, emotion, ethics and reason are used as interconnected, essential components in assessing knowledge claims" (Collins, 1991, p. 219).
Only one of the authors identifies as African American. However, we are all women who come to our scholarly work from a social justice stance that is very much informed by the intersection of our multiple identities-social justice female leaders working in contexts that support marginalized groups. As we embrace the communities for which we work, our identities are steeped in the challenges our students and educational communities face. It is in fact that saturation that informs our liberatory research practices; we all bear witness to the resilience and shared humanity palpable in our educational organizations. | 2021-07-25T16:22:14.036Z | 2021-04-30T00:00:00.000 | {
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264180935 | pes2o/s2orc | v3-fos-license | Therapeutic efficacy of omalizumab in children with moderate-to-severe allergic asthma combined with chronic sinusitis
Background Omalizumab has been approved for treating moderate-to-severe asthma in children aged over 6 years. Its application to asthmatic children with other allergic diseases has been rarely explored. The present study aims to explore the therapeutic efficacy of omalizumab in children with moderate-to-severe allergic asthma combined with chronic sinusitis. Methods The clinical data of children diagnosed with moderate-to-severe allergic asthma combined with chronic sinusitis and treated with omalizumab between September 2020 and April 2022 were retrospectively analyzed. Lung function indexes such as Childhood Asthma Control Test (C-ACT) scores, fractional exhaled nitric oxide (FeNO), and forced expiratory volume in the first second (FEV1) percent predicted (FEV1%pred), small airway function indexes, and the clinical symptoms of chronic sinusitis were analyzed. Results A total of 26 children were observed for 16 weeks. After 16 weeks of omalizumab treatment, the significantly increased C-ACT scores (15.57 ± 3.25 points vs. 24.98 ± 5.21 points, F = 15.7112, P < 0.001) and decreased FeNO (31.55 ± 15.57 ppb vs. 19.86 ± 9.80 ppb, F = 4.4265, P = 0.0022), compared with those at baseline, were suggestive of well-controlled symptoms of asthma and improved lung function. FEV1%pred and FEV1/forced vital capacity (FVC) ratio (the ratio of the forced expiratory volume in the first 1 s to the forced vital capacity) increased after omalizumab treatment, although no significant differences were detected (P = 0.9954 and 0.9382, respectively). Peak expiratory flow (PEF) percent predicted (PEF%pred) and forced expiratory flow at 75% of FVC (FEF75%), 50% of FVC (FEF50%), and 25%–75% of FVC (FEF25%–75%) significantly increased after omalizumab treatment (P = 0.0477, <0.001, <0.001, and <0.001, respectively). Visual analog scale scores significantly decreased after omalizumab treatment (6.40 ± 2.98 points vs. 0.85 ± 0.40 points, t = 27.2419, P < 0.001), suggesting alleviation in the clinical symptoms of chronic sinusitis. Conclusion In this study, it was found that omalizumab can effectively alleviate clinical symptoms and improve lung function and quality of life in children with moderate-to-severe allergic asthma combined with chronic sinusitis.
Introduction
Allergic asthma, a type of asthma caused and/or triggered by inhaled allergens, is characterized by recurrent wheezing, coughing, shortness of breath, and chest tightness.Allergic asthma is the most common type of childhood asthma, with an increasing prevalence in recent years (1,2).Eosinophilia, mast cell activation, and immunoglobulin E (IgE) elevations caused by allergen exposures are important events during the onset of allergic asthma (3).Childhood asthma often accompanies other allergic diseases such as allergic rhinitis, sinusitis, eczema, atopic dermatitis, and chronic urticaria, which seriously affects the quality of life of children and their parents and brings challenges to clinical management.
Omalizumab is the first targeted drug for the treatment of allergic asthma.It is a recombinant humanized monoclonal antibody that selectively binds to IgE to inhibit its combination with high-affinity receptors on the surface of effector cells, thus preventing effector cell degranulation, inflammatory factor release, and inflammatory cell recruitment (4).The therapeutic efficacy of omalizumab on childhood asthma has been extensively validated (3,5), although its application in the treatment of children with asthma, combined with other allergic diseases, has been rarely explored.In the present study, we retrospectively analyzed the efficacy of omalizumab in children with moderate-to-severe allergic asthma, combined with chronic sinusitis, thus providing clinical experiences for clinical management.
Study subjects
Children diagnosed with moderate-to-severe allergic asthma and chronic sinusitis were retrospectively recruited in the study at the Allergic Disease Clinic, Department of Pediatrics, BenQ Medical Center, Nanjing.They were treated with omalizumab between September 2020 and April 2022.The inclusion criteria included (i) children aged ≥6 and <18 years; (ii) diagnosis of allergic asthma in accordance with the Guidelines for the Diagnosis and Treatment of Bronchial Asthma in Children (2016 Edition) (6), where asthma well controlled by step 3 therapy was defined as moderate (2, 6) and that well controlled (or still uncontrolled) by step 4-5 therapy was defined as severe (6).Chronic sinusitis was diagnosed on the basis of symptoms and signs, combined with the results of nasal endoscopy and computed tomography (CT) scans (7,8).All recruited children showed no nasal polyps; (iii) a positive allergy test result for serum IgE, a positive skin prick test result, or a positive blood test result for allergen-specific IgE (sIgE) (2); and (iv) children or their guardians were able to cooperate with the questionnaire survey.The exclusion criteria included children (i) exhibiting allergy to the active ingredients of omalizumab or any other excipients; (ii) who had complicated conditions such as chronic lung disease, pneumonia, pneumothorax, thoracic deformity, heart failure, and other diseases that may affect cardiopulmonary function; and (iii) who had received a course of omalizumab treatment lasting for less than 4 months.Written informed consent was obtained from the guardians or parents of the recruited children before providing the first omalizumab treatment.
Study design
The dosage and frequency of omalizumab injections were determined on the basis of instructions for the use of omalizumab, body mass index (BMI), and baseline serum IgE.In general, omalizumab was subcutaneously injected every 2-4 weeks at 150-600 mg per administration.Information on Childhood Asthma Control Test (C-ACT) scores, fractional exhaled nitric oxide (FeNO), lung function indexes, and visual analog scale (VAS) scores for chronic sinusitis was collected before administering omalizumab injections in the 1st, 4th, 8th, 12th, and 16th weeks.Incidentally, all these evaluations are part of our routine practice during follow-up.
Efficacy evaluation of allergic asthma
The therapeutic efficacy of omalizumab on allergic asthma in children aged 6-11 years and those older than 12 years was assessed using the C-ACT and ACT, respectively.In addition, FeNO and lung function indexes such as forced expiratory volume in the first second (FEV 1 ) percent predicted (FEV 1 % pred), forced expiratory volume in the first second (FEV 1 )/forced vital capacity (FVC), and small airway indexes were measured.
Evaluation of chronic sinusitis
The subjective symptoms of chronic sinusitis in children, such as nasal congestion, runny nose, cough, and headache, were assessed using a VAS scale of 0-10 points and classified into mild (≤3 points), moderate (3 < VAS scores ≤ 7 points), and severe (>8 points) (7).
Statistical analyses
Statistical analyses were performed using SPSS 20.0.Measurement data that were normally distributed were expressed as x + s, and differences between and among groups were analyzed by using the paired t-test and repeated-measures ANOVA, respectively.A score of P < 0.05 was considered statistically significant.
Population characteristics
A total of 26 eligible children with moderate-to-severe allergic asthma, combined with chronic sinusitis, were recruited, of which 14 were boys and 12 were girls aged between 6 years and 5 months and between 14 years and 9 months, respectively.The median total serum IgE was 490 (117-1,389) IU ml −1 .Subcutaneous injections of omalizumab were administered in all recruited children as follows: eight children were administered with a dose of 300 mg every 4 weeks, six with a dose of 150 mg every 4 weeks, five with a dose of 450 mg every 4 weeks, five with a dose of 300 mg every 2 weeks, and two with a dose of 600 mg every 4 weeks.Adverse events were not reported during the treatment period.A CT scans of the sinuses were performed in 12 children as follows: five children were scanned for paranasal sinusitis of four-paired sinuses; three for maxillary sinusitis, ethmoid sinusitis, and sphenoid sinusitis; three for maxillary sinusitis and ethmoid sinusitis; and one for maxillary sinusitis.All children had moderate-to-severe asthma, with a course ranging from 6 months to 3 years.Routine treatment of asthma, such as the use of inhaled and intranasal corticosteroids, nasal irrigation, and mucolytics, was provided before administering the omalizumab injections.However, the clinical symptoms of asthma and sinusitis still recurred or persisted.
Therapeutic efficacy of omalizumab on allergic asthma in children
This study enrolled two children older than 11 years.After the omalizumab injections were administered, their ACT scores demonstrated a significant improvement.In one child aged 12 years and 2 months, the ACT score increased from 18 to 24 points, while in a 14-year and 9-month-old child, the score increased from 21 to 25 points.The C-ACT scores of the remaining 24 children younger than 11 years significantly improved after 16 weeks of omalizumab treatment compared with the baseline (P < 0.05).FeNO in all recruited children significantly reduced after omalizumab injections (P < 0.05).Lung function indexes, such as FEV 1 %pred, FEV 1 /FVC, Peak expiratory flow (PEF) percent predicted (PEF%pred), forced expiratory flow at 75% of FVC (FEF 75% ), 50% of FVC (FEF 50% ), and 25%-75% of FVC (FEF 25%-75% ), all showed favorable changes after administering the injections, although significant differences were detected only in the last three indexes mentioned (P < 0.05, Table 1).Inhaled corticosteroids (ICSs) were withdrawn in 10 children after 12 weeks of omalizumab treatment, and they were withdrawn in the remaining 16 children after 16 weeks of treatment.
Therapeutic efficacy of omalizumab on chronic sinusitis in children
After 16 weeks of omalizumab treatment, the clinical symptoms of sinusitis in children significantly improved.Withdrawal of nasal corticosteroids was achieved in 12, nine, and five children after omalizumab treatment for 8, 12, and 16 weeks, respectively.The VAS scores of the clinical symptoms of chronic sinusitis significantly reduced after omalizumab treatment in all recruited children (P < 0.05, Table 2).A CT scan re-examination of the sinuses was performed in seven children after 16 weeks, all indicating a significant alleviation in chronic sinusitis.Figure 1 shows representative CT scans of sinuses in a 9-year-old boy.
Adverse events of omalizumab
Local and systemic adverse events were not reported during the 16-week course of omalizumab treatment.Liver and renal dysfunctions were not detected during the regular follow-up testing.
Discussion
Childhood asthma is mainly controlled by medications of ICSs, ICSs combined with long-acting β2 agonists (ICSs-LABAs), and leukotriene receptor antagonists (LTRAs).However, it has been found that the symptoms in some asthmatic children have not been well controlled by these drugs.A multicenter study has reported that asthma is controlled in 19.9% of children aged 2-16 years (9).Children with asthma usually suffer from many comorbidities such as allergic rhinitis, sinusitis, atopic dermatitis, food allergy, obesity, and gastroesophageal reflux disease, which potentially influence the clinical management of asthma and thus pose greater challenges in controlling asthmatic symptoms (10).
Chronic sinusitis and asthma are common heterogeneous diseases in children sharing the same pathogenesis.Sinusitis is an inflammation of the nasal cavity and sinus mucosa.Asthma is a chronic inflammatory disease of the lower respiratory tract.Closely linked to allergen exposures, both sinusitis and asthma often coexist and interrelate with each other (11).At present, the causal relationship between sinusitis and asthma is controversial.However, previous findings have validated their epidemiological correlation (12,13).Approximately 40% of adults and children with asthma suffer from the comorbidity of sinusitis, and the incidence rate of sinusitis is even higher in patients with severe asthma (14).
For children and adolescents with poorly controlled asthma, nasal endoscopy is recommended to identify occult or obvious sinusitis, and CT examinations are performed if necessary (15).However, the clinical symptoms of upper airway inflammation in children with allergic asthma are usually poorly controlled because of the lack of clinical experience in the management of chronic sinusitis.Intranasal corticosteroids and nasal irrigation with topical antibiotics or normal saline are common therapeutic strategies for chronic sinusitis in children (16).Nevertheless, a single treatment usually cannot reap an acceptable therapeutic outcome because of the complicated pathogenesis of sinusitis and persistent inflammatory response.
Precision medicine and immunotherapy have transformed the treatment of airway inflammatory diseases (17).Omalizumab, a recombinant humanized anti-IgE monoclonal antibody, is the first targeted drug for the treatment of asthma.After 10 years of its clinical application, omalizumab has been validated for its efficacy in controlling the symptoms of asthma, preventing the aggravation of asthma, and lowering the medical utilization and the rate of class absence (18).Previous studies have demonstrated that omalizumab treatment alleviates not only asthma symptoms but also the outcomes of asthma comorbidities (14,19).Similar to the immune process of asthma and allergic rhinitis, IgE is of great significance in the Th2 responses of chronic sinusitis.Meanwhile, serum IgE level is correlated with the inflammation severity of the mucous membrane lining the sinuses.Considering the pathogenic similarities shared by asthma and chronic sinusitis, omalizumab injections also provide clinical benefits to patients with chronic sinusitis (20).In December 2020, the Food and Drug Administration (FDA) approved omalizumab for the treatment of chronic rhinosinusitis with nasal polyps (CRSwNP) (21).All recruited asthma children had complications of chronic sinusitis.Their nasal symptoms of sinusitis were recurrent with a longperiod course ranging from 6 months to 3 years, even after ICSs and nasal irrigation were used, which significantly influenced the quality of life.After omalizumab treatment, significantly decreased VAS scores were indicative of relieved symptoms of sinusitis.A total of 12 children achieved withdrawal of intranasal corticosteroids after 8 weeks of omalizumab treatment.The sinuses of seven children were re-scanned using CT after 16 weeks of treatment, suggesting an obvious relief in inflammation in the sinuses.However, we were unable to provide the Lund-Mackay score to quantitatively reflect the change in chronic sinusitis before and after treatment.At 16 weeks of omalizumab treatment, all recruited children withdrew intranasal corticosteroids.So far, they have been regularly followed up in our pediatric outpatient clinic, and they have been presenting stable symptoms of allergic asthma and chronic sinusitis.Some children were also given intranasal corticosteroids during allergy seasons, and their symptoms could be well controlled within 1 week.Sui et al. (22) consistently reported the acceptable efficacy and safety of omalizumab in the treatment of asthma combined with refractory sinusitis in Chinese patients.An anti-IgE therapy is recommended for children with allergic asthma combined with chronic sinusitis because they are generally poorly responsive to conventional treatment.
Several multicenter, randomized, double-blinded controlled studies have shown that ICSs, combined with omalizumab, can effectively control the clinical symptoms of moderate-to-severe allergic asthma in children and reduce the incidence of an acute attack of asthma (23,24).In the present study, significantly increased C-ACT and ACT scores after omalizumab injections suggested that omalizumab treatment effectively relieved asthma Japanese research team demonstrated that omalizumab treatment did not significantly improve FEV 1 %pred and FEF 25%-75% pred in asthmatic children (27).In the present study, both FEV 1 %pred and FEV 1 /FVC increased after 16 weeks of omalizumab treatment, although no significant differences were detected.PEF%pred, FEF 75% , FEF 50% , and FEF 25%-75% in children significantly increased after omalizumab treatment, suggesting that omalizumab mainly improved the small airway function.In addition, the significantly decreased FeNO after omalizumab treatment could be attributed to the removal of eosinophilic airway inflammation.Taken together, omalizumab injections effectively relieved the symptoms of asthma and sinusitis and improved lung function and quality of life in children with moderate-to-severe allergic asthma, combined with chronic sinusitis, with no local and systemic adverse events.Notably, this study was limited by a small sample size, a short follow-up period, and a lack of the Lund-Kennedy score for objectively evaluating endoscopic mucosal morphology of sinusitis.The small sample size and the selection of patients with only moderate asthma may limit our results about the impact of omalizumab on some clinical parameters such as lung function FEV1%pred and FEV1/FVC.Therefore, our findings should be further validated in a large-scale study using a rigorous study design.
TABLE 1 C
-ACT scores, FeNO, and lung function indexes before and after omalizumab treatment (n = 26).
a C-ACT scores were graded in 24 children younger than 11 years.
TABLE 2
VAS scores for assessing the clinical symptoms of chronic sinusitis before and after omalizumab treatment (n = 26).andimproved the quality of life of affected children.The role of omalizumab in enhancing the lung function of asthma children has been found to be controversial.Oliveira et al. (25) reported that omalizumab treatment increases FEV 1 in obese patients with severe asthma.Licari et al. (26)revealed that FEV 1 % pred in children with severe allergic asthma increases from 79% to 91% after 12 months of omalizumab treatment.However, a FIGURE 1Representative CT scans of sinuses in a 9-year-old boy with moderateto-severe allergic asthma combined with chronic sinusitis before and after 16 weeks of omalizumab treatment.CT scans of the sphenoid sinus and ethmoid sinus before (A) and after (B) omalizumab treatment.CT scans of the frontal sinus before (C) and after (D) omalizumab treatment.CT scans of the maxillary sinus before (E) and after (F) omalizumab treatment.Red arrows indicate sinusitis.symptoms | 2023-10-18T15:15:12.078Z | 2023-10-16T00:00:00.000 | {
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53043063 | pes2o/s2orc | v3-fos-license | Propofol is a more effective and safer sedative agent than midazolam in endoscopic injection sclerotherapy for esophageal varices in patients with liver cirrhosis : a randomized controlled trial
Objective : The efficacy of sedation during endoscopic injection sclerotherapy (EIS) for esophageal varices (EVs) in patients with liver cirrhosis remains unclear. The aim of this study is to compare the efficacy and safety between propofoland midazolam-based sedation for EIS. Methods : Twenty-three patients with EVs were prospectively and randomly assigned to midazolam-based (Midazolam group) or propofol-based (Propofol group) sedation. All patients underwent a number connection test (NCT) to evaluate minimal hepatic encephalopathy (MHE) on the day before and at 2 and 24 hours following EIS. The primary endpoint was exacerbation of MHE after EIS, which was defined as deterioration of the NCT. The secondary endpoints were postoperative awareness, technical success rate, frequency of body movement, patient and operator satisfaction, cardiorespiratory dynamics during EIS, and adverse events. Results : Exacerbations of MHE at 2 hours after EIS compared with those before EIS were not significantly different between the two groups. In both groups, the deterioration of NCT scores before and 2 hours after EIS was observed (Propofol group : 60.0 vs. 70.0 s, P = 0.026 ; Midazolam group : 42.5 vs. 67.0 s, P = 0.002). There were no significant differences in awareness, technical success rate, or patient satisfaction. However, the frequency of body movement in the Propofol group was significantly lower than that in the Midazolam group (1 vs. 4, P = 0.045), and operator satisfaction in the Propofol group was significantly higher than that in the Midazolam group (P = 0.016). No adverse events were observed. Conclusions : Propofol-based sedation exacerbated MHE after EIS similarly to midazolam-based sedation in patients with liver cirrhosis. However, propofol-based sedation provided stable sedation with a lower frequency of body movements and high operator satisfaction.
Introduction
Endoscopic injection sclerotherapy (EIS) using ethanolamine oleate (EO) as an intravariceal scle-rosant is an established treatment for esophageal varices (EVs) in Japan 1,2) . EIS is a technically demanding and lengthy procedure involving injection into the variceal lumen and requires appropriate se-dation. Midazolam is the main sedative agent used in endoscopic procedures 3) . However, it is difficult to achieve stable maintenance anesthesia through the intermittent intravenous administration of midazolam 4) . Recently, an increasing number of reports have been published on the use of propofol for various endoscopic procedures 5 -10) . Propofol has a rapid onset and short duration of action 11) . Therefore, propofol can be continuously administered, enabling the constant maintenance of the depth of sedation. However, propofol has a narrow safety range that can result in the rapid depression of cardiovascular function 12,13) .
Many patients who undergo EIS suffer from liver cirrhosis. Patients with liver dysfunction are at increased risk of sedationrelated adverse events, such as cardiorespiratory depression, particularly when they receive sedation using midazolam 14,15) . Propofol, however, has a favorable pharmacokinetic profile and does not require any dose adjustment in patients with liver cirrhosis 16,17) . In addition, the main problem with sedation in patients with liver cirrhosis is the potential influence on minimal hepatic encephalopathy (MHE). MHE is defined as a condition in which patients demonstrate quantifiable neuropsychological defects in psychometric tests, such as the number connection test (NCT), yet these individuals show a normal mental and neurological status upon clinical examination [18]. Sedation using midazolam has been reported to exacerbate MHE in patients with liver cirrhosis 19 -23) . In contrast, sedation using propofol has not been reported to exacerbate MHE 21 -26) . However, previous studies have only reported the use of sedation for esophagogastroduodenoscopy (EGD) or endoscopic variceal ligation (EVL). The efficacy and safety of sedation for EIS remain unclear. In addition, there have been no studies comparing propofol with midazolam as sedation for EIS. Therefore, the aim of this prospective study is to compare the efficacy and safety between propofolbased and midazolambased sedation for EIS.
Study Design and Patients
We conducted a prospective, randomized controlled parallel group trial at Fukushima Medical University Hospital between April 2015 and October 2016. Patients with EVs and liver cirrhosis who were scheduled for treatment with prophylactic EIS were randomly assigned to one of two groups : mid-azolambased (Midazolam group) or propofolbased sedation (Propofol group) (simple randomization, allocation ratio 1 : 1). Randomization was performed by sequentially opening numbered opaque envelopes with computergenerated group allocation cards in a random sequence. Although the operator and assistant were not blinded, the patients were blinded to their allocation. The randomization list was stored in the laboratory and completely concealed from the operator.
The following inclusion criteria were considered : 1) patients with liver cirrhosis who were scheduled for treatment with prophylactic EIS for EVs ; 2) patients without a history of treatment or bleeding of EVs ; 3) patients between 20 and 80 years of age ; 4) patients with a performance status of 0 ; and 5) patients who provided consent to receive EIS and participate in this study. Indications for prophylactic EIS were moderate or largesized varices (F2 or F3) and/or red color signs on varices diagnosed based on endoscopic findings according to the Japan Research Society for Portal Hypertension 27) . The diagnosis of liver cirrhosis was based on history, serologic testing, radiologic imaging, and liver histology when available. The following exclusion criteria were considered : 1) American Society of Anesthesiologists classification III, IV, or V ; 2) severe hepatic disorder (Child -Pugh C classification and serum total bilirubin of 4 mg/dL or greater) ; 3) hepatocellular carcinoma with portal vein invasion ; 4) alcohol intake during the past 2 weeks ; 5) use of illicit drugs or drugs that act in the central nervous system, such as benzodiazepines ; 6) clinically overt hepatic encephalopathy (HE) ; 7) dementia ; 8) neurologic diseases, such as Alzheimer's disease or Parkinson's disease ; 9) renal failure (serum creatinine > 2 mg/dL) ; 10) allergy to midazolam or propofol ; and 11) patients who did not elect to undergo EIS. There was no modification of these criteria during this study period.
This study was conducted according to the Declaration of Helsinki, approved by the Ethics Committee of Fukushima Medical University (approval No. 2058) and registered in the University Hospital Medical Information Network (UMIN) as number UMIN 000017173. All the patients provided written consent to participate in the study.
EIS Procedures
EIS was performed using a GIF -Q240X endoscope (Olympus Medical Systems Corp., Tokyo, Japan) with an oral side balloon (CREATE MEDIC Sedation for EIS Co., Ltd., Yokohama, Japan) and a 23 -or 25 -gauge injection needle (TOP Corporation, Tokyo, Japan). The EIS procedure was performed by intravariceal injection of 5% EO (Oldamin ; ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) under fluoroscopic guidance. EO was injected to fill the varices to the supplying vessels, such as the left gastric vein. The puncture was repeated for multiple varices until the injectable varices disappeared or the maximum amount of EO (0.4 mL/kg) was injected. All EIS procedures were performed by a single expert physician (K.W.) who was a qualified doctor accredited by the skill qualification system of the Japan Society for Portal Hypertension and had extensive experience in performing EIS over 100 times.
Monitoring and Sedation Protocols
During EIS, patients received oxygen via a nasal cannula at a rate of 2 L/min. Blood pressure (BP) was immediately measured after sedation, then every minute until the insertion of the endoscope and every 5 minutes thereafter. Heart rate (HR) and oxygen saturation (SpO2) were also continuously monitored. Sedation was administered by an independent physician with expertise in anesthesia and who was not involved in the endoscopic procedures.
In the Midazolam group, anesthesia was induced by a bolus intravenous (IV) infusion of 15 mg of pentazocine (Sosegon ; Astellas Pharma, Tokyo, Japan) as an analgesic plus 2.5 -5 mg of midazolam (Midazolam ; Sandoz, Tokyo, Japan). In the event of body movement or signs of discomfort, an additional bolus IV infusion of 2.5 mg of midazolam was administered. Body movement was defined as movement disturbing the procedure and requiring control by a third person, such as a nurse. If cardiopulmonary suppression, such as hypotension with systolic BP < 80 mmHg or oxygen desaturation < 90%, was observed, then the amount of drip infusion or flow volume of oxygen was increased. When needed, flumazenil (Flumazenil ; Maruishi Pharmaceutical, Osaka, Japan) was administered as a reversal agent. After the completion of EIS, patients recovered from anesthesia with the administration of 0.5 mg of flumazenil.
In the Propofol group, anesthesia was induced by a bolus IV infusion of 15 mg of pentazocine plus 20 mg of propofol (1% Propofol ; Maruishi Pharmaceutical, Osaka, Japan) followed by a continuous infusion of propofol using an infusion pump. The rate was maintained at 3 -5 mg/kg/h during EIS. In the event of body movement or signs of discomfort, a bolus IV infusion of 20 mg of propofol was administered, and when needed, the continuous infusion rate was increased. If cardiopulmonary suppression was observed, the amount of drip infusion or flow volume of oxygen was increased, and the maintenance dose was reduced by 1 mg/kg/h. After the completion of EIS, the continuous propofol infusion was discontinued to allow the patient to recover from anesthesia. In both groups, 7.5 mg of pentazocine was administered every 30 min during EIS.
The sedation depth in both groups was adjusted to "deep sedation", under which spontaneous breathing was maintained, according to the "Practice Guidelines for Sedation and Analgesia by Non -Anesthesiologists" published by the American Society of Anesthesiologists 28) .
Number Connection Test
The NCT consisted of documenting the time required to sequentially connect randomly placed circles labeled from 1 to 25. Different patterns were used to eliminate the learning effect of repetitive testing. The total time required was assessed using a stopwatch by a single examiner who was blinded to the randomization group and not involved in the endoscopic procedures. If the patient could not complete the NCT within 30 seconds, the result was defined as MHE 29) . The NCT was performed the day before and at 2 and 24 hours after EIS to evaluate exacerbation of MHE.
Outcomes
The primary endpoint was exacerbation of MHE after EIS, which was defined as deterioration of the NCT. The secondary endpoints were postoperative awareness, technical success rate, frequency of patient body movement during EIS, patient and operator satisfaction, cardiorespiratory dynamics during EIS, and adverse events. Postoperative awareness was evaluated at 2 and 24 hours after EIS according to the Observer's Assessment of Awareness/Sedation Scale (OAAS : 1 point, awake ; 2 points, somnolent/drowsy ; 3 points, responsive to loud or repeated verbal stimuli ; 4 points, responsive to physical/painful stimuli ; and 5, no response to physical/painful stimuli) 3) . Technical success was defined as an injection of EO from the varices to the supplying vessels under fluoroscopic guidance. Patient and operator satisfaction were evaluated on a 10 -point visual analog scale, where 1 point indicated the worst and 10 points indicated the best. This assessment was performed using a questionnaire after EIS. Adverse events related to sedation were defined as hypotension with a systolic BP (SBP) < 80 mmHg, SpO2 < 90%, or bradycardia with a HR < 45 beats/min. The characteristics of each patient were obtained before EIS, and procedural details were prospectively recorded in a database.
Sample Size Calculation and Statistical Analysis
Based on the results of previous studies, NCT scores were worse in greater than 70% of patients after sedation using midazolam 19,20) . However, NCT scores were not worse after sedation using propofol 21 -23,25) . The required sample size was calculated as 10 patients for each group to detect a significant difference with a power of 0.8, a type I error of 0.05, and a type II error of 0.02 using Open -Epi version 3.0 (free and open source software available from http://www.openepi.com/Menu/OE_Menu.htm). Considering dropout cases, the inclusion number for this study was set at 23 patients for both groups.
Normally distributed data are reported as the means (± standard deviation [SD]), and data that were not normally distributed are reported as the median with a range. Changes in NCT scores before and after EIS were analyzed using the Wilcoxon's signedrank test. Continuous variables were analyzed using the Student's ttest or the Mann -Whitney U test. Categorical variables were analyzed using the Chisquared (χ 2 ) test. Differences were considered statistically significant at P < 0.05. This analysis was performed using SPSS software (version 21 for windows ; IBM Corp, Armonk, NY, USA).
Patient Characteristics
A total of 23 consecutive patients with EVs were enrolled in this study. Twelve patients were assigned to the Midazolam group, and 11 patients were assigned to the Propofol group, as shown in Figure 1. The patient characteristics are summarized in Table 1. There was no significant difference in age, gender, cirrhosis etiology, Child -Pugh classification, and variceal size between groups.
Outcomes
All patients in the Propofol group had MHE, which was defined as being unable to complete the NCT within 30 seconds before EIS. Ten patients (83.3% : 10/12) in the Midazolam group had MHE before EIS. All patients in both groups had MHE 2 hours after EIS. NCT scores at 2 hours after EIS were compared to scores before EIS and expressed as the differences between the values before EIS and the values at 2 hours after EIS. These scores were not significantly different between groups (Table 2). In both groups, significant deterioration of NCT scores between before and 2 hours after EIS was observed (Propofol group : P = 0.026, Midazolam group : P = 0.002), which indicated propofol also exacerbated MHE similar to midazolam. However, no significant difference was observed in NCT scores before and at 24 hours after EIS in both groups (Figure 2a and b, Table 2). In both groups,
137
Sedation for EIS there were no significant differences in NCT scores before and at 2 or 24 hours after EIS ( Table 2). None of the patients developed overt hepatic encephalopathy after EIS.
In terms of awareness, there was no significant difference between groups in OAAS at 2 hours after EIS ( Table 2). The technical success rate, procedure time, and total amount of EO injected were not significantly different between groups ( Table 3). The frequency of body movement during EIS was Table 3). There was no difference in patient satisfaction between groups ; however, operator satisfaction in the Propofol group was significantly higher than that in the Midazolam group (10 vs. 7, P = 0.016, Table 3). Procedure discontinuation was not necessary in any case.
In terms of cardiorespiratory dynamics, there were no differences in the minimum values of SBP, SpO2 and HR during EIS. Changes in the minimum SBP compared with baseline in the Propofol group were significantly higher than those in the Midazolam group (P = 0.009, Table 3). However, none of the patients experienced adverse events. EIS was safely performed in all cases when sedative administration was performed by physicians who were dedicated to sedation with an expertise in anesthesia.
Discussion
To the best of our knowledge, this study is the first trial to compare propofolbased sedation with midazolambased sedation for EIS in patients with Fig | 2018-11-09T23:10:04.989Z | 2018-10-21T00:00:00.000 | {
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184486955 | pes2o/s2orc | v3-fos-license | Comparative Study of Two Chondroitin Sulfate/Dermatan Sulfate 4-O-Sulfatases With High Identity
Chondroitin sulfate/dermatan sulfate (CS/DS) sulfatases are potential tools for structural and functional studies of CD/DS chains. In our previous study, a CS/DS 4-O-endosulfatase (endoVB4SF) was identified from a marine bacterium (Wang et al., 2015). Herein, another CS/DS 4-O-sulfatase (exoPB4SF) was identified from a Photobacterium sp. ExoPB4SF shares an 83% identity with endoVB4SF but showed strict exolytic activity. Comparative studies were performed for both enzymes on the basis of biochemical features, substrate-degrading patterns and three-dimensional structures. exoPB4SF exhibited a wider temperature and pH adaptability and better thermostability than endoVB4SF. Furthermore, exoPB4SF is a strict exolytic sulfatase that only releases the sulfate group from the GalNAc residue located at the reducing end, whereas endoVB4SF preferentially removed sulfate esters from the reducing end toward the non-reducing end though its directional degradation property was not strict. In addition, the structure of endoVB4SF was determined by X-ray crystallography at 1.95 Å. It adopts a globular conformation with two monomers per asymmetric unit. The exoPB4SF structure was constructed by homology modeling. Molecular docking results showed that although the residues around the catalytic center are conserved, the residues at the active site of endoVB4SF adopted a more favorable conformation for the binding of long CS/DS chains than those of exoPB4SF, which may explain why the two highly homogenous sulfatases possessed different action patterns. The results of this study provide insight into the structure-function relationship of CS/DS endo- and exosulfatases for the first time.
INTRODUCTION
Chondroitin sulfate (CS) and dermatan sulfate (DS) are glycosaminoglycan (GAG) sidechains of proteoglycans that are widely present in the extracellular matrix and on cell surfaces (Iozzo, 1998). The backbone of CS chains is composed of -[GlcUA-β1-3-GalNAc]-disaccharide units linked by β1-4-glycosidic bonds, while in DS, the β-D-glucuronic acid (GlcUA) residues are transformed to α-L-iduronic acid (IdoUA) residues through the action of glucuronyl C5 epimerase. Thus, CS and DS usually exist in a hybrid form of CS-DS (Sugahara et al., 2003). The backbone of CS/DS can be modified by various sulfotransferases to produce many different disaccharide structures with unique sulfation patterns, such as the following common CS disaccharide units: O unit (GlcUAβ1-3GalNAc), C unit (Linhardt and Hileman, 1995). CS/DS chains are structural materials in connective tissues and are key functional molecules regulating various physiological and pathological processes (Linhardt and Toida, 2004), such as cell division, central nervous system development, viral and bacterial infections, and tumor progression (Hardingham and Fosang, 1992;Faissner et al., 1994;Wang et al., 2016). Many studies have shown that CS/DS chains exert these functions through interactions with various proteins, such as adhesion molecules, growth factors and cytokines, where the sulfation pattern of CS/DS chains is crucial for these interactions (Malavaki et al., 2008;Mizumoto et al., 2015;Djerbal et al., 2017).
CS/DS sulfatases specifically catalyze the hydrolysis of sulfate groups from CS/DS chains and are potential tools for structural and functional studies of CS/DS (Sugahara and Kojima, 1996;Ulmer et al., 2014;Wang et al., 2015). These enzymes belong to the conservative arylsulfatase family, whose members carry a C α -formylglycine (FGly) residue generated by the oxidation of a conserved cysteine/serine residue at the active site of the enzyme and are essential for the degradation of CS/DS in various organisms, from bacteria to animals (Knaust et al., 1998;Boltes et al., 2001;Berteau et al., 2006). In humans, deficiencies in these enzymes results in a series of physical disorders, such as multiple sulfatase deficiency (MSD) (Czyzewska and Dzialoszynski, 1978;Dierks et al., 2003). Recently, a CS/DS sulfatase was shown to participate in the axon regeneration process of central nervous system injures in mammals through the modification of CS/DS chains (Pearson et al., 2018). However, despite being an important class of enzymes, only a few CS/DS sulfatases from prokaryotes and eukaryotes have been studied in detail, including N-acetylgalactosamine-4-O-sulfatase (Wang et al., 2015), N-acetylgalactosamine-6-O-sulfatase and 4,5 hexuronate-2-O-sulfatase (Sugahara and Kojima, 1996;Myette et al., 2003;Ulmer et al., 2014).
Most of the identified CS/DS sulfatases are exo-type enzymes, which hydrolyze sulfate esters from the ends of CS/DS poly-/oligosaccharides. The catalytic mechanisms of these enzymes have been well elucidated through structural and biochemical studies of several enzymes, including three human CS/DS exosulfatases: ARSB (4-O-sulfatase) (Bond et al., 1997), GALNS (6-O-sulfatase) (Rivera-Colon et al., 2012) and IDS (iduronate-2-O-sulfatase) (Demydchuk et al., 2017); and two bacterial 4,5 hexuronate-2-O-sulfatases from Pedobacter heparinus (Raman et al., 2003) and Bacteroides thetaiotaomicron (Ulmer et al., 2014). In contrast, endo-type CS/DS sulfatases, which can effectively remove sulfate groups within the CS/DS chains, are rare, with only two endo-4-O-sulfatases having been identified from Bacteroides thetaiotaomicron (Ulmer et al., 2014) and Vibrio sp. (Wang et al., 2015). Compared to exosulfatases, CS/DS endosulfatases are more potent tools for structural and functional studies of CS/DS. However, the detailed action pattern and catalytic mechanism of these enzymes remain to be investigated using biochemical and crystallographic methods.
In this study, a novel exo-type CS/DS 4-O-sulfatase (exoPB4SF) was identified from a newly isolated bacterium, Photobacterium sp. FC615. Interestingly, this enzyme shares high sequence identity (83%) with the endo-type 4-O-sulfatase (endoVB4SF) from Vibrio sp. (Wang et al., 2015), providing an ideal model for investigating the different mechanisms of action between exo-and endosulfatases. Herein, a comparative study was performed on the basic biochemical features, substratedegrading patterns and catalytic mechanisms between the two 4-O-sulfatases exoPB4SF and endoVB4SF. The results of this study showed that the CS/DS endosulfatase endoVB4SF has a directional tendency during hydrolysis of sulfate groups, and the crystal structure of CS/DS endosulfatase was determined for the first time to elucidate its mechanism of action.
Materials
The strains and plasmids used in this study are listed in Table 1
Preparation of the Hexasaccharide A-A-A
An aliquot of commercial CS-A (10 mg) was partially digested with 10 U of highly purified rHCLase (Han et al., 2014) in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) in a total volume of 4 ml at 37 • C for 10 min. The reaction sample was heated in boiling water for 10 min and then cooled in ice-cold water for 10 min. The digest was fractionated on a Superdex TM Peptide 10/300 GL column (GE Healthcare) with 0.2 mM NH 4 HCO 3 as the eluent. The hexasaccharide fraction was pooled and further separated on a YMC-Pack PA-G column (YMC-Pack PA, Kyoto, Japan), eluted with a linear gradient from 16 to 460 mM NaH 2 PO 4 over 60 min at a flow rate of 1.0 ml/min at room temperature. The subfractions were pooled and desalted using a Superdex TM Peptide10/300 GL column followed by repeated lyophilization. The disaccharide compositions of these hexasaccharide subfractions were analyzed by digestion with the CSase ABC followed by HPLC separation on a YMC-Pack PA-G column as described above. The hexasaccharide fraction that yielded a single disaccharide 4,5 HexUAβ1-3GalNAc(4S) was the A-A-A hexasaccharide.
Sequence Analysis of the Genes and Proteins of exoPB4SF and endoVB4SF
The promoter motifs of the 5 -flanking DNA region upstream to the open reading frames (ORFs) were identified using Primer Premier version 5.0 (PREMIER Biosoft International, Palo Alto, CA, United States) and the Promoter 2.0 Prediction Server. The GC content of the ORFs were calculated using Bio-Edit version 7.0.5.3. The molecular masses of the proteins were estimated using the peptide mass tool on the ExPASy server of the Swiss Institute of Bioinformatics. Secretion signal peptides were identified using the SignalP 4.1 server. An online similarity search of the protein sequence was performed using the BLASTp algorithm. Multiple sequence alignments and phylogenetic analysis were performed using MEGA version 7.0. Sequences of endoVB4SF (GenBank TM accession number: AJK90566) from Vibrio sp. FC509 (Wang et al., 2015), 4,5 hexuronate-2-O-sulfatase (GenBank TM accession number: AXP31968) from Photobacterium sp. FC615, 4,5 hexuronate-2-O-sulfatase (GenBank TM accession number: WP_015808635) from Pedobacter heparinus , 4,5 hexuronate-2-O-sulfatase (GenBank TM accession number: NP_810509), N-acetylgalactosamine-4-Osulfatase (GenBank TM accession number: AAO78455) and N-acetylgalactosamine-6-O-sulfatase (GenBank TM accession number: NP_812245) from Bacteroides thetaiotaomicron (Ulmer et al., 2014) were downloaded from the NCBI protein sequence database.
To express the sulfatase exoPB4SF in E. coli, the full-length gene encoding exoPB4SF was amplified using the primer pair 5 -GAATTCTTGACGGGCAATCAGCCCGCTG-3 (forward) and 5 -CTCGAGGGTATAGGCATTAAGTACACTGG-3 (reverse) and high-fidelity PrimeSTAR TM HS DNA polymerase. Next, the PCR product was inserted into the expression vector pET-30a (+) (Invitrogen). The exoPB4SF protein with a His6 tag added at the C-terminus was expressed as previously described for endoVB4SF (Wang et al., 2015). For protein purification, the bacterial cells were harvested by centrifugation at 6,000 × g for 15 min, washed twice using ice-cold buffer A (50 mM Tris-HCl and 150 mM NaCl, pH 8.0), resuspended in buffer A, and disrupted by sonication (50 repetitions, 5 s) in an ice-cold environment. After centrifugation at 15,000 × g, 4 • C for 30 min, the supernatant was loaded onto a nickel affinity column (nickel-Sepharose TM 6 Fast Flow resin, GE Healthcare) and was further purified via ion exchange (Source 15Q HR 16/10, GE Healthcare) and size-exclusion chromatography (Superdex TM 200 10/300 GL, GE Healthcare) in 10 mM Tris-HCl and 100 mM NaCl, pH 8.0. The purity of the enzymes was assessed by SDS-PAGE using 13.2% polyacrylamide gels followed by staining with Coomassie Brilliant Blue. Protein concentrations were determined using the Folin-Lowry method (Bramhall et al., 1969).
Activity of exoPB4SF and endoVB4SF Toward Unsaturated Disaccharides and Polysaccharides
To determine the substrate specificities of the enzymes, different types of unsaturated disaccharides were dissolved in deionized water to prepare stock solutions (200 pmol/µl), including the C, A, D, E, and T units. Each stock solution (10 µl) was mixed with 30 µl of 150 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0), 40 µl of deionized water and 10 µl of appropriately diluted enzyme (0.5 µg/µl), and the solutions were incubated at 30 • C for 12 h. Each enzymatic reaction included a negative control in which active enzyme was substituted with boiled inactive enzyme. The enzyme-treated disaccharide samples were heated in boiling water for 10 min and then cooled in ice-cold water for 10 min. After centrifugation at 15,000 × g for 15 min, the supernatants were collected and labeled with 2-AB in the presence of NaBH 3 CN, as described by Bigge et al. (1995). Free 2-AB was removed by extraction with chloroform. All of the samples were analyzed by anion-exchange HPLC on a YMC-Pack PA-G column eluted with a linear gradient from 16 to 460 mM NaH 2 PO 4 over 60 min at a flow rate of 1.0 ml/min at room temperature. The eluates were monitored by measuring the absorbance using a fluorescence detector at excitation and emission wavelengths of 330 and 420 nm, respectively. Disaccharides were identified and quantified by comparison with CS-derived authentic unsaturated disaccharides.
To assess the enzymatic activities of endoVB4SF and exoPB4SF toward CS/DS polysaccharides, CS-A and DS polysaccharides were used as substrates for enzyme digestion. CS-A (30 µg) and DS (30 µg) was digested with exoPB4SF (5 µg), respectively, in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) in a total volume of 100 µl, at 30 • C for over 72 h, with the addition of 5 µg of fresh enzyme every 24 h two times. Subsequently, the reaction samples were heated in boiling water for 10 min and then cooled in ice-cold water for 10 min. Next, the sample was digested with the CSase ABC for disaccharide composition analysis. The digest was analyzed by gel filtration chromatography on a Superdex TM Peptide 10/300 GL column. The mobile phase was 0.20 M NH 4 HCO 3 at a flow rate of 0.4 ml/min, and the eluted fractions were monitored at 232 nm using a UV detector.
To analyze the enzymatic activity of exoPB4SF toward DS tetrasaccharides, 15 µg of DS tetrasaccharides [ 4,5 HexUAα1-3GalNAc(4S)β1-4IdoUAα1-3GalNAc(4S)] were digested with exoPB4SF (5 µg) in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) in a total volume of 100 µl at 30 • C for over 6 h. Subsequently, the reaction samples were heated in boiling water for 10 min and then cooled in ice-cold water for 10 min. After centrifugation at 15,000 × g for 15 min, the supernatants were analyzed by anion-exchange HPLC on a YMC-Pack PA-G column eluted with a linear gradient from 16 to 460 mM NaH 2 PO 4 over 60 min at a flow rate of 1.0 ml/min at room temperature. The eluates were monitored by measuring the absorbance using an ultraviolet detector at 232 nm.
Biochemical Characterization of endoVB4SF and exoPB4SF
The biochemical characteristics of endoVB4SF were reported in a previous study (Wang et al., 2015). To determine the optimal temperature of exoPB4SF, an aliquot of the A unit (10 nmol) was incubated with 0.1 µg of exoPB4SF in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) in a total volume of 100 µl at temperatures ranging from 0 to 70 • C for 10 min. After the optimum temperature was determined, the optimal pH assay was carried out in buffers at different pH values, including 50 mM HAc-NaAc buffer (pH 5.0-6.0), 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 6.0-8.0), and 50 mM Tris-HCl buffer (pH 7.0-10.0) at 30 • C for 10 min. The effects of chemical agents, including various metal ions, EDTA and DTT (5 mM) on the reaction rate of exoPB4SF were investigated under the optimal reaction conditions (50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer pH 8.0 at 30 • C). Finally, to investigate the thermostability of exoPB4SF, the enzyme was preincubated in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) from 0 to 60 • C for 0.5-24 h, after which the residual enzymatic activity was determined under the optimum conditions. All reactions were performed in triplicate, and enzyme-treated samples were analyzed by anion-exchange HPLC on a YMC-Pack PA-G column as described above. The eluates were monitored by measuring the absorbance at 232 nm.
The enzymatic activity of exoPB4SF against the A unit was determined under the optimum conditions. An aliquot of the A unit (30 nmol) was incubated with 1 µg of exoPB4SF in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) in a total volume of 500 µl at 30 • C. At various time intervals (up to 30 min), 30 µl aliquots were withdrawn in duplicate, boiled for 10 min, and then cooled in ice-cold water for 10 min. After centrifugation at 15,000 × g for 15 min, the supernatant fluid of each digest was collected and analyzed using anion-exchange HPLC on a YMC-Pack PA-G column. One unit of enzyme was defined as the amount of enzyme required to produce 1 µmol of free sulfate per minute.
Determination of the Catalytic Direction of endoVB4SF and exoPB4SF
To determine the substrate-degrading orientation of endoVB4SF, aliquots of the hexasaccharide A-A-A (2 µg) purified from CS-A were treated with 0.1 µg of endoVB4SF in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) in a total volume of 240 µl at 30 • C. At the indicated time points, an aliquot of 30 µl was withdrawn and immediately heated at 100 • C to denature the enzyme. The digests were labeled with 2-AB and analyzed by anion-exchange HPLC on a YMC-Pack PA-G column as described above. To further determine the structures of the digested mixtures, A-A-A (10 µg) was partially digested by endoVB4SF, and the digest was fractionated on a YMC-Pack PA-G column. Two intermediates (S2 and S1), with a final product S0, were individually pooled and desalted for sequencing. The disaccharide compositions of the three products (50 pmol each) were first analyzed through CSase ABC digestion and 2-AB labeling followed by anion-exchange HPLC on a YMC-Pack PA-G column as mentioned above. To analyze the disaccharide located at the non-reducing end of S2 or S1, the sample (50 pmol) was labeled with 2-AB and purified by paper chromatography (Wang et al., 2017), after which the 2-AB-labeled sample was digested with CSase ABC and analyzed by HPLC. To investigate the sequence of the tetrasaccharide at the non-reducing end of the hexasaccharide, the sample was reduced with sodium borohydride and then digested with an exo-type CS/DS lyase HCDLase (Wang et al., 2017) at 30 • C in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) for 6 h. The digest was labeled with 2-AB and analyzed by HPLC as described above.
To investigate the action pattern of exoPB4SF, a similar timecourse assay and product-sequencing analysis was performed for exoPB4SF as described for endoVB4SF.
Crystallization, Data Collection and Structure Refinement
endoVB4SF was purified and concentrated to ∼10 mg/ml. Crystallization conditions were first screened by sitting drop vapor diffusion using crystallization screening kits (Hampton Research) at 20 • C. Crystal optimization was performed using the hanging drop vapor diffusion method (McPherson and Gavira, 2014). The hanging drop was prepared by mixing equal volumes of the protein solution and the reservoir solution containing 100 mM HEPS (pH 7.5) and 20% (w/v) PEG 10000. For data collection, crystals were flash-frozen in liquid nitrogen, with 15-20% (v/v) ethylene glycol used as cryoprotectant. The X-ray diffraction data sets were collected at 100K on the beam line BL17U at the Shanghai Synchrotron Radiation Facility (Shanghai, China) equipped with an ADSC Q315r CCD-detector.
The X-ray diffraction data were integrated and scaled using the HKL-2000 program suite (Otwinowski and Minor, 1997). The endoVB4SF structure was resolved by molecular replacement using Phaser from the CCP4 suit of programs (Winn et al., 2011) using the sulfatase (PDB code: 2QZU) as the search model. Model building and refinement was performed using COOT (Emsley and Cowtan, 2004) and PHENIX (Adams et al., 2002), respectively. The final R-values were Rwork = 18.54% and Rfree = 21.40% based on a subset of 5% of the reflections.
The diffraction data collection and refinement statistics are listed in Table 2. The final model was checked and validated using PROCHECK (Laskowski et al., 1996), which indicated that the model was of good quality. Structure graphics were illustrated with the PyMOL (Lill and Danielson, 2011) molecular visualization system. The atomic coordinates and structure factors of endoVB4SF were deposited in the Protein Data Bank with accession code 6J66.
The crystallization conditions of exoPB4SF were screened using the same method for endoVB4SF. The exoPB4SF crystalized in reservoir solution containing 0.19 mM CYMAL-7, 0.1 M HEPES (pH 7.5) and 40% (w/v) PEG 400, but the crystal was snowflake-shaped and small. Although the crystallization conditions were optimized by adjusting pH and concentrations of PEG 400, the quality of crystals was not good enough for structure determination even using fragments of exoPB4SF. Therefore, its three-dimensional structure was constructed through structural modeling using ITASSER (Zhang, 2008;Roy et al., 2010;Yang et al., 2015).
Enzyme-Substrate Molecular Docking
The structures of endoVB4SF and exoPB4SF were manipulated using the "Protein Preparation Wizard" workflow in Maestro. The primary manipulations involved the removal of all water molecules, protonation, and optimization based on the OPLS_2005 force field (Petrovic et al., 2010). The potential acting site near the metal ion was discovered by the SiteMap module, after which a docking grid was generated using the "Receptor Grid Generation" module in Maestro (Schrödinger LLC, 2015b). The grid encloses a box centered on the binding site with a dimension of 10 × 10 × 10 (x × y × z, Å). A scaling factor of 0.8 was set for van der Waals radii of receptor atoms, with a partial atomic charge of less than 0.15. Standard precision of Glide docking procedure (Glide-SP) was used for docking the compounds to the binding site of protein (Schrödinger LLC, 2015a), and the best docking pose of the compound was retained based upon Glide scoring function (G-score).
Comparison of the Gene and Protein Sequences of exoPB4SF and endoVB4SF
The putative CS/DS 4-O-exosulfatase gene exopb4sf (GenBank TM accession number: MK282892) from Photobacterium sp. FC615 is 1470 bp in length and has a GC content (G + C%) of 49%. The exopb4sf gene encodes a protein of approximately 56.1 kDa composed of 490 amino acids. The isoelectric point (pI) of exopb4sf is 5.2, and the SignalP 4.1 analysis showed that the exopb4sf protein does not contain an N-terminal signal peptide. In contrast, the CS/DS 4-Oendosulfatase gene endovb4sf (GenBank TM accession number AJK90566) from Vibrio sp. FC509, which shares high sequence identity (83%) with exopb4sf (Supplementary Figure S1), is 1566 bp in length, encoding a 60 kDa protein that contains an N-terminal signal peptide of 19 amino acids (Wang et al., 2015).
The results of a neighbor-joining phylogenetic tree analysis showed that exoPB4SF clustered to form a single clade with 4-Osulfatase (Figure 1), resulting in it being preliminarily identified as CS/DS 4-O-sulfatase.
Recombinant Expression of exoPB4SF and endoVB4SF in E. coli
The full length sequences of the exoPB4SF and endoVB4SF genes were amplified directly from the genomic DNA of Photobacterium sp. FC615 and Vibrio sp. FC509, respectively and were recombinantly expressed as described previously (Wang et al., 2015). The results of an SDS-PAGE analysis indicated that BL21 (DE3) cells harboring the pET-30a-exopb4sf and pET-30a-endovb4sf plasmids yielded ∼4.5 and ∼5 mg/liter of soluble protein with the correct molecular mass, respectively. The crude enzymes were extracted from cultures of the host cells and further purified as described under "Experimental Procedures." Finally, the purity and concentrations of both enzymes was up to 95% and ∼2 mg/ml, respectively (Supplementary Figure S2). The purified enzymes were stored at −80 • C for the subsequent biochemical and crystallographic studies.
Comparison of exoPB4SF and endoVB4SF Activities Toward Unsaturated Disaccharides and Polysaccharides
To compare the specific activities of exoPB4SF and endoVB4SF toward CS/DS oligo-and polysaccharides, five types of unsaturated CS disaccharides ( C, A, D, E and T) with distinct sulfation patterns were individually treated with or without exoPB4SF and endoVB4SF. The results showed that both exoPB4SF and endoVB4SF could effectively remove the 4-O-sulfate group from the GalNAc residue of A to yield a nonsulfated O (Figures 2B,G) but no effect was observed toward C with a 6-O-sulfate group (Figures 2A,F). Furthermore, for disulfated disaccharides, both enzymes could completely transfer E with 4-O-and 6-O-sulfate groups at the GalNAc residue to C (Figures 2D,I), although neither enzyme affected D with 2-O-and 6-O-sulfate groups at the 4,5 hexuronate and GalNAc residues, respectively (Figures 2C,H). In addition, both enzymes did not show any activity toward heparin/heparin sulfate disaccharides (data not shown). Taken together, these results clearly show that exoPB4SF, similar to endoVB4SF, is a CS/DS 4-O-sulfatase that specifically hydrolyzes the 4-O-sulfate group at GalNAc residues. Notably, unlike endoVB4SF, exoPB4SF could not hydrolyze the 4-O-sulfate group of T with three sulfate groups at C4 and C6 of GalNAc and C2 of 4,5 hexuronate ( Figures 2E,J), suggesting that the high sulfation of T strongly inhibits the activity of exoPB4SF but not endoVB4SF.
To investigate if exoPB4SF can hydrolyze the 4-O-sulfate groups within CS/DS chains, the bovine cartilage-derived polysaccharide CS-A which contains about 67% of A unit and some minor disaccharides C unit and O unit, and DS which FIGURE 1 | Phylogenetic analysis of the characterized bacterial CS/DS sulfatases. The numbers on the branches indicate the bootstrap confidence values from 1,000 replicates. The bar is equal to the distance corresponding to 1 amino acid substitution per 10 amino acid residues. The neighbor-joining tree was constructed using MEGA. Frontiers in Microbiology | www.frontiersin.org contains more than 90% A unit (Wang et al., 2015) were exhaustively treated with this enzyme, and the disaccharide composition of the resulting mixture was analyzed by HPLC as described under "Experimental Procedures." The results showed that the proportion of non-sulfated disaccharide O was not significantly increased by the treatment with exoPB4SF (Supplementary Figure S3A), indicating that the 4-O-sulfate groups of GalNAc residues within CS/DS polysaccharide chains cannot be effectively removed by exoPB4SF. Thus, exoPB4SF is a strict exolytic 4-O-sulfatase in contrast with the endolytic activity of endoVB4SF (Supplementary Figure S3B) (Wang et al., 2015). However, exoPB4SF could hydrolyze the C-4 sulfate on the reducing end of the DS tetrasaccharides [ 4,5 HexUAα1-3GalNAc(4S)β1-4IdoUAα1-3GalNAc(4S)] indicating that exoPB4SF is an exolytic sulfatase that could act on oligosaccharides (Supplementary Figures S3C,D).
Comparison of the Basic Enzymatic Properties of exoPB4SF and endoVB4SF
To compare the basic enzymatic properties of exoPB4SF and endoVB4SF, the effects of temperature, pH and chemical agents on the activity of exoPB4SF were investigated using A as substrate. Similar to endoVB4SF (Wang et al., 2015), exoPB4SF exhibited the maximal rate at 30 • C (Figure 3A), but it possessed FIGURE 3 | Biochemical characterization of recombinant exoPB4SF. (A) Effect of temperature. The enzyme activity of exoPB4SF was determined using the disaccharide A as substrate in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) at different temperatures for 10 min. The data are shown as the percentages of the activity obtained at 30 • C (100%). (B) Effect of pH. The activity of exoPB4SF toward A was measured in buffers with pH values from 5 to 10 at 30 • C for 10 min. The data are shown as the percentages of the activity obtained in NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) (100%). (C) Effects of chemical agents. The activity of exoPB4SF against A was measured in NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) containing 5 mM of various chemical agents at 30 • C for 10 min. Data are shown as the percentage of the activity of that obtained in the buffer without tested other agents. (D) Thermostability of exoPB4SF. The enzyme in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) was preincubated for 0-24 h at temperatures from 0 to 60 • C, and the residual activity toward A was estimated at 30 • C. The data are shown as the activity relative to that of untreated exoPB4SF. Error bars represent means of triplicates ± SD. a high relative activity (>60%) over a wide range of temperatures, from 10 to 50 • C, which was notably different from the high sensitivity of endoVB4SF to different temperatures (Wang et al., 2015). The optimal pH value for exoPB4SF activity was pH 8.0 in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (Figure 3B), which was the same as that observed for endoVB4SF (Wang et al., 2015). However, it should be noted that the relative activity of exoPB4SF in 50 mM Tris-HCl buffer was low but relatively stable over a pH range of 8.0-10.0 (Figure 3B), whereas that of endoVB4SF quickly decreased as the pH increased from 8.0 to 10.0 in the same buffer (Wang et al., 2015). In addition, the enzymatic activity of exoPB4SF was significantly promoted by some monovalent and divalent cations, including Na + , Li + , K + , Mg 2+ , and Ca 2+ (Figure 3C), whereas no metal ion notably stimulated the activity of endoVB4SF (Wang et al., 2015). The divalent metal ionchelating agent EDTA strongly inhibited the activity of exoPB4SF ( Figure 3C) but did not significantly affect that of endoVB4SF (Wang et al., 2015). In contrast, the reducing agent DTT significantly promoted the activity of both enzymes ( Figure 3C). Thermostability analyses revealed that exoPB4SF retained 80% activity when incubated in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) at 30 • C for 24 h (Figure 3D), demonstrating that exoPB4SF was more stable than endoVB4SF, the catalytic activity of which decreased dramatically when incubated in the same buffer at 30 • C for 4 h.
The enzymatic activity of exoPB4SF was determined under the optimum conditions of 30 • C in 50 mM NaH 2 PO 4 -Na 2 HPO 4 buffer (pH 8.0) using A unit as a substrate. The specific activity of exoPB4SF was 1.37 units/mg of protein, slightly lower than that of endoVB4SF (2.01 units/mg) (one unit of enzyme was defined as the amount of enzyme required to produce 1 µmol of free sulfate per minute) ( Table 3). However, the wider temperature and pH adaptability and much better thermostability of exoPB4SF indicated that its structure may be more stable than that of endoVB4SF.
Frontiers in Microbiology | www.frontiersin.org assay, the hexasaccharide A-A-A was treated with exoPB4SF or endoVB4SF for varying times, and the digests were individually analyzed by HPLC on a YMC-Pack PA-G column. As shown in Figure 4, the parental hexasaccharide A-A-A (S3) was quickly transformed to S2 (Figures 4A,B), after which the S2 peak gradually decreased as two earlier-eluting peaks, S1 and S0, correspondingly increased (Figures 4C-F). As the treatment time increased, the intermediates S2 and S1 were finally transferred to the final product S0 (Figures 4G,H). These results suggested that the sulfate groups of the parental hexasaccharide A-A-A were sequentially removed by the action of endoVB4SF. Thus, we presumed that endoVB4SF hydrolyzed the sulfate esters from substrates regularly, not randomly. To elucidate the action pattern of endoVB4SF toward CS chains, the intermediates (S2 and S1) and the final product (S0) were individually collected and desalted for structural analysis through enzymatic digestion followed by HPLC analysis (Figures 5A,E,H). The results of the disaccharide composition analysis showed that S2 and S1 were composed of O and A units in molar ratios of 1:2 and 2:1, respectively (Figures 5B,F), while S0 contained only O (Figure 5I), indicating that they were the di-, mono-and non-sulfated products derived from the digestion of A-A-A by endoVB4SF. To further determine the disaccharide sequences of the intermediate S2, it was labeled with 2-aminobenzamide (2-AB) followed by digestion with the CSase ABC. Subsequently, the digest was relabeled with 2-AB and then analyzed by HPLC. Because CSase ABC cannot cleave the tetrasaccharide portion on the reducing end of hexasaccharide after 2-AB-labeling (Wang et al., 2017), the digestion of 2-AB-labeled hexasaccharide with the CSase ABC will produce a disaccharide from non-reducing end and a tetrasaccharide from reducing end. A disaccharide peak corresponding to the elution position of the disaccharide A standard and a presumed tetrasaccharide peak were detected in the digests of 2-ABlabeled S2 after 2-AB relabeling, indicating that the disaccharide on the non-reducing end was the 4-O-sulfated A unit for S2 ( Figure 5C). Based on these results, the sequences of S2 could be deduced as A-(A-O/O-A). To further confirm the sequence of the tetrasaccharide on the reducing side of S2, S2 was reduced with sodium borohydride and digested with an exo-type CS/DS lyase HCDLase (Wang et al., 2017). After 2-AB labeling, the digest was analyzed by HPLC. Due to the reduction with sodium borohydride, the disaccharide released from the reducing end of S2 could not be labeled with 2-AB, and thus only the disaccharides from the tetrasaccharide portion on the non-reducing side could be labeled and detected using a fluorescence detector. The results showed that only the 2-AB-labeled A unit could be detected from the digest of S2 reduced with sodium borohydride (Figure 5D), indicating that the tetrasaccharide portion on the non-reducing side of S2 had a sequence of A-A. These results allowed the sequence of S2 to be deduced as A-A-O. Notably, when the injected sample was reduced, the 2-AB-labeled S1 appeared as two peaks, S1-1 and S1-2, in molar ratios of approximately 2.4:1 (Figure 5E), indicating the presence of two different structures. The HPLC analysis showed that the mixture resulting from the digestion of 2-AB-labeled S1 with the CSase ABC followed by 2-AB labeling again separated into four fractions, including A, O-O, O and A-O units in a molar ratio of approximately 2.4: 2.4: 1: 1 (Figure 5G), indicating that S1 contained two structures, S1-1 and S1-2, which were A-O-O and O-A-O, respectively. Taken together, the digestion of hexasaccharide S3 ( A-A-A) showed that endoVB4SF preferentially and sequentially hydrolyzes sulfate esters from the reducing ends of CS/DS chains. In contrast, the digestion of A-A-A with exoPB4SF only yielded a single product (S2'), which eluted at the same position as S2, even after extending the treatment time to 12 h (Figure 6). The final product S2' was pooled and sequenced using the same methods as those used for S2. The results showed that the only product, S2' , had the same structure as S2 ( A-A-O) (data not shown), suggesting that exoPB4SF is an exolytic sulfatase that can only hydrolyze the 4-O-sulfate esters of GalNAc residues located at the reducing end of CS chains.
Crystal Structure of endoVB4SF and Structural Modeling of exoPB4SF
Although the crystal structures of several CS/DS exosulfatases have been resolved, this is not the case for CS/DS endosulfatases. In this study, the crystal structure of endoVB4SF was obtained and refined at 1.95 Å resolution. EndoVB4SF adopts a globular conformation with two monomers per asymmetric unit (Figures 7A,B). The refined structure revealed that each endoVB4SF monomer folded into α/β topology containing 12 β-strands (β1-β12) and 10 α-helices (α1-α10) organized into two domains ( Figure 7C). In the larger N-terminal domain (residues 20-420), the major 8-stranded β-sheets (β4, β3, β5, β1, β6, β7, β2, and β8) are sandwiched by α-helices α1, α4, α5, and α6 on one side and α-helices α2, α3, and α7 on the other side, forming the catalytic domain of the enzyme as observed in other sulfatases (Hanson et al., 2004). In the 8-stranded β-sheets, seven of them are in the parallel orientation except for strand β7, which is antiparallel to the others. In the smaller C-terminal domain (residues 421-521), four antiparallel β-strands (β9-β12) are flanked by two α-helices (α9 and α10) located on the solventexposed surface. The N-and C-terminal domains are closely integrated together through hydrophobic interactions to form an extensive positively charged groove, which may be important for the binding of the substrates.
Although various crystallization approaches were attempted, we failed to obtain a well-diffracted crystal for exoPB4SF. Considering that exoPB4SF shares a very high sequence identity (>80%) with endoVB4SF, its three-dimensional structure was constructed through homology modeling using I-TASSER (Figures 7D,E). The final model with the highest C score (=1.11) was selected from the 5 top models predicted. The estimated TMscore and RMSD was 0.87 ± 0.07 and 4.9 ± 3.2 Å, respectively. These parameters indicate that our structure model of exoPB4SF is of good validity.
According to the endoVB4SF structure, the two molecules in the asymmetric unit form a dimer, primarily through interactions between the C-terminal domains. The interface is approximately 1714.6 Å 2 as predicted by PISA (Krissinel and Henrick, 2005). Size-exclusion chromatography analysis revealed that endoVB4SF primarily existed as a dimer in solution, the formation of which was significantly inhibited by the addition of DTT, indicating the existence of a disulfide bridge between the monomers at the dimer interface ( Figure 7F). Compared to exoPB4SF, endoVB4SF has an extra Cys27 residue closely following the signal peptide at the N-terminal end. To investigate if this extra Cys residue is involved in the formation and stability of dimers, Cys27 was replaced by Ala (C27A) through site-directed mutagenesis. Sizeexclusion chromatography analysis showed that the dimerforming capacity of the endoVB4SF-C27A mutant was notably reduced compared to the wild-type enzyme (Figure 7F), suggesting that the Cys27 residue is involved in forming endoVB4SF dimers through the formation of intermolecular disulfide bridges between the monomers. Nevertheless, the mutation did not significantly affect the enzymatic activity toward the disaccharide A or the polysaccharide CS-A (data not shown). To further investigate the role of Cys residue in the dimerization, the Gln5 of exoPB4SF, corresponding to the Cys27 in endoVB4SF, was mutated to Cys. The result showed that most of the mutant exoPB4SF-Q5C formed into dimers (Figure 7F), which confirmed the important role of Cys27 for the dimerization of endoVB4SF.
Active Sites of endoVB4SF and exoPB4SF
The active site of endoVB4SF is located in a pocket at the carboxyl end of the central parallel portion of the β sheets. The pocket is primarily formed by the amino acid residues of loops from the N-terminal domain, including Asp56, Glu57, Leu95, Cys96, Pro98, Arg100, Asn117, Lys158, His160, His263, Asp343, His344, Lys356, and Asn357. The predominance of basic residues in the active site, such as Arg100, Lys158, His160, His263, His344, and Lys356, generates a positively charged surface to facilitate the binding of negatively charged CS/DS substrates. Notably, the residue Cys96 in the N-terminal end of α2-helix is posttranslationally modified to FGly and plays a key role in the catalytic mechanism of this enzyme. Notably, all of these active-site residues are highly conserved in various sulfatases from different species. The equivalent residues involved in the catalytic center of endoVB4SF and exoPB4SF are listed in Table 4. Furthermore, a metal cation modeled as calcium was observed in the vicinity of the FGly residue and was coordinated by six atoms: four oxygen atoms from the side chains of Asp56, FGly96, and Asp343; one nitrogen from the side chain of His344; and one water molecule. A divalent metal cation, such as Ca 2+ or Mg 2+ , has been believed to be important for the catalytic activity of sulfatases (Bruce et al., 1985;Myette et al., 2009;Rivera-Colon et al., 2012). To comprehensively analyze the tertiary structure of the exo-and endosulfatases, the structure of another exolytic 4-O-sulfatase ARSB (PDB code: 1fsu) (Bond et al., 1997) was downloaded and aligned with those of exoPB4SF and endoVB4SF. Through structural alignments, we observed that the orientations of the amino acid side chains at the substrate binding cavities were different in the tertiary structures of endoVB4SF, exoVB4SF and ARSB, despite the active sites of these enzymes exhibiting highly conserved secondary structures (Figure 8), indicating that the substrate-binding capacity of endo-and exo-4-O-sulfatases may be different. Pro98 Pro67 Arg100 Arg69 Asn117 Asn86 Lys158 Lys127 His160 His129 Arg179 Arg148 Trp181 Trp150 Thr201 Thr170 Asp203 Asp172 His263 His232 Ser264 Ser233 Tyr301 Tyr270 Asp343 Asp312 His344 His313 Thr355 Thr324 Lys356 Lys325 Asn357 Asn326
Substrate Docking Simulations
Docking simulation studies were performed to better understand the substrate binding modes and catalytic mechanisms of endo-and exo-4-O-sulfatases. (Figures 9A,B). The docking scores for A and A-A-A to endoVB4SF were −5.24 and −7.18 kcal/mol, respectively. The lower score for A-A-A suggests that endoVB4SF has a higher binding affinity toward hexasaccharide. The endo-4-O-sulfatase-hexasaccharide docking complex revealed that additional amino acids, including a series of positively charged residues, are involved in binding to the hexasaccharide A-A-A (Figures 9C,D).
With respect to exoPB4SF, the exo-4-O-sulfatase-disaccharide binding mode assay revealed that the disaccharide A was docked well into its active site (Figures 9E,F), with a docking score of −4.59 kcal/mol. However, the hexasaccharide A-A-A could not properly dock into the active site of exoPB4SF, which may be attributable to the spatial arrangement of the amino acids in the active site. Furthermore, we observed that the substrate was fitted into the active site with the saccharide chain approximately parallel to the β-sheet core of exoPB4SF but perpendicular to the β-sheet core of endoVB4SF. These differences in substrate binding modes may lead to the different substrate length selectivity and endo-/exolytic activities observed for endoVB4SF and exoPB4SF.
DISCUSSION
CS/DS sulfatases are key enzymes involved in the degradation of CS/DS and are potential tools for structural and functional studies of CS/DS. However, only a few of CS/DS sulfatases have been identified from mammals and bacteria, most of which are exosulfatases, except the two endo-type sulfatases recently identified from Bacteroides thetaiotaomicron (Ulmer et al., 2014) and Vibrio sp. (Wang et al., 2015). The action pattern of CS/DS sulfatases, particular endosulfatases, has rarely been investigated. In the present study, the substrate-degrading modes of the 4-Oexosulfatase exoPB4SF and the 4-O-endosulfatase endoVB4SF, which exhibit high sequence identity, were comparatively studied through biochemical and structural analyses. The newly identified exoPB4SF from Photobacterium sp. shares 83% sequence identity with endoVB4SF from Vibrio sp. (Wang et al., 2015), but compared to endoVB4SF, exoPB4SF is a strict exolytic 4-O-sulfatase exhibiting a number of different features such as a wider temperature and pH adaptability and better thermostability which make it excellent tool for oligosaccharides analysis. The remarkable difference between the two highly homologous enzymes suggests that the biochemical and enzymatic features of CS/DS sulfatases are determined by both their primary and tertiary structures.
The results of previous studies showed that the N-acetylgalactosamine-4-O-sulfatase and N-acetylgalactosamine-6-Osulfatase from Proteus vulgaris could release the 4-O-and 6-O-sulfate groups from the GalNAc residues of disaccharides located at the reducing ends of various CS/DS hexasaccharides, respectively, but they had difficulty or were even unable to act upon the corresponding sulfate groups of disaccharides located in the middle and non-reducing ends of hexasaccharides (Sugahara et al., 1994;Sugahara and Kojima, 1996). The same direction selectivity was observed for endoVB4SF and exoPB4SF acting upon the CS-A-derived hexasaccharide A-A-A, but endoVB4SF could easily remove the internal 4-O-sulfate groups of A-A-A due to its endolytic activity. Interestingly, the endo-type sulfatase endoVB4SF was observed to preferentially hydrolyze the sulfate esters from the reducing to non-reducing ends of the hexasaccharide A-A-A, suggesting that endosulfatases act upon CS/DS chains in a more orderly manner than endolytic lyases, which typically cleave CS/DS chains at random.
The crystal structure of endoVB4SF revealed that it adopts a globular conformation with two monomers per asymmetric unit, consistent with the results of a biophysical characterization of this enzyme (Neira et al., 2016). The results of a size exclusion chromatography analysis showed that unlike endoVB4SF, exoPB4SF existed as monomer in solution. Furthermore, the replacement of Cys27 to Ala significantly reduced the proportion of dimeric endoVB4SF in solution, suggesting that the Cys27 in endoVB4SF facilitates and stabilizes the dimer formation through the formation of a disulfide bridge, which was confirmed by the observation that the addition of DTT effectively inhibited endoVB4SF dimerization. In addition, we found that once the Gln5 of exoPB4SF, corresponding to the Cys27 in endoVB4SF, was mutated to Cys the mutant exoPB4SF-Q5C tended to form dimers. These findings suggest the important role of Cys residue in the oligomerization of some sulfatases, which is inconsistent with the results of a previous biophysical study (Neira et al., 2016). Notably, the endoVB4SF-C27A mutation could not completely inhibit dimerization, indicating that there some other interaction, such as a hydrophobic interaction, is involved in the dimerization process. Dimerization has also been observed in other sulfatases, although its biological significance is no clear (Lukatela et al., 1998;Rivera-Colon et al., 2012). We also observed that the inhibition of dimerization by site-directed mutagenesis did not influence the enzymatic activity of endoVB4SF. Thus, the exact biological function of dimer formation remains to be further elucidated.
Arylsulfatases have been reported to possess the signature motif Cys-X-Pro-X-Arg at the active site (Barbeyron et al., 2016), where the Cys residue is specifically recognized and oxidized to the aldehyde hydrate FGly [-CH(OH) 2 ], catalyzed by a formylglycine-generating enzyme. Both endoVB4SF and exoPB4SF possess the Cys-X-Pro-X-Arg motif in their primary structures and, the tertiary structure revealed that the Cys96 residue in the active site of endoVB4SF was modified to the FGly residue with two geminal hydroxyl groups, indicating that CS/DS sulfatases possess the same maturation mechanism as other arylsulfatases. The FGly and other residues form the catalytic site of endoVB4SF. A bivalent metal ion was observed to be in close proximity to the hydroxyl group of FGly, which was coordinated by the residues Asp56, FGly96, Asp343, and His344. These active site residues are highly conserved in exoPB4SF and other sulfatases identified from bacteria and mammals. The highly conserved conformation of active sites suggests that endoVB4SF and exoPB4SF have the same catalytic and transesterificationelimination mechanisms as other reported sulfatases, such as ARSB (Bond et al., 1997;Waldow et al., 1999;Boltes et al., 2001). In this catalytic mechanism, the sulfate-sulfur on a saccharide residue is attacked by one of the hydroxyl groups of the FGly residue to generate a sulfated enzyme intermediate and an alcohol group, after which a sulfate group is released and the aldehyde of FGly is simultaneously regenerated through an intramolecular rearrangement. Structural alignment among endoVB4SF, exoPB4SF and ARSB shows that their catalytic cavity is rich in positively charged amino acids such as lysine, arginine and histidine, which form a positively charged catalytic region that facilitates the binding of negatively charged sugar chains. Furthermore, the catalytic cavity of endoVB4SF and exoPB4SF form a long groove while the catalytic cavity of ARSB is partially blocked by the irregular crimp from the C-terminal domain leading to a narrow substrates binding site. This difference may affect the substrate-binding and catalyzing ability of ARSB but it need to be further investigated.
Although exoPB4SF and endoVB4SF were suggested to share the same catalytic mechanism as other sulfatases, the docking results revealed that their capacities to bind substrates were different depending on the length of the CS/DS oligosaccharides. The docking models showed that small-sized substrates such as disaccharides could properly dock into the catalytic sites of both enzymes, and five highly conservative residues (FGly65, Asn86, Lys 127, His232 and Lys325 for exoPB4SF; and FGly96, Asn117, Lys158, His 263 and Lys 356 for endoVB4SF) were involved in the binding and catalysis of disaccharides (Supplementary Figures S4A,B). In contrast, large-sized substrates such as hexasaccharides could easily dock into the catalytic pocket of endoVB4SF but not exoPB4SF. The docking model of hexasaccharide-endoVB4SF revealed that additional residues were involved in the substrate binding process (Supplementary Figure S4C), where six C-terminal residues (Tyr301, Asn357, Phe433, Tyr436, Arg443, and Lys458), located on the rim of the catalytic pocket of endoVB4SF, were involved in the hexasaccharide-binding process. Interestingly, these residues are also conserved in exoPB4SF. We found that the extension direction of the side chain of these amino acids around the catalytic cavity were different. Furthermore, electrostatic surface potential diagram revealed that the catalytic center of endoVB4SF carries higher positive charges, which may facilitate the binding of negatively charged sugar chains. Therefore, we propose that the primary factor influencing the substrate selectivity of endoVB4SF and exoPB4SF is the conformation of the catalytic center in the tertiary structure. The residues at the active site of endoVB4SF adopt a more favorable conformation for the interaction of the enzyme with longer CS/DS chains than that of exoPB4SF, which may explain why endoVB4SF exhibits significant endolytic activity but exoPB4SF does not.
Overall, the action patterns of endoVB4SF and exoPB4SF were detailed characterized in this work, which will facilitate their use in structural and functional studies of CS/DS chains. The structure of endoVB4SF and molecular modeling of exoPB4SF provide structural and biochemical insight into the interaction of CS/DS chains with endo-/exo-type sulfatases, which should be instructive for gaining an understanding of the different catalytic modes between glycosaminoglycan endo-and exosulfatases.
DATA AVAILABILITY
The datasets generated for this study can be found in NCBI GenBank and PDB, GenBank accession number: MK282892, PDB code:6J66.
AUTHOR CONTRIBUTIONS
SW conducted most of the experiments, analyzed the results, and wrote the manuscript under the guidance of FL and LG. TS performed the X-ray crystallography and structural refinement for the endoVB4SF structure. JG and JH conducted experiments for expressing the sulfatases. FL conceived the idea for the project and wrote the manuscript. All authors revised and edited the manuscript and read and approved the final manuscript. | 2019-06-12T15:03:55.407Z | 2019-06-12T00:00:00.000 | {
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13156412 | pes2o/s2orc | v3-fos-license | Clinical Holistic Medicine: The Patient with Multiple Diseases
In clinical practice, patients can present with many different diseases, often both somatic and mental. Holistic medicine will try to see the diseases as a whole, as symptoms of a more fundamental imbalance in the state of being. The holistic physician must help the patient to recover existence and a good relationship with self. According to the life mission theory, theory of character, and holistic process theory of healing, recovering the purpose of life (the life mission) is essential for the patient to regain life, love, and trust in order to find happiness and realize the true purpose of life. We illustrate the power of the holistic medical approach with a case study of an invalidated female artist, aged 42 years, who suffered from multiple severe health problems, many of which had been chronic for years. She had a combination of neurological disturbances (tinnitus, migraine, minor hallucinations), immunological disturbances (recurrent herpes simplex, phlegm in the throat, fungal infection in the crotch), hormonal disturbances (14 days of menstruation in each cycle), muscle disturbances (neck tensions), mental disturbances (tendency to cry, inferiority feeling, mild depression, desolation, anxiety), abdominal complaints, hemorrhoids, and more. The treatment was a combined strategy of improving the general quality of life, recovering her human character and purpose of life (“renewing the patients life energy”, “balancing her global information system”), and processing the local blockages, thus healing most of her many different diseases in a treatment using 30 h of intense holistic therapy over a period of 18 months.
INTRODUCTION
From the biomedical perspective, a disease is a well-defined entity that can be understood in its own right and treated independently of the person and almost independently of other diseases and conditions in this person. A physical disease (for example, an infection) can be treated independently of a mental disease (for example, depression) naturally taking into consideration the interactive effect of any drugs. There has been very little research on how to treat the patient with many diseases (a recent search on www.pubmed.gov did not reveal any controlled clinical studies with such patients). From the holistic medical perspective, diseases, if not caused by genes or by traumatic impacts from the outside (like asbestos, oncogenic tar from smoking, etc.), are caused by lack of coherence[1,2] due to inner conflicts that disturb the fine order of the organism and its cells [3,4,5,6,7,8,9,10].
When a patient, such as the one we are going to present in this paper, has a combination of neurological disturbances (tinnitus, migraine with severe scotomata, minor hallucinations), immunological disturbances (recurrent herpes simplex 1 and 2 on lip and labia, phlegm in the throat, fungal infection), hormonal disturbances (14 days of menstruation in each cycle), muscle disturbances (neck tensions), psychic disturbances (tendency to cry, inferiority feeling, mild depression, feelings of desolation, anxiety), abdominal complaints, hemorrhoids, and more, the approach of a biomedically trained physician would be to start from an end, treating what is possible and leaving the rest untreated. When the patient has a moderate number of diseases, this strategy often works wonders, but when she has 20 problems, it mostly will not work. Biomedicine does not seem effective with this group of patients.
From a holistic point of view, all the organ systems are intimately connected and they share the same information system [3,4,5,6,7,8,9,10,11,12,13,14,15,16] or they share the same "life energy". So, the many diseases are a function of the person being low on energy or being disturbed informationally. The cause is due to fundamental inner conflicts that bind the energy in blockages that are held by the different tissues and these are revealed by the various symptoms. Interestingly, there are two kinds of causal patterns in holistic medicine: a positive pattern of a blockage giving a local disease and a negative pattern of the body and organism being drained of its energy. The multidisease syndrome is often caused by a combination of the two. Tinnitus is a typical manifestation of the negative pattern, as is severe chronic pain in the bladder and kidney. Cancer is a typical manifestation of the positive pattern. When it comes to the holistic treatment of the diseases, a negative pattern must be handled in a long, systematic process of rebuilding the person as her true self. A positive pattern is often easier and faster to treat as the whole existence is less affected. Whatever the chosen course of the treatment, it always starts with the physician's love or intense care and goodness for his patient, inviting the patient's trust and allowing the physician to hold and cure [17].
CLINICAL HOLISTIC MEDICINE
The life mission theory [18,19,20,21,22,23] states that everybody has a purpose of life or huge talent. Happiness comes from living this purpose and succeeding in expressing the core talent in your life. To do this, it is important to develop as a person into what is known as the natural condition, a condition where the person knows himself and uses all his efforts to achieve what is most important for him. The holistic process theory of healing [17,24,25,26] and the related quality of life theories [11,12,13] state that the return to the natural state of being is possible whenever the person gets the resources needed for existential healing. The resources needed are "holding" in the dimensions of awareness, respect, care, acknowledgment, and acceptance with support and processing in the dimensions of feeling, understanding, and letting go of negative attitudes and beliefs. The preconditions for holistic healing to take place are trust and the intention for the healing to take place. Existential healing is not a local healing of any tissue, but a healing of the wholeness of the person, making him much more resourceful, loving, and knowledgeable of himself, his own needs, and his wishes. In letting go of negative attitudes and beliefs, the person returns to a more responsible existential position and an improved quality of life. The philosophical change of the person healing is often a change towards preferring difficult problems and challenges instead of avoiding difficulties in life [3,4,5,6,7,8,9,10]. The person who becomes happier and more resourceful often also becomes more healthy, more talented, and more able to function [27,28,29].
A CASE STUDY ON MULTIPLE DISEASE
When we began our professional career in general practice, we soon discovered that it was fairly safe to ask the patient the relevant question: "What can you do to get better?" This simple question would often make the patient become distant, silent, and pensive and so an important process had begun. In the case of the following patient, a 42-year-old female artist whom we will call Mia, we found that additional measures were required. Following 10 years of illness in the form of various ailments ranging from migraine and herpes simplex 1 and 2 to treatment-resistant genital warts and depression, she had come to accept that there was no solution to her problems and merely wanted the standard medication for her symptoms. However, she had a problem that worried her most -her tinnitus had become worse, much worse in fact, and would it be at all possible to do something about it?
Despite her age, Mia was in very poor condition physically and mentally, but she possessed something special: alertness and an interest in the spiritual world. She wanted to develop as a person and that meant that she was ready to assume responsibility and take the rather bitter, holistic medicine that the clinic could offer her. Not once during the period when she came to the clinic, which lasted over a year, was she prescribed a single pill. She met the therapist (SV) in a good and sincere way and after her first appointment, she went home with four exercises that she was very keen on doing for her next appointment 14 days later. Exactly 14 days later, she arrived with ten closely written A4 pages. She surprised the therapist because she provided complete solutions to all tasks, which she had taken very seriously and worked on with great determination. Clearly, she regarded the treatment as a real opportunity to recover, primarily from her debilitating tinnitus. She understood the working methods of the holistic clinic, believed in them, and was very motivated.
Processing her painful personal history took her directly to her life purpose. Following this acknowledgment, her art began to flourish and grow like never before. Suddenly, she could do things that she had not been able to do before and her art expressed her new state of acceptance and understanding of good and evil, beautiful and ugly, muck and mire, and sky and light. Occasionally, the therapist appeared as a counselor in Mia's dreams. At some point, she revealed that she was in love with the therapist, which is common when a patient experiences genuine help. Fortunately, it will pass away, but it can be quite a strain at the time. When she acknowledged her life purpose, Mia became able to manage on her own and able to develop further without our help. The therapeutic work of guiding her through the pain that made her ill and blocked her enjoyment of life and self-expression is finished. Her body and soul have largely healed, her tinnitus is almost gone, and she cannot hear it at all most of the time. Obviously, this patient may become physically ill again, but her resistance and inner equilibrium appear to be much greater than before, so next time she is likely to recover much faster.
It is worth noticing how the pain shifts from body to mind and existence in the course of the conversations. By the 12th conversation, she was on the verge of becoming psychotic. By the 18th conversation, after about 1 year of therapy, the patient no longer had any physical complaints, but still struggled with difficult existential problems. We talked about the world being "evil" and she was coached to see the world as both good and evil. Taking responsibility for our lives means that we can relate to both good and bad things in the world in a constructive and clear way. Following a total of 23 conversations over a period of 18 months, she gained control of all her talents, became happy, and is functioning well with a new job. Below are the chart abstracts of the 23 conversations.
Female, aged 42 years, with tinnitus, migraine, herpes simplex 1 and 2, lower back pain, treatment-resistant genital warts, sun allergy, depression, etc.: First quality-oflife (QOL) conversation: Presents with multiple problems: Menstruate 14 days in each cycle, suffers from migraine with severe scotomata blinding her for up to 20 minutes, herpes simplex 1 and 2 on lip and on labium minus and labium majus, both on left side, neck tensions, phlegm in the throat, tendency to cry, inferiority feeling, fungal infection in the crotch, haemorrhoids and severe abdominal complaints previously but not at present, tinnitus, tendency towards depression, occasional minor hallucinations in the form of symbols, signs and aura vision which she cannot control, feelings of desolation, anxiety….. Spent one year in a foster home as an infant, then adopted. Present either in her head/rational mind or in her body and sexuality or in her mind and the spiritual dimension. By contrast, she is rarely centred in her heart/soul, although she considers herself an emotional person. Social: Has had a boyfriend for one year, whom she has known for 17 years. Has recently abandoned her lover. On examination: neither an acute nor a chronic affliction, not psychotic, but skilled at escaping from herself and from emotional pain. All symptoms might have the same cause. EXERCISE: the patient is instructed to search for such a cause. In addition, she will compile a complete list of all symptoms and their causes, as far as possible.
Second QOL conversation: Arrives with about ten written pages -well done. She has sorted out all her symptoms and feels that it is very much about her not being allowed to be the woman she is. She feels as though something is missing in her history, so the next exercise is to go home and write down her relevant history. Since many of her problems have occurred in connection with a relationship, she will focus on that. She has realised that all her problems relate to "suffering in her senses": eyes, ears, mouth, neck, skin and reproductive organs. Next appointment in two weeks. Patient wrote concerning EXERCISE:1. Suggested common cause: entire organism craving attention by displaying symptoms. There appears to be a need for care and love. MENTALLY ORIENTED: 19 years old and in steady relationship with routine sex. Often, the sexual activity moves into the head, and I lose contact with my entire body, the sexual desire. Then present in head and crotch.
SELF-FOCUS: Self-absorbed due to ailments. HIGH ENERGY/COLLAPSE: 22 years old, nervous breakdown due to working overtime and insomnia, later tendency to work more than body and mind can handle, then collapse from exhaustion or depression.
"HYPERSENSITIVITY": From childhood, exposed to anger and hysteria. Later, hypersensitive to other people's attitudes and moods. Sense other people's moods and become affected as if they were my own.
WITHDRAWAL: Childhood, withdrew into myself and my own physical space to have peace and quiet. Later, I need to be alone in my physical space following gettogethers, otherwise I feel invaded.
HEAVY DEMANDS: Childhood, very responsible and helpful. Now I make excessive demands on myself, to be attentive and helpful in relation to other people. TENSION: Shyness from childhood, reduced self-esteem, feeling of being different. Adult shyness, feels the body become tense because it wants to escape the situation, because it feels insecure.
SHYNESS/OVER-CONFIDENCE: Puberty, developed a tough personality through idolisation. Now I can display over-confidence in tight situations, I become tired afterwards.
This list is a fine example of how we become ill when we simply do not have the vital energy to stay in one piece when we are young. Many patients disclose a similar list when forced to compile a complete Ventegodt and Merrick: Clinical Holistic Medicine (26) TheScientificWorldJOURNAL (2005) 5,[324][325][326][327][328][329][330][331][332][333][334][335][336][337][338][339] list of all symptoms and ailments. Usually, the regular physician sees only the tip of the iceberg and the patient sees only one symptom at a time and does not realize that there may be 20 different symptoms appearing one after the other.
Third QOL conversation: Good progress in understanding the problems. Patient can now partly control tinnitus herself. There is no love in her life. She does not want to be vulnerable and is therefore unable to give and accept love. She protects herself by being masculine and domineering. EXERCISE: Accept your vulnerability and responsiveness, love the vulnerability of femininity. Take stock of your current symptoms. For next time: Give-and-take flow must be restored.
Fourth QOL conversation: The patient relates that a lot is happening inside her in terms of order and understanding. She believes her ailments derive from the suppression of the vulnerable woman, which she has worked on since last time. Brings full-size picture of herself as vulnerable. Has been angry, had a fit where she smashed a door. Has celebrated her femininity and wearing a dress for the first time. Six out of 13 physical symptoms have improved (migraine, herpes both on lips and genitals, which she usually has for months, did not develop beyond a herpes "tickle" at the sites without atrovir, no longer troubled by "black spots" in her visual field, low back pain and fungal infection in the vagina have improved and genital warts "now look white as though they are about to go away", three of the physical symptoms have become worse (urge to void, tension in head and neck and tinnitus), while the rest are unchanged (phlegm in the throat, protracted menstruation, childlessness and pigmentary disturbance). The six psychological symptoms have improved substantially -easier to fall asleep, not so depressed, no episodes of paranoia, not so forgetful and absent-minded, does not think so much about being the rejected child, and is not "manic". EXERCISE: Enjoy being a woman, be good to yourself, but accept the suffering that sometimes emerge.
Fifth QOL conversation: She is feeling much better -the following have disappeared: migraine, herpes simplex 1 and 2, black spots in the visual field, low back pain, fungal infection in the vagina. Improvement: tension in neck/head, phlegm in the throat, urge to void and pigmentary changes disappearing from her hands, patient says. Psychologically, four of the six symptoms are gone: sleeping problems, depression, feeling of being a rejected child (patient dreamed that she took care of it), she is less forgetful and absentminded, but her paranoia is unchanged. New symptom: Has a sensation of fullness in the stomach. Patient feels disabled by her childhood and we discuss the nature of the damage. "I have lost contact with myself". EXERCISE for next time: Write one A4 page about each question: Who are you? What are you? Where are you? Why are you? If the patient can handle this difficult exercise, I (SV) think we will make a breakthrough soon.
Sixth QOL conversation: Patient says it is as though a miracle has happened. She has worked on who and what and how she is and brings many closely written pages. She has had some problems with her boyfriend, however, who has moved in with her. She makes sexual demands on him, and he backs off. We talk about her making demands, because she feels that "she has a need" and about the dependency being a manifestation of her escaping from herself instead of being at ease in her own centre. When confronted with this, the patient cries. We talk about being at ease in one's centre and being balanced. EXERCISE: Sit and be who you are. Stop making demands, stop criticising and controlling. Centre physically, in terms of your thoughts, in terms of your emotions. Seventh QOL conversation: Patient has gone through a long and significant, painful process, dealing with the early loss of both father and mother. She was placed in a foster home. We discuss that. EXERCISE: Write down all the essential events from that time in details. Search for your life-denying decisions. All negative feelings are rooted in a statement (e.g. "I'm going to be sick", "I feel like dying") that says it all. Find it.
Eighth QOL conversation: Patient has had great success in touring as an artist and sold nearly everything she has painted. Is happy on arrival. Has established contact with the evil inside her. EXERCISE: Describe in detail all the evil in your life, including the circumstances of your conception (rape).
Ninth QOL conversation: Patient has relived a vast amount of evil since last. Subsequently, she has had a vision, where -perhaps God -showed her as a ray of light in the darkness, whose purpose was to transform the darkness. We talk about her personal mission, which she has formulated first as "I am the light transforming the darkness", then as "I am light in the darkness." Then she becomes very sad and ends up curling into a foetal position, in contact with the events where she denied her mission. EXERCISE for next time: Find all the events in your life, when you have denied being a light in the darkness. What did you decide?
Tenth QOL conversation: Patient is very sad on arrival. "I miss my mother," she says. We work on that statement: "I miss you, Mum," and the patient cries. She has had three mothers, first a biological mother who gave her up to a foster home, when she was a few months old. Then a mother in the form of the caregiver at the foster home and finally an adoptive mother. Early on she made the following decisions: The man/father does not see me and the woman/mother is going through hell, because of the man. EXERCISE: Let go of these statements, when you have found their exact wording. Finally, the patient has a very severe headache, treated with massage of very tense neck muscles, a hard knot on the right side corresponding to cervical vertebrae C4, C5 and C6.
Eleventh QOL conversation: Severe problems with anxiety and pity for her partner. Is very frightened and wants to sell the flat, which he will not move out of, to get him out. He nearly killed an ex-girlfriend, when she left him, she was hit on the back of the head with a bottle and hospitalised with head injury. She does not want to involve the police, which is what I recommend. EXERCISE: The exercise for next time is to accomplish a respectful and proper farewell and make arrangements for the break-up and for her partner to move out. We discuss her lacking grounding, and the solar plexus and heart being weak and needing attention.
Twelfth QOL conversation: Patient has been moving at a very fast pace and was apparently close to going insane yesterday. Today she is more grounded and possible to talk to. We talk about her feeling a fundamental pain in facing reality, that is, the pain residing in her personal mission: being a light in the darkness. I recommend her to see a private psychiatrist, which she rejects. I give her the following EXERCISE: she has to make a determined effort to become grounded and slow down, otherwise things may easily go wrong. When the emotional pain moves from the body to existence and cannot be accommodated there, it often returns to the mind, which easily becomes overloaded: the patient becomes psychotic. It is important to understand this mechanism to allow the treatment to last for as long as the patient needs and to avoid any unnecessary human tragedies. To prevent psychoses, the physician has to slow down the over-zealous patient, for example by instructing the patient only to work on herself for 30 minutes a day; then, the patient should turn to other activities when she can no longer handle the responsibility for the emotional pain uncovered by the exercises and homework.
Thirteenth QOL conversation: Patient has broken up with her partner and now wants to live in celibacy for two years. I suggest six months. She has produced a fantastic picture of positive and joyous fertilisation. She is working on very early and harsh rejection by her parents, which is healed by her reconnecting to humanity in her art, which now deals with people and bodies. Lying on the couch, she cries and talks about when she was let down. "I gave up," she says, and I let her enter the sensation of giving up and "dying" on the couch. In my assessment, the patient is making good progress, processing ever deeper and more painful layers of her consciousness. Her tendency to become psychotic has decreased.
Telephone conversation 1: Patient feels well, needs grounding, has dropped her other alternative therapist and is going for Rosen sessions. Wants to go into gestalt therapy. Can return to me in three months or if required.
Telephone conversation 2: Things are going well in the Rosen therapy, working on her relationship to muck -physically and figuratively. Works on the statement, "I don't want to grow up". Stopped growing at 13 years. Watched Tin Drum and broke down completely because of that statement.
Telephone conversation 3: Very happy about sessions with gestalt therapist, who "has so many colleagues" that help her. "The other day a pixie removed a load of muck from my third eye," the patient says, and "I feel so happy every day." "I don't feel ill anymoresometimes I hear a tiny noise in my ear, but I know why it's there, so it goes away." Apparently, the patient has recovered and is now working on her personal development and will therefore finish the gestalt sessions. Can continue for as long as she needs with Rosen therapy. To return to me for check-up/adjustment in about three months. Patient draws a full-size picture of herself, and as she develops herself during therapy the picture transforms from shadow through pain and agony, 'naked' and 'skeletal' into a vivid, lovely and life-affirming picture, the primal mother, Eve.
Fourteenth QOL conversation: Brings a list of 30 negative statements, which she has to let go of. I help her with the first one. "I don't receive anything" -let go with roll. Then on the couch, where "heart blockage" is loosened in the spine (T12). We work on a shift in perspective: from false to true self. EXERCISE: Let go of all the statements one by one until they have all been released. Return in a month with the list, organised by chakras.
Fifteenth QOL conversation: Feels very bad, new gestalt emerging: my task is "to balance yin and yang", I am in this world to create harmony between man and woman. We work on the gestalt with time line therapy ranging from conception to just after her birth, we go through it three times, the patient is first her mother, then her father, then both to begin with and she sees numerous diabolic images, where father and mother rip off each other's flesh and skin, like classic devil scenarios from medieval painting. Has to stop here, but should continue. Believed the gestalt's statement to be the second mission in therapy and that is correct. First mending statement, it would seem, where the pain around her becomes internalised. Next appointment ASAP.
Sixteenth QOL conversation: We continue and process the event from conception to birth four times. The patient enters something very dark, first massive catharsis, then it crystallises apparently as an attempt at abortion with a knitting needle through the brain, foetus 2-3 months old. Patient cooperates with her mother, who wants her dead and commits suicide in terms of energy, constantly sets aside her own life to become quiet, easy and tractable and not difficult or demanding when she was born. Patient sees herself as grey and dead at birth. Process to be continued at another time; the most emotional parts appear to have been removed from the event, but still no absolute clarity. If the patient knows her life purpose, formulated at conception according to the purpose of life theory, it is possible to review the patient's entire life from conception to the present by means of time line therapy. It often promotes a patient's sense of responsibility for his or her own life when all life events are seen in the right context, based on the purpose of being here, which is the meaning of life.
Seventeenth QOL conversation: What is responsibility: "That is when you act in relation to your goal," the patient suggests. I suggest that it is about being a causal soul in relation to your world and enduring the suffering by fighting for the development of your determination. "I am my body," the patient says -long talk about the difference between having and being. Has begun working with great discipline for eight hours a day, which is new to her, feels like an isolated bubble, which I consider a step in the right direction. Patient makes a prayer to God to be allowed to reconnect. We talk about divine creativity; talking about it, she does not seem to fully understand it. EXERCISE: Cosmological upgrading: describe your cosmology based on you as a responsible, causal soul (you are the cause). EXERCISE: Let your pictures make a real difference to people's lives. Then they are sure to sell.
Eighteenth QOL conversation: Has no longer any physical complaints, but is still struggling with existential matters. We talk about the world being evil and the patient cries. "Many people are worried about me and believe that it is not good for me to see you," she says. Who, I ask, and the patient finds it difficult to come up with any names. "Does anyone mind you letting go of your negative perception of the world as an evil place," I ask her, and the patient cannot think of anyone. We work on the statement "the world is evil", and I coach her to see the world as both evil and good (the divine perspective). At first, the patient is unable to say it out loud -"I am breaking a vow of silence to lots and lots of creatures in Heaven, I am going insane," she says. "You are already crazy, there is no need to be scared," I say. We talk about letting go of her standpoints, so that she can sink into life and let "life carry her" instead of her carrying life. Cries and moans, lets go of the evil world. To return in two weeks. EXERCISE: Look at everything as 'both-and', not 'either-or'.
Nineteenth QOL conversation: Has done her exercise very well. Patient "tries to trust in me", tries not to be controlled by her emotions, and so on. Strong defences. Does not want to make a plan for her life and that way assume responsibility. Enter your position as a free soul and formulate your wishes concisely and the road to fulfill them. Abandon the strategy of floating in a stream of emotions. It will take you nowhere. Hardly any progress since her healing session. Life and death are the same, she says, when I point out to her that she may be heading for her own death. Patient takes no responsibility. EXERCISE: Make a plan for the next five years of your life, your personal life, working life, social life, etc. Specified goals at 3 months, 6 months, 12 months, 24 months, 36 months, etc.
Twentieth QOL conversation: Has made a fine plan as agreed for her working life, personal and social life. The very first day after her last appointment she started to feel better, and today she is not suffering at all. She is aware that she is still like a girl in puberty, and we talk about being an adult -and about the significance of becoming 'reparented' in therapy and finding one's bearings as an adult. Has had breathing difficulties. "Would you like to call your mother and say goodbye?" I ask her. "Uh, don't say that -fear of death?!" Yes, I say. And press my hands against her chest. Hold your breath. She holds her breath and starts to cry. "Here is the black hole I don't want to go into anymore," she says. "But now you are in the soul perspective, and now you can let go of your negative decisions." "Alright then." "I want to die," she repeats, going back in time to when she was about to be conceived and ended up dying as a free soul and becoming the soul she has been ever since. As she says "I want to die", it is as though she vanishes in a haze, and I have to slap her face a few times to make her return. She becomes satanically angry and I ask her whether she would prefer exorcism. She repeats it and lets go and ends up having a smiling mouth on her chest, where there used to be oppressive fear of death. EXERCISE: Let go of your statements. First repeat it and understand where it comes from. Do not let go until you have acknowledged it clearly. Those you cannot handle, you can bring to me. Begin with the easiest ones. Promises me copies.
Twenty-first QOL conversation: Patient broke down the other day at the social security office and finally managed to ask for help. She could not bear the thought of having employment found for her, because she is working on her art and herself. Cried incessantly and felt completely powerless. Finally, the good mother in the form of society was there for her. That is understandable considering what she has been through. /Suspected post-traumatic stress disorder/. Medical certificate form 205 -three months. Partially unfit for work. Believes herself that she is able to work for a few hours a day. Arrives with 30 negative statements: The world is evil. I am evil. I don't receive anything. I become wrong. I am worthless. I am all wrong. EXERCISE: Is to convert all these statement into a positive life philosophy. Stating the opposite is not enough (for example "I am wrong" becomes "I am right"), she has to be genuinely creative.
Twenty-second QOL conversation: Has worked very well with life philosophy -36 statements about herself have found a healthier replacement. Now has to work on her attitudes to the outside world and later also to life inside herself. She suffers from anxiety/paranoia, for instance when she is in brightly lit rooms with many strangers around her. "I steal", "Other people hurt me" etc. has to be turned into the opposite. EXERCISE: Three A4 pages about her perception of herself, the outside world and lifethe three main elements of life philosophy. Compile a list of all the old statements and write them next to the new and truer ones. She is in existential pain, and we talk about this pain being a basic condition that can only be counterbalanced by the fundamental enjoyment contained in life. PLAN: Gestalt therapy/holotropy can be applied now.
Twenty-third QOL conversation: Has written a book about her process that will be finished soon. We talk about having a narrator in the book explain what happens in each chapter in the present tense. Still has problems with sexuality and femininity -perceives herself as androgynous -"I am yin and yang," she says. I believe a female therapist can help her, so: PLAN: My gestalt therapist, psychotherapy/holotropy can be applied nowfor the male/female/sexuality field. Process: "Aspects of life purpose (the Swiss knife)". We draw her 'life purpose flower' with petals: knowledge, insight, creativity, love, colours and energy, joy, play, craziness and wildness, consciousness, exchange, intelligence, life, including sex, etc. EXERCISE: Discover all the petals in the flower = your talents, write 4-7 lines about each quality.
COMMENTS
The most important aspects of this case history are the following: • Recognition of illness. The patient was suffering physically from 14 diseases or disorders: Migraine, herpes simplex 1 and 2 (mouth and genitals), tension in neck and head, often phlegm in her throat, black interfering spots in the visual field, tinnitus, protracted and heavy menstruation, frequent low back pain, fungal infection in the vagina almost constantly, treatment-resistant genital warts, annoying urge to void, plus sun allergy, pigmentary disturbance, and involuntary childlessness. • Psychologically she had six problems: sleeping problems, minor episodes of depression, paranoia, tendency to be forgetful and absent-minded, painful feeling of having been rejected as a child, and tendency towards mania. • Spiritually she described: longing for more contact with her own essence. • Sexually she described: mentally oriented (often being inside her head) and varying sexual desire. • Emotionally she described: overwhelming shyness, instability, low stress threshold and tendency towards nervous breakdown/collapse, often after working overtime. • Socially she described: hypersensitive to other people's attitudes and moods, tendency to withdraw from social relations, excessive demands on herself, tension, alternating between shyness and sudden overconfidence. The patient's list of complaints would not be unusually long for a patient 30 years older, but she was far too young to be so troubled. It contained about 20 different issues, all intertwined. • Causal explanation. It turns out that the 20, seemingly independent, ailments all derived from one and the same source deep down in the patient's existence. When this source, invariably old pain, was identified and integrated, all the ailments disappeared. This is genuinely effective medicine. The patient herself had thought about a possible underlying cause and, in that connection, she obtained an important insight: all her physical complaints were somehow related to her skin and the other sensory organs. Her suggestion of a central, common cause was: "The entire organism calls for attention by displaying symptoms. There appears to be a need for care and love". Finally, everything was gathered in an impressive diagram of the interrelations. She went from being unloved, through failing to thrive, to escaping from failing to thrive, and onwards through great (heavenly) expectations to merciless disappointment. • Ambition, trust, and persistence. The patient had to work herself into her problems with her senses and sensitivity by reliving all the often-disappointing and painful relationships in her life. I gave her a new exercise, namely, to write a detailed autobiography focusing on love and sexuality. For patients like her, this exercise may take up 100 closely written pages. The patient also did this exercise in 14 days. • Searching for truth. The patient was asked: "Who am I? What am I? Where am I? Why am I?" The focus was on life purpose, which the patient was asked to find. She took this task seriously and returned two weeks later and said: "It is as though a miracle has happened." These are her answers to the four questions: 1. Who am I? "I am like the cucumber plant growing in my garden, a strong survivor with difficult growing conditions. I am a soul in the shape of a person, a woman with a physical presence, a name, an age and a history. I contain all opportunities and potentials." 2. What am I? "A free human being, going back to search for my soul, a self less and less overshadowed by the ego." 3. Where am I? "I am in the working process in my spiritual workshop, sometimes a very lonely place to be in and very self-focused." 4. Why am I here?" "Because the universe wants me to be on Earth." "My person is needed in this world, I am here for myself and for others." "I am here now, conceived through rape, constricted in a corset during my entire creation. Unwanted, rejected, thrown back and forth. Ignored." • Responsibility for existential pain. We can now uncover the earliest and most painful aspects of her personal history. She was conceived under the most dramatic circumstances, when her mother was raped. She was unwanted and adopted as an infant. She was placed in a foster home and suffered terribly. She encountered the pain both at home on her own and together with me at the clinic. She wrote about it: "Pain, pain. Yes, I see that I am at the centre, back at the centre. I see that I have been there many times before drawn into it by numerous therapists. I see that I must stay there, at the centre of the pain ... I went by train to see the doctor, preoccupied with my pain; a woman stops and wants to help me. It touches me to see how much shows up when I do not escape. The bird in my garden is not afraid of me. God shows me love. I can begin to feel love for other people." • Healing through love and contact with God. The patient is now ready for the classic exercise called "sitting" (Vipassana): sitting and feeling herself and letting go of the tension in her body, making room for emotions and letting her thoughts be clouds drifting in the wind, while she is witnessing everything and noticing how her body is breathing by itself.
Over the course of the therapy, many things happened in her dreams that she wrote about manically and filled several thick diaries with writings about love and her new contact with God. It turned out that the terrible experience of being conceived through rape contained something very beautiful, namely, that her very life purpose had become to bring goodness into all the evil, to bring light into the darkness, apparently as a result of the rape. The reason for the patient's recovery was that the inner and outer connection was restored, so that all the cells in her body once more could receive all the information required to be healed. Externally, she has the experience of being reunited with the universe or, subjectively, God. Internally, she was reunited with life or, subjectively, her soul and its purpose for being here. In our view, it is also possible to give a purely biological description: Everything is interrelated, at all levels, information flows freely inside the organism and between the organism and the outside world and all parts of the body and mind returned to their natural and healthy condition.
DISCUSSION
According to the purpose of life theory, the gestalts with repressed existential pain can be placed either in the body or in the mind and brain. In practice, all patients who are very ill display symptoms from both body and mind. Statistically, there is a close relationship between perceived physical health and perceived mental health. This can be investigated by having patients assess these health factors themselves, for example, in a questionnaire as we did in the Quality of Life Survey. According to the purpose of life theory [20] and theory of character [30], mental illness is a direct consequence of denial of our life purpose, where we destroy our own being, doing, or having. As with mental illness, physical illness appears to be highly sensitive to the restoration of life purpose. It is difficult to explain this in an exact manner because the mechanism by which gestalts -the frozen, painful now -are displaced from the whole to a part is not thoroughly understood by science. Indeed, the organization of the flow from the cells in the organism to the conscious mind is not thoroughly understood.
However, it would appear that cells merge their internal information systems and fields of consciousness when they join and form tissues and organs, which in turn merge into organ systems, which in turn are united in the body as a whole. The unity is not complete, however, since each level of the body and all its parts retain some degree of autonomy and a constant, active, and alert willingness to help. A major or minor part of the body can, therefore, spontaneously be assigned the function of accommodating intense emotional pain, the purpose being the survival of the organism as a whole. A good theory on exactly how this takes place has yet to be found. It is quite odd that our tissues and organs are independent and intelligent, cooperative and able to make autonomous decisions. In principle, it is no more odd than the assumption that the brain, which is also such an organ consisting of cells, is able to make decisions on its own, which very few people would doubt! The brain and the bowel, for example, are really not that different. Obviously, their decisions relate to different things, but they invariably serve the totality, its survival, and development in life.
When cells and tissues assume the task of accommodating a gestalt instead of attending to their original functions in the body, this will cause severe disruption. For example, in connection with the immune system, such blockages can provide the tissue with a status of nonself, which means that it falls victim to an autoimmune attack. When the cells are withdrawn from the totality, the control that normally prevents the cells from dividing uncontrollably disappears, leading to the development of the disease referred to as cancer. Depending on which cells are withdrawn from the organism and in what way, a wide range of different diseases occur. This theory would be strange, complex, and poor if it were not supported directly by experience.
Our experience with applied holistic medicine founded on the purpose of life now dates back several years. Apparently, patients who are willing and able to sort out their lives so thoroughly that they find themselves again, and can contain their life purpose and live accordingly, will recover almost irrespective of the nature of their disease. Unfortunately, we do not yet have enough examples to provide sufficiently strong evidence to support this hypothesis. We have only worked in depth with about 30 patients suffering from completely different diseases and ailments, so this field still requires a great deal of research, in terms of both quality and quantity. Having said that, any person who breaks through to his or her life purpose and is able to live accordingly will, in our clinical experience, also recover, regardless of the nature of the particular disease -even in the case of very serious and potentially life-threatening diseases [31,32]. When we have worked with the patients according to the purpose of life theory and have made them break through and acknowledge their life purpose, very often great -and in rare cases almost miraculous -things have happened to the patients with very different diagnoses such as chronic pain, tinnitus, cancer, and sclerosis. Scientifically, it may appear surprising that a psychosomatic approach is effective in virtually any disease, but that appears to be the case. Thus, the door has been opened to a new era of psychosomatic medicine of the type that we refer to as holistic medicine or, to be more precise, consciousness-based medicine.
CONCLUSION
Sometimes the patient has many different diseases, both somatic and mental. Biomedicine will normally see and treat each disease individually and, with many diseases and effects of the many drugs used, that can become a burden in itself, preventing the patient from getting better. With holistic medicine, all the diseases are seen as whole, as symptoms of a more fundamental imbalance in the state of being. What becomes the focus of the holistic treatment is the intention to understand the cause of all the different diseases and, thus, to be able to cure the patient. The disease is caused by the patient's poor quality of life and the fundamental existential imbalances.
The holistic physician must help the patient to recover his or her existence and create a good relationship with self. The life mission theory, the theory of character, and the holistic process theory of healing stress that recovering the purpose of life (the life mission) and the physical, mental, and spiritual character is what is needed for the patient to regain life, love, and trust in order to find happiness and realize the true purpose of life.
We illustrate the power of holistic medicine with a case study, an invalidated female artist, aged 42 years, who suffered from multiple health problems, many of which had been chronic for years: tinnitus, 14-days menstruation in each cycle, migraine with severe scotomata blinding her for up to 20 min, often | 2018-04-03T00:37:14.078Z | 2005-04-12T00:00:00.000 | {
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243809250 | pes2o/s2orc | v3-fos-license | Extraction and Nano-Sized Delivery Systems for Phlorotannins to Improve Its Bioavailability and Bioactivity
This review aims to provide an informative summary of studies on extraction and nanoencapsulation of phlorotannins to improve their bioavailability and bioactivity. The origin, structure, and different types of phlorotannins were briefly discussed, and the extraction/purification/characterization methods for phlorotannins were reviewed, with a focus on techniques to improve the bioactivities and bioavailability of phlorotannins via nano-sized delivery systems. Phlorotannins are promising natural polyphenol compounds that have displayed high bioactivities in several areas: anticancer, anti-inflammation, anti-HIV, antidiabetic, and antioxidant. This review aims to provide a useful reference for researchers working on developing better utilization strategies for phlorotannins as pharmaceuticals, therapeuticals, and functional food supplements.
Last by not least, phlorotannins are strong natural antioxidants [55][56][57][58][59][60]. Dieckol can suppress UV-B radiation induced photo-oxidative stress in human fibroblast cell line and reduce cell damage [55]; diphlorethohydroxycarmalol (DPHC) isolated from I. okamurae can scavenge UV-B radiation induced ROS in human fibroblast cells [57]; and dieckol can reduce cell damage induced by UV-B radiation both in vitro (HaCaT cells) [61] and in vivo (zebra fish) [59]. Phlorotannins can also potentially be used as functional ingredients in skin care products to offer protection against photo-induced skin damages.
Although phlorotannins have many attractive biofunctionalities, as members of the polyphenol family, phlorotannins also suffer from low bioavailability, which limits their utilization. Polyphenols' low bioavailability is mainly due to their low absorption in the human gastrointestinal (GI) tract following consumption and their extensive biotransformation within the gut, which may lead to their rapid clearance from the body (Figure 1 [62]). To improve the bioavailability of phlorotannins, nano-sized delivery systems have been investigated [26,61,[63][64][65][66][67][68][69][70]. Figure 2 showed a schematic representation of examples of these systems [71]. These nano-enabled approaches could protect phlorotannins against transformation in the gut; they can also improve delivery/absorption of phlorotannins [61]. Nano-phlorotannin systems were also shown to enhance the functionality of the phlorotannins. For example, gold nanoparticle-phlorotannin and silver nanoparticle-phlorotannin complexes have been investigated by serval researchers [26,61,[63][64][65][66][67][68][69][70]; they were shown by DPPH assays to have elevated antioxidant activity. Nano-enabled improvement on bioavailability and bioactivity may offer a more effective way for the utilization of phlorotannins in pharmaceutical, food, and biochemical industries.
Extraction and Purification of Phlorotannins
The operation of phlorotannin extraction aims to achieve two major goals: one is to extract as high a quantity of phlorotannins, primarily from brown algae, as possible; another is to maintain high bio-integrity and bioactivities of the phlorotannins [7][8][9][10][11][12]. These two goals could be in conflict: phlorotannins are unstable polyhydroxylated molecules, and they can easily be oxidized [17]. Considering the different types of phlorotannins and their susceptibility to various physical/chemical processing, development of different isolation/extraction methods suitable for different types with specific biofunctionality [17] remains a challenge [18,19]. A quick summary of the pros and cons of common isolation/extraction methods reported by different research groups follows.
HPLE methods: HPLE is generally considered environmentally friendly to an extent, if water is used instead of organic solvents [2,27,29,31,32]. Low cost is another advantage of HPLE [28,30]. HPLE can also be scaled up to a certain extent to go beyond lab scale, but its yield as of today is still not high [29]. Another restriction of HPLE is that it requires the solvent to be kept in liquid state during the entire extraction process, which can be an operational challenge [29]. In addition, macroalgae might contain heavy metal [72], which could be extracted together with phlorotannins by HPLE [72] and become enriched.
HPLE-RP methods: to reduce the risk of heavy metal being enriched along with phlorotannins, resin purification (RP) methods were introduced [33,34]. With RP, heavy metal content in the phlorotannins extracted from macroalgae can be dramatically reduced [33] Kim et al. [33] compared four types of resins: HP-20, SP-580, XAD-7HP, and XAD-2. All four types of resins displayed effective purification of phlorotannins [33]. Among them, HP-20 resins displayed the highest absorption and desorption capacities, and this led to the best purification performance.
NEDES extraction: Another new development in phlorotannin extraction was the use of deep eutectic solvents (DESs). DES is a new class of ionic liquid (IL), typically formed by mixing choline chloride with hydrogen bond donors [81]. Environmentally friendly natural DESs (NADESs) were first explored for extracting active compounds from plant raw material [82,83]. Oblichinskaya et al. reported the first effort of using NADES to extract phrolotannins from brown algae F. vesiculosus L. and A. nodosum (L.) Le Jolis [84]. Ten NADES with different compositions were studied for their efficiency of extracting phlorotannins. They found that aqueous solutions of NADES based on choline chloride and lactic acid were the most efficient, comparable to those of traditional extractants such as Me 2 CO 3 and EtOH and almost 10 times more efficient than pure NADES. As NADESs are clean green solvents, they could become an alternative to organic solvents to reduce the environmental footprint of phlorotannin extraction.
Following extraction, purification and characterization are the next steps. Molecular size-based separation is often used to purify different types of phlorotannins: these methods usually involve dialysis or ultrafiltration (UF) or a combination of the two [85]. Dialysis is an approach to isolate phlorotannins by different molecular weight [79,86,87]. The proper selection of membrane pore size is key to separating high molecular weight (MW) phlorotannins from low MW ones [79,86,87]. As for the UF system, with a fixed flow rate, UF can have a better performance [85]. The capacity of the UF system reported in the literature is still limited to lab-scale operations, ranging from 100 mL to 10 L [85]. One advantage of the UF system is its wide range of MW cutoff (MWCO), but industrial-ready UF for phlorotannins remains to be developed for large-scale applications [85].
After purification, NMR spectroscopy, mass spectrometry, and advanced chromatography are three common tools to evaluate the phlorotannin product to characterize the level of polymerization and the molecule weight distribution [88]. Phlorotannins could be indirectly quantified via the Folin-Ciocalteu (F-C) method, which measured the antioxidant activities in a sample. Although the F-C method is not selective for phlorotannins, it could yield quick readings with a simple operation, short turnaround time, and high throughput [20,79,[89][90][91]; hence, it has been used for rapid determination of phlorotannin content in brown algae extracts [92]. In addition, computational chemistry was another useful tool [93][94][95]. Computational chemistry method can provide a cost-efficient supplementary tool to NMR spectroscopy [93][94][95][96]. Density functional theory (DFT) quantum chemical calculation was introduced to determine electronic structure of phlorotannins [96]. Natural bond orbital (NBO) was introduced to determine the donor acceptor stabilization energy caused by intramolecular and intermolecular hydrogen bonds in phlorotannins [96].
Bioactivities of Phlorotannins
As a group of tannins, phlorotannins from brown algae were shown to be effective in regulating several biochemical processes linked to the disruption of homeostasis in major chronic diseases [18,39]. They have been revealed to have a range of diverse biological functions and activities, such as antibacterial [7], anti-inflammatory [8], antioxidant [9], antidiabetic [10], anti-HIV [11], and anticancer [12,97,98] activities. They can form complexes with pro-oxidant proteins, chelate metal ions, or directly trap reactive oxygen species (ROS) to modulate cellular responses to stresses and/or injuries [99]. The most studied bioactivity of phlorotannins is their ability to scavenge radicals due to donation of their hydrogen atoms or electrons [100][101][102][103][104][105], which can be evaluated via 2, 2-diphenyl-1picrylhydrazyl (DPPH) radical-scavenging assay [88,106,107], ferric reducing/antioxidant power (FRAP) assay [108], dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay [109], trolox equivalent antioxidant capacity (TEAC) assay [110], and electron spin resonance spectroscopy [107]. Table 2 listed some of reported anti-inflammation effects of phlorotannins from literature. This list is by no means comprehensive, but it showcases the great potential of phlorotannins as anti-inflammation agents. In addition, phlorotannins isolated from brown algae have also been widely reported to have anticancer bioactivities. For example, eckol from brown algae was shown to suppress tumor growth in vivo in S180 xenograft-bearing mice [46].
Nano-Sized Delivery Systems for Phlorotannins to Enhance Bioavailability and Bioactivities
Although phlorotannins have all the highly desirable bioactivities and functionalities summarized in Section 3, their utilization as pharmaceuticals, therapeuticals, and food supplements is often limited due to their low bioavailability. Phlorotannins are unstable in the acidic condition of the gut and the alkaline condition of the small intestine; if not protected, they could easily be transformed and/or metabolized [138]. Phlorotannins are water soluble, but their absorption in the human gastrointestinal (GI) tract following consumption could be low, as evidence suggested that phlorotannins were mainly absorbed after metabolized in the large intestine [139], not in the stomach and the small intestine. A number of strategies have been used to increase the chemical stability of phlorotannins and to improve their transportation/absorption in the GI tract via nano-sized delivery systems that involved the encapsulation of phlorotannins in nanovectors, which offer protection against chemical/enzymatic transformation. These nano-systems could improve the distribution and overall bioavailability and bioactivity of the phlorotannins [65]. Such systems differ for the internal structures (e.g., core-shell-like vs. embedding matrixes) and the physical/chemical states of the encapsulated active ingredients (i.e., the phlorotannins), and their encapsulation efficiencies (EE, defined as the experimental loading/theoretical loading × 100%) were different. Effective drug delivery systems usually require an EE to be higher than 60%, which could be used as a reference to evaluate various phlorotannin nano-delivery systems.
Nano-sized delivery systems utilize nanoparticles or nanostructures with diameters ranging from 1-1000 nm to encapsulate/carry phlorotannins as the "payload". These nanoparticles, due to their size, exhibit properties and phenomena attributable to their dimension [140]. Nano-sized encapsulation systems can be made via several different approaches based on chemical, physical, and physiochemical principles. Chemical encapsulation (e.g., interfacial and in situ polymerization methods) usually requires the polymerization of monomers that cross-link with the payload [141]; physical encapsulation, on the other hand, usually uses already formed polymers (and natural polymers) to form a matrix in which the payload can be entrapped via various methods including air suspension, pan coating, spray drying, spray congealing, and micro-orifice systems [65]. Physiochemical methods (e.g., coacervation, phase separation, complex emulsion, meltable dispersion, and nanoprecipitation) aim to form stable nano-sized payload-carrying systems through particle size reduction processes [142]. Nanoencapsulation techniques, especially physical and physiochemical methods, have been [140] widely investigated for improving bioavailability and bioactivities of polyphenols by protecting the polyphenols against digestive transformation/degradation, reducing their toxicity and improving their water solubility [65]. Various nanocarrier systems, including cyclodextrins, nanospheres, nanocapsules, solid lipid nanoparticles, liposomes, and micelles, have been explored for polyphenols in general. Here, we will review a few specific cases in which phlorotannins were the payloads.
Shibata et al. [71] studied phlorotannins extracted from brown algae E. bicyclis, E. cava, and E. kurome; they showed that, with a liposome carrying system, the nanoencapsulated phlorotannins showed potent inhibition of phospholipid peroxidation at 1 µM, had significant radical scavenging activities against the superoxide anion (50% effective concentration values: 6.5-8.4 µM) and DPPH (50% effective concentration values: 12-26 µM), and were more effective compared to ascorbic acid and α-tocopherol. Furthermore, a nanocarrier system of phlorotannins and soybean protein prepared by co-extrusion was shown to have a pronounced DPPH radical scavenging activity. The authors concluded that the nanoencapsulated phlorotannins could be a good functional food or supplement with anti-inflammatory activity.
Physical encapsulation was also applied to phrolotannins. In a recent effort, Cuong [69] reported nano phlorotannin powder made by spray-drying of phlorotannins isolated from S. serratum grown in Vietnam. The optimum spray drying condition was established as follows: a carrier-to-solution ratio of 10%, compressed air pressure of 0.8 bar, liquid feed speed of 10 mL min −1 , and inlet temperature of 110 • C. Under these conditions, the antioxidant activity of phlorotannin powder possessed total antioxidant activity at 4.347 ± 0.018 g ascorbic acid equivalent 100 g −1 DP, reducing power activity at 9.390 ± 0.024 g FeSO 4 equivalent 100 g −1 DP and DPPH free radical scavenging activity at 70.02 ± 0.26%. The phlorotannin content and antioxidant activities of the nano-sized powder particles were affected by the spray drying condition (p < 0.05). These particles were nano-sized with morphology of irregular shapes. These nano phlorotannin powders displayed antioxidant activities for potential application in pharmaceuticals and nutritional supplements. In another effort [81], electrospun polycaprolactone (PCL)/phlorotannin micro/nanofibres containing different phlorotannin (from E. cava) concentrations (1, 3, and 5 wt%) were fabricated. Due to their hydrophilicity and water absorption ability, phlorotannin-containing fibrous mats exhibited outstanding wettability compared with pure PCL fibrous mats. The biocompatibility of the mats was examined in vitro using osteoblast-like cells (MG63). The phlorotannins were shown to promote alkaline phosphatase (ALP) activity and calcium deposition, which could induce bone regeneration. Cell viability (at 5 wt% phlorotannin), total protein content, ALP activity, and calcium deposition were all higher with PCL/phlorotannin mats than with pure PCL mats. These results suggest that electrospun nano PCL/phlorotannin fiber is a promising bioactive material for enhancing bone tissue growth. Lin et al. also demonstrated phlorotannin-encapsulating nanofibers with blended sodium alginate (SA) and poly(ethyleneoxide) (PEO) as the matrix via electrospinning, The optimum blending ratio to produce quality nanofibers was found to be 50:50:10 (SA/PEO/Ph); the nanofibers were shown to have an average diameter of 331 nm. The fibers were then used as an antibacterial reagent against S. enteritidis on chicken at 4 and 25 • C with great performance; the cell count drastically decreased from 6.20 to 3.28 Log CFU/g at 4 • C and decreased from 8.80 to 2.53 Log CFU/g at 25 • C. Their application significantly increased the shelf-life of chicken, which suggested that these nanofibers could be good food packaging materials due to the activities of the phlorotannins. Cui et al. [67] used a natural polymer (Momordica charantia polysaccharide (MCP)) as a nanofiber matrix to encapsulate phlorotannins via cold plasma treatment. The successfulness of PT loading into MCP nanofibers was confirmed using SEM, AFM, TGA, and DSC, as shown in Figure 3. After cold plasma treatment, the release efficiency of PT from the nanofibers was enhanced by 23.5% (4 • C) and 25% (25 • C). In addition, both antioxidant and antibacterial activities of the MCP-phlorotannin nanofibers were markedly improved. These nanofibers could be a novel functional food packaging material. In this paper [67], the authors reported that both MCP and phlorotannin exhibit antidiabetic and antioxidant properties. They demonstrated that an MCP nanofiber-phlorotannin complex can enhance antioxidant effects. Thus, this MCP-phlorotannin complex was also used to make novel and enhanced food packaging materials.
Solid dispersion technique is another method that is widely used for generating nanocapsulation systems for wrapping a core material (the payload) with functionality (e.g., phlorotannin) inside a coating material. This technology can not only mask or retain flavor and increase solubility of the payload, but also protects it from degradation by environmental factors, and controls its release at the target site [82]. In a recent report, Qi et al. [66] investigated the potential of using polyvinylpyrrolidone (PVP) as the coating to produce nanocapsulated PVP-phlorotannin complexes (Figure 4) with improved bioactivities and bioavailability. Different loading ratios of PVP vs. phlorotannin were investigated, and an optimum ratio of 8:1 (w/w) was established. As shown in Figure 5, the results indicated that the PVP-phlorotannin nanoparticles showed a slow and sustained kinetic release of phlorotannin in simulated gastrointestinal fluids; they were non-toxic to HaCaT keratinocytes (i.e., skin cells), and they could reduce the generation of endogenous reactive oxygen species (ROS). A PVP-based solid dispersion system would be a good choice for making better nano-sized delivery systems for phlorotannins in medicine, food, and cosmetics [66,83]. An interesting development with phlorotannin nano carriers was the synthesis of metallic nanoparticles (typically Au and Ag), with phlorotannins serving as the reducing agent. In these processes, the metallic nanoparticles formed have phlorotannin coating, hence can serve as delivery vehicles for phlorotannins (PhTs). Kim et al. [68] demonstrated that Au nanoparticles were formed within 1 min while mixing phlorotannins extract from E. cava with chloroauric acid at 80 • C, and the Au-PhT nanoparticles were 30 ± 0.25 nm and displayed promising antimicrobial activities. Machado et al [70] compared phlorotannins extracts from two macroalgae, C. baccata (CB) and C. tamariscifolia (CT), for their abilities to form gold nanoparticles; the results showed that CT possess three times more reducing power, almost four times more phenolic content, and four times more DPPH scavenging activity than CB, and the gold nanoparticles produced presented a non-cytotoxic profile in lower concentrations in mouse cell line L929 and human cell line BJ5-ta, which were efficient in cell regeneration, although with some differences between both species. The CT-gold nanoparticle complex had significantly better antioxidant bioactivity than the CB-gold nanoparticle complexes. Shim et al. [69] reported a green synthesis process for silver nanoparticles. The phlorotannin-silver nanoparticle complex has an average size of around 40 nm with spherical structure. These phlorotannin-silver nanoparticle complexes displayed antibacterial and antioxidant bioactivities. In particular, they exhibited a strong apoptotic anticancer activity against human cervical cancer cells that was not observed for phlorotannin, which suggested that the synergistic effects of phlorotannins and silver nanoparticles could further enhance their anticancer activities ( Figure 6). Abedel-Raouf et al. [74] reported that phlorotannin isolated from G. elongate can be utilized to synthesize phlorotannin-gold nanoparticle complexes. Several shapes of phlorotannin-gold nanoparticles were formed, as shown by TEM. Triangular, truncated triangular, hexagonal, and rod shapes were detected. The sizes of these phlorotannin-gold nanoparticle complexes were around 3-77 nm. Antibacterial bioactivity was tested against E. coli, K. pneumoniae, and MRSA. Phlorotannin-gold nanoparticle complexes of 13 nm demonstrated the best antibacterial performance. It could be concluded that phlorotannininduced Au and/or Ag nanoparticles could potentially become effective antibacterial, antiviral, and antioxidant reagents [75].
Conclusions
In this review, we summarized extraction techniques and bioactivities of phlorotannins and the strategies that have been explored via utilization of nano-sized delivery systems to improve their bioavailability and bioactivities. With nano-carriers protecting phlorotannins against GI tract biotransformation and degradation, phlorotannins can be released in a controlled manner to enhance their therapeutic functionalities, which included anticancer, anti-inflammatory, anti-HIV, antidiabetic, antioxidant, antibacterial activities, etc. This review serves as a roadmap for the development of more effective phlorotannin utilization strategies to fully take advantage of the tremendous potential of these algae-derived natural compounds as drugs and nutritional supplements for the pharmaceutical, food, and cosmetics industries.
Conflicts of Interest:
The authors declare no conflict of interest. | 2021-11-07T16:08:47.853Z | 2021-11-01T00:00:00.000 | {
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51741315 | pes2o/s2orc | v3-fos-license | Addiction Treatment Aftercare Outcome Study
Aftercare is crucial once an individual has completed drug or alcohol treatment and is in recovery. There is a continuity of care that should be followed once initial treatment is completed. This usually involves a lower level of treatment such as outpatient care and a sober living environment. In order to assess the efficacy and benefit of our addiction treatment program, we investigate a set of patients in which addiction treatment outcome and rehabilitation is determined for patients who have completed treatment and followed up. We determine abstinence rates and identify predictors of treatment outcome.
Introduction
Addiction is a serious problem affecting between 20 million and 40 million individuals in the United States.The economic impact on the country is estimated to be $200 billion dollars per year in terms of lost productivity, health-related treatment costs, and criminal justice expenses [1].The Minnesota Model of addiction treatment utilizes the Twelve-Step Facilitation program from Alcoholics Anonymous.This approach began at the state hospital in Willmar, Minnesota in 1950 and spread to the Hazelden Foundation and eventually throughout the country [2].Hazelden's model has been assessed to determine the outcome for patients.An analysis of 1083 male and female patients identified a 1-year abstinence rate of 53% [3].
A recent study investigating the outcome for 284 patients receiving treatment for addiction in outpatient treatment, residential treatment and sober living environments identified 1-year abstinence rates of 16.8%, 11.7%, and 23.8% respectively.When considering across all three treatments, 1-month abstinence rate was 74.6%; 3-month abstinence was 63.7%; 6-month abstinence was 55.7%; and 1-year abstinence rate was 42.1% [4].In a separate study, the efficacy associated with self-help groups and psy-chotherapy was explored in a group of 200 outpatients (28.5% attended self-help groups and 14.5% participated in group therapy).The results indicated that 1-month abstinence was 19.3%; 6-month abstinence was 22.8%; and 1-year or longer abstinence was 43.9% [5].
Similar rates of abstinence have been reported for substance specific treatment programs.For example, in a study of 202 outpatients treated for tobacco-dependence, 43% abstained for at least 7 days after completing the treatment program [6].Some studies have sought to investigate demographic features that may be associated with treatment success.In one study, African American and Latino smokers of menthol cigarettes exhibited lower 1-month abstinence (30% and 23%, respectively) when compared to white smokers (43%) of menthol cigarettes [7].
Efforts aimed at developing successful treatment programs for addiction have led to the exploration of invasive brain based methods of treatment.In a study of five patients undergoing deep brain stimulation of the nucleus accumbens for the treatment of alcohol dependence, 40% exhibited long-term abstinence [8].A treatment method designed to accomplish surgical ablation of the nucleus accumbens was performed in 1167 patients in China.Within a set of 272 patients undergoing this invasive surgical treatment, 5-year abstinence was documented as 58%.In another group of 150 cases, the non-relapse rate was reported as 50% [9].
In order to assess the efficacy and benefit our addiction treatment program, we investigate a set of patients in which addiction treatment outcome and rehabilitation is determined for patients who have completed treatment and followed up.We determine abstinence rates and identify predictors of treatment outcome.
Detoxification
Patients are assessed for treatment based on a dual diagnosis consisting of substance abuse coupled with mental health co-morbidity.Upon admission into the program patients undergo detoxification.Nurses acquire relevant intake history including what substance is used, how much of the substance is used, duration of use for each substance and frequency.That information is used to customize a patient specific detoxification protocol, conditions and length.Detox for each patient is individualized by MD's who are board certified in both addiction medicine and psychiatry.The resulting individualized detox tapers utilize four FDA approved addiction drugs: naltrexone, antabuse, acamprosate, and vivotrol.Taper duration ranges from 1 -2 weeks and may be extended if necessary.Pharmacological therapies used during treatment include Naltrexone which is FDA approved for treating alcoholism and administered orally.Vivitrol is a longer acting opioid antagonist given via injection once a month.Antabuse is an acetaldehyde dehydrogenase inhibitor used to reduce craving and acamprosate stabilizes glutamate and GABA neurotransmitters during alcohol withdrawal.Additional medications may be included in treatment and thereafter, provided they have been proven to be effective in addiction clinical studies.
Aftercare
A component of the treatment program includes facilitating an effective aftercare plan for each patient.This is accomplished using teams of doctors and treatment program support staff in structured interactions with patients.The goal of an aftercare plan is to seamlessly transition a patient into an independent substance free lifestyle.Aftercare is typically successful when a patient fully participates in the plan-facilitation process.
Completing a drug/alcohol treatment program is a feat but the battle is still being fought which is why aftercare is important in maintaining sobriety.
Aftercare Follow Up
Once patients exit the program, a follow up plan is enacted lasting up to a year.During this time patients are contacted by phone and asked questions about substance use, treatment efficacy and adherence to the aftercare plan.Responses to phone calls are recorded in patient records.
Data Analysis
Seventy-two patients were included in this study.Demographic data for patients was collected and analyzed to determine average age for all patients and for males and females.This data was used to characterize treatment outcome by age and gender over 12-month post-treatment period.Treatment outcome was analyzed to identify any relationship between treatment length and treatment efficacy.
In order to identify predictors of addiction relapse, three different levels of treatment success based on stringency of aftercare follow up data.Three different definitions for treatment success were defined and the treatment outcome data was analyzed under each of the different stringencies.The results of this analysis were used to identify predictors of addiction relapse following treatment.The average age of male patients who successfully completed treatment was 26.7 years (s.d.= 8.6 yrs) while the average age of female patients who successfully completed the treatment was 31.8 years (s.d.= 12.9 yrs).Interestingly, the average age of men who did not successfully complete treatment was 35.6 years (s.d.= 10.7 yrs) while the average age of women who failed to complete treatment successfully was 28.8 years (s.d.= 10.5 yrs).
Statistical Analysis of Patient Demographic Factors Associated with Treatment Efficacy
We chose to investigate if patient gender or age was a factor associated with treatment outcome.In our study, a total of 72 patients underwent clinical treatment for addiction for which 32 were male and 40 were female.The average age of males was 30.0 years (s.d = 10.4 yrs) and the average age of females was 30.7 years (s.d.= 12.2 yrs).There was no statistically significant difference between the average age of males and the age of females in our study population (p-value = 0.3966, alpha = 0.05).
The average age of all patients who successfully completed the treatment was 29.2 years (male and female, n = 45, s.d.= 11.6 yrs) while the average age of all those who failed to complete treatment successfully was 29.0 years (male and female, n = 27, s.d.= 10.6 yrs).There was no statistically significant difference between the average ages of patients who successfully completed treatment versus those for which treatment was not successful (p = 0.52972, alpha = 0.05).
As no significant difference was identified between the average age of patients by gender, or by treatment outcome, we next wondered if there was a significant difference between the average age of males patients who successfully completed treatment (n = 23, avg = 26.7 yrs, s.d.= 8.6 yrs) versus the average age of female patients who successfully completed the treatment (n = 22, 31.8 yrs, s.d.= 12.9 yrs).Again, although the p-value was just barely larger than 0.05, there was no statistically significant difference between the average age of successfully treated men versus women (p = 0.06551, alpha = 0.05).In a similar manner, we tested to see if there was a significant difference between the average age of male patients (n = 32 − 23 = 9, avg = 35.6 yrs, s.d.= 10.7 yrs) versus female patients (n = 40 − 22 = 18, avg = 28.8yrs, s.d.= 10.5 yrs) for whom treatment was not successful.Likewise, there was no statistically significant difference detected (0.93256, alpha = 0.05).
Overall we found no significant differences between the patient demographic factors of gender and age with 12-month treatment outcome.
Statistical Analysis of Clinical Treatment Length with 12-Month Treatment Efficacy
We next wanted to investigate whether the length of the treatment program (30 days vs. more than 30 days) was significantly associated with treatment efficacy.Our initial analysis revealed that of the 53 patients undergoing the 30-day treatment program, 29 patients (54.7%) exhibited a successful 12-month treatment outcome.In contrast, among the 19 patients that participated in a treatment program lasting more than 30 days, 16 patients (84.2%) experienced a successful 12-month treatment outcome.To assess whether the treatment efficacy was significantly different between treatment length, we performed a two-sided Z-Test for population proportions using an alpha = 0.05, which resulted in a p-value of 0.0226.This result indicates that within our clinical addiction treatment program, treatment lengths greater than 30 days were significantly associated with better treatment outcome.
Patient 12-Month Treatment Efficacy by Addiction Type
Patients were treated for a number of chemical dependencies including alcohol, amphetamine, benzodiazepines, and opioids.Among the 72 total patients, 29 were treated for alcohol dependency (13 males, 16 females), 2 were treated for amphetamine dependency (2 males, 0 females), 13 were treated for benzodiazepine addiction (5 males, 8 females) and 28 were treated for opioid dependency (12 males, 16 females).The overall 12-month treatment success rate across all patients was 62.5 percent.Overall, treatment success varied from a high of 100% for amphetamine addiction, to 69.2% for benzodiazepine dependency, with 64.3% percent for opioid dependency, and the lowest treatment success rate of 55.2% percent for alcohol addiction.
Statistical Analysis of Patient Addiction Type with 12-Month Treatment Efficacy
Within our after-care study, the number of patients being treated for alcohol dependency (n_TXsuccess = 16, n_total = 29) and opioid dependency (n_TXsuccess = 18, n_total = 28) was nearly identical.The remaining patients (n_TXsuccess = 11, n_total = 15) represent 13 with benzodiazepine addiction and just 2 with amphetamine addiction.We first decided to perform statistical analysis to compare treatment efficacy between alcohol addiction and opioid addiction.Using the two-sided Z-Test for population proportions with an alpha = 0.05, produced a p-value of 0.48392.This result indicates that, within our clinical addiction treatment program, there was no statistically significant difference between 12-month treatment efficacies for patients treated for alcohol dependency versus patients treated for opioid dependency.
Next we tested to see if there was a significant difference between the 12-month treatment efficacy between the remaining patients (amphetamine and benzodiazepine dependencies) compared to those with alcohol dependency.The two-sided Z-Test for population proportions with an alpha = 0.05 produced a p-value of 0.242 indicating that there is no difference between treatment outcome between patients with alcohol dependency compared to those with amphetamine or benzodiazepine dependency.
Similarly, we tested to see if there was a significant difference in 12-month treatment efficacy between the amphetamine/benzodiazepine dependent patients compared to the opioid dependent patients.Once again, we employed the two-sided Z-Test for population proportions with an alpha = 0.05 and obtained a p-value of 0.5485, indicating that there is not a significant difference between treatment outcome for patients treated for opioid dependency versus patients treated for amphetamine/benzodiazepine dependency.No significant difference between patient addiction type and 12-month treatment outcome was identified.
Analysis of Treatment Success Using Three Distinct Stringency Models for Efficacy
Since our study design employed self-reporting from patients who suffered from addiction, we hypothesized that it was likely that a patient might misrepresent abstinence during the 12-month after-care follow up period.As we reviewed the phone call responses from the patients, it became clear that some patients were readily available for our phone calls, while other patients consistently failed to answer the phone.Initially, we struggled with how to best to identify legitimate reasons for missing a follow-up phone call, versus intentional avoidance associated with substance abuse relapse.Although there is no perfect strategy for accurately classifying missed phone calls, we decided to perform three separate analyses of the data using a sliding scale of stringency for assessing treatment success.Specifically, we assessed treatment efficacy at 1-month, 3-month, 6-month and 12-month time points for each of our levels of stringency based explicitly on how we interpreted unanswered phone calls.
The most stringent level of treatment success was designed to assess the lowest possible success rate, under the most rigorous criteria for considering treatment successful.
In this model, only patients who answered the phone and explicitly stated that they remained substance free ("Y") for entire post-treatment interval were classified as successfully treated at each of the four time points.Under this most stringent model, 63.8% of patients exhibited success at the 1 month time point.While only 51.4% were successful at 3 months, with 38.9% successful at 6 months and just 23.6% remaining substance free 12 months after completing the treatment program.
For the moderate stringency model, we relaxed the inclusion criteria classifying patients as successful with the following three conditions for classifying a patient as successfully treated: 1) no "N" responses at all; 2) no more than 4 "NA" responses in any 12-month period; and 3) no "NA" responses allowed if they were immediately followed by a "N" response in the very next phone call.Under these criteria, 81.9% of patients were successful at 1-month, 72.2% were successful at 3-month, 62.5% were successful at 6-month and 48.6% reported success at the 12-month time point.
Finally, in the least stringent model for treatment success, our only exclusion criteria for success was any patient that replied "N" to the question of being substance free within the time period being assessed.Under this model, 95.8% of patients were successful at the 1-month time point, 86.1% were successful at the 3-month time point, 83.3% were successful at the 6-month time point and 69.4% were successfully substance free 12-month following treatment.
Patterns of Post-Treatment Patient Behavior with 12-Month Treatment Efficacy
In the course of this study we analyzed 3240 clinical data points (72 patients × 15 time points × 3 stringency models) in an attempt to assess the efficacy of our addiction treatment program.Interestingly, the process of defining the inclusion and exclusion criteria for treatment success across the different stringency models offered a unique opportunity to characterize trends in patient post-treatment behavior with 12-month treatment outcome.We reasoned that certain behavioral patterns in the after-care follow up phase of our study might have prognostic value for inferring the likelihood of treatment success or failure.Ultimately, we are interested in identifying any factors associated with treatment outcome that can prove informative in identifying patients at risk for poor treatment response.
We chose to base our analysis of patient behavioral patterns using only the data contained in the most stringent model for assessing treatment outcome; in which only patients who always answered the phone and explicitly stated that they remained substance free ("Y") for the entire duration of the post-treatment interval (1-month, 3-month, 6-month & 12-month) were classified as successfully treated.We reasoned that markers of poor treatment outcome are more easily identifiable when the classification of poor patient response is as broad and sensitive as possible.
In order to identify putative patterns of patient behavior during the after-care portion of the study, we counted the total number of "no telephone answers" (NA) and "negative responses to substance free question" (N) for each of the 72 patients.The number of "NA's" ranged from 0 (18 patients), to 15 (1 patient).Similarly, the number of "N" responses ranged from 0 (47 patients) to 4 (4 patients).The average number of "NA's" per patient was 3.55 with a standard deviation of 3.92, while the average number of "N" responses per patient was 0.681 with a standard deviation of 1.165.Based on the relationship between "NA" and "N" across the patients, we decided to investigate the feasibility of using the frequency of "NA's" among patients as a prognostic marker for "N" responses.
To quantify the value of specific frequencies of "NA's" as predictors of treatment outcome, we calculated the sensitivity and specificity for specific frequencies of NA among patients.Within our data set, all patients with 0 NA's also had 0 "N" responses.
We next considered patients for which at least 1 NA occurred during the 12 month post-treatment period.The sensitivity for NA ≥ 1 was 1.00 while the specificity was 0.383.Similarly, the sensitivity associated with NA ≥ 2 was also 1.00 while the specificity increased to 0.574.When we calculated the sensitivity and specificity calculations for NA ≥ 3, we obtained a sensitivity of 0.92 and a specificity of 0.894.Finally, we calculated a specificity of 0.8 and a sensitivity of 0.936 for NA ≥ 4.During the analysis we noticed that increasing the frequency of NA's resulted in fewer true positives and more false negatives.Subsequently we selected NA ≥ 3 as the optimal balance between sensitivity and specificity because when NA ≥ 3, true positives were 23 and true negatives were 42 while there were only 3 false positives and 5 false negatives.
Relative Risk of Addiction Relapse for Patients with Specific Post-Treatment Behavior
Given the results of sensitivity and specificity calculations for different frequencies of "NA" as behavioral markers for treatment response, we identified a value of NA ≥ 3 as exhibiting an optimal balance of high sensitivity and specificity.Subsequently, we were curious to discover if patients having at least 3 occurrences of NA during the 12-month post treatment period were at an increased relative risk for substance abuse relapse compared to patients having fewer than 3 NA's in the same period.
A total of 28 patients exhibited at least three instances of NA in the 12-month post treatment period.Of these, 23 patients exhibited evidence of unsuccessful treatment response resulting in an incidence of 23/28 = 0.8214.In contrast, 44 patients exhibited fewer than 3 NA instances during the 12 month post treatment period.Among these, just 2 patients experienced poor treatment outcome, associated with an incidence of 0.04545.We calculated the relative risk of poor treatment outcome by dividing the incidence of poor treatment outcome among patients with NA ≥ 3 (0.8214) by the incidence of poor treatment outcome among patients for whom NA < 3 (0.04545).
The corresponding relative risk of substance abuse relapse following treatment is 18.1 for patients who fail to answer the phone at least three times during the follow up period compared to patients who only failed to answer the phone either 0, 1 or 2 times during the entire 12-month post treatment period.These results strongly suggest that behavioral patterns of patients associated with non-answered phone calls exhibit a much greater relative risk of substance abuse relapse.
Discussion
This study investigated the efficacy and benefit of our addiction treatment program on patients who completed treatment and participated in aftercare follow-up.Specifically we conducted an aftercare follow study to determine abstinence rates and identify predictors of treatment outcome.Our results demonstrated that patients undergoing the 30 days treatment program exhibited a 54.7% treatment success rate while patients treated for more than 30 days experienced a success rate of 84.2% (p-value = 0.0226).
Our results indicated no statistically significant difference in treatment outcome across addiction types of amphetamine addiction, benzodiazepine dependency, opioid dependency, and alcohol addiction.
As part of the analysis, we developed three stringency models of treatment outcome This study was data intensive as 3240 clinical data points (72 patients × 15 time points × 3 stringency models) were analyzed in an attempt to assess the efficacy of our addiction treatment program.In the course of this comprehensive analysis, we were able to identify trends in patient post-treatment behavior with 12-month treatment outcome.Specifically we counted the total number of "no telephone answers" (NA) and "negative responses to substance free question" (N) for each of the 72 patients.
We chose to base our analysis of patient behavioral patterns using only the data contained in the most stringent model for assessing treatment outcome based on the rationale that poor treatment outcome is most easily identified when the classification of poor patient response is as broad and sensitive as possible.Subsequently only patients who always answered the phone and explicitly stated that they remained substance free ("Y") for the entire duration of the post-treatment interval (1 month, 3 months, 6 months & 12 months) were classified as successfully treated.
In order to determine if there was any specific behavioral pattern of post-treatment follow-up that might be indicative of poor treatment outcome, we selected NA ≥ 3 as an aftercare response pattern that provided the optimal balance between sensitivity and specificity because when NA ≥ 3, treatment outcome true positives and true negatives were maximized while false positives and false negatives were minimized.
The corresponding relative risk of substance abuse relapse following treatment is 18.1 for patients who fail to answer the phone at least three times during the follow-up period compared to patients who only failed to answer the phone either 0, 1 or 2 times during the entire 12-month post treatment period.These results suggest that aftercare follow-up can identify patients at risk for relapse or additional treatment.
Recovery is an ongoing process once a client leaves treatment.Clients who adhere to their discharge plan and immerse themselves in recovery related activities and lifestyle are likely to achieve sobriety for longer periods of time if not indefinitely.Clients who remain in treatment longer have higher success rates.Most clients ideally just want to return to normality once they've left inpatient treatment.At Inspire Malibu drug and alcohol treatment center, we offer aftercare follow-up as a courtesy to our clients once they leave treatment.While our clients are in treatment, we develop a rapport that allows us to maintain an aftercare relationship where we document their progress and sobriety.
Duration of treatment is optimized for each patient based a variety of factors including types of substances abused, and frequency/duration of abuse history.Typical treatment durations last 30 -45 days and may extend as long as 90 days.Treatment consists of various modalities of science and evidence based therapies.Patients attend group therapy 3 -4 times daily and individual sessions 4 -5 times per week.
A
total of 72 patients underwent clinical treatment for addiction for which 32 were male and 40 were female.The average age of all patients was 30.36 years (s.d.= 11.3 years) with very similar ages between the genders.The average age of males was 30.0 years (s.d = 10.4 yrs) and the average age of females was 30.7 years (s.d.= 12.2 yrs).Overall, the average age of patients who successfully completed the treatment was 29.2 years (s.d.= 11.6 yrs) while the average age of those who failed to complete treatment successfully was 29.0 years (s.d.= 10.6 yrs).
Of the 72 patients who participated in the treatment program, 53 had a treatment length of 30 days while just 19 underwent treatment for more than 30 days.Patients participating in the longer treatment program included those in a 33-day program (n = 1), 35-day program (n = 3), 40-day program (n = 2), 45-day program (n = 9) and a 60-day program (n = 4).Patients undergoing the 30 day treatment program exhibited a 54.7% treatment success rate.In contrast, patients that participated in a treatment pro-gram lasting more than 30 days experienced a success rate of 84.2%.
8% of patients exhibited success at the 1 month time point; 51.4% were successful at 3 months; 38.9% were successful at 6 months and 23.6% remained substance free 12 months after completing the treatment program.The rationale for choosing multiple levels of stringency in this study was to accurately and objectively assess the efficacy of our addiction treatment program.The criteria for characterizing success can sometimes be subjective based on the inclusion criteria chosen.It is common for studies to report the treatment outcome in the most favorable manner.We believe that such reporting makes it difficult to compare studies across treatment programs.Accordingly, we created multiple definitions of treatment success and assessed our data under each stringency model.Regardless of the stringency level selected, we identified a trend of higher treatment at earlier time points (1 month and 3 months) versus later time points (6 months and 12 months).
The moderate stringency model was constructed as an intermediate model of treatment success.Treatment outcome results were 81.9%, 72.2%, 62.5%, and 48.6% for the 1 month, 3 months, 6 months and 12 months aftercare time points respectively.The high stringency model produced the lowest possible success rate using the most rigorous criteria for considering treatment success.Under the high stringency model 63. | 2017-08-14T22:47:32.064Z | 2017-01-01T00:00:00.000 | {
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248302512 | pes2o/s2orc | v3-fos-license | Construction of life-and-death education contents for the elderly: a Delphi study
Background Life-and-death education is intimately related to palliative-and-hospice care. It should be implemented among groups of all ages, especially for the elderly. This study aims to establish expert consensus on a set of scientific and systematic life-and-death education contents for the elderly and provide reference for the practice on the elderly. Methods This study designed three rounds of expert consultation by using a Delphi method. A panel of 22 experts from the fields of palliative-and-hospice care, life-and-death education, geriatric nursing, humanities and ethics, and geriatric psychology participated in the study. Results This study finally reached expert consensus on the contents of life-and-death education for the elderly, containing 4 first-level items, Life-and-death literacy promotion in the elderly; Life-and-death concept establishment of the elderly; Life-and-death planning of the elderly; Life-and-death thoughts of the elderly with affiliated 9 second-level items, and corresponding 23 detailed third-level items. Conclusions The life-and-death education contents for the elderly offer a basis for publicity for health professionals, promote dialogues on death, preparation, and planning for death and dying. The life-and-death education contents system was clear in coherence containing definite and comprehensive contents, which enriched life-and-death education resources globally. The results could assist in the planning of palliative-and-hospice care services to improve quality of death of the elderly.
Background
The population and proportion of the elderly have increased rapidly in recent years, and virtually all countries are experiencing population ageing [1]. According to the World Health Organization (WHO), the number of the global population aged over 60 and above is estimated to be doubled to two billion in 2050, and 80% of the elderly are in low and middle-income countries [2]. China is now one of the fastest aging countries [3]. The results of China's seventh census indicate that the population aged above 60 is approximately 264 million people [4].
With the expansion of people's life span, the quality of life and death are of vital significance [3]. Medicine is both scientific and humanistic [5] because it not only cures diseases and prolongs life, but also brings about dignity and rights of decision to the elderly. To improve the quality of life of the elderly worldwide, WHO has proposed programs of healthy ageing, active ageing, and others [3]. Palliative-and-hospice care has become one of the principal measures to ease the pressure on medical care and to improve quality of life of the older people [6]. Palliative-and-hospice care provides physical, psychological, social, and spiritual care as well as humanistic care for end-of-life or elderly patients before their deaths, so that their pains and discomfort symptoms can be alleviated, Lei et al. BMC Public Health (2022) 22:802 by improving the quality of life and helping patients relax and leave with dignity [7,8]. With the promotion and application of this mode of care, the topic of death is inevitable for the elderly. On the other hand, as the age increases, the body experiences weakness in both physical strength and decline in physical abilities [9]. Moreover, experience of the loss of important persons occurs more frequently [10]. The factors turn death into an inescapable fact for the elderly. Additionally, the threats of chronic diseases and infectious diseases, such as cancer, COVID-19, and Ebola keep reminding us that death is close at hand [11]. If older people know less about death, they are more likely to experience aggravated suffering about death, which is not conducive to the physical and mental health of them [12].
Unfortunately, the topic of death is often avoided or denied in eastern and western cultures [13]. Human attitudes and behaviors are deeply rooted in culture [14]. Actually, most of people are usually negative to the topic of death. They are afraid of mentioning it and reluctant to discuss dying matters [15]. It is therefore that people focus more on life planning, healthy birth, excellent education, and a better life whereas death is rarely mentioned. China ranks 9th from the bottom in the 2015 Quality of Death Index Ranking released by The Economist Intelligence Unit [16], which also reveals that Chinese people are far from the goal of good quality of death. Regarding the issue of death, Western society has carried out research related to thanatology as early as the 1920s [17]. Death awareness campaigns arose in the United States in the 1960s [18]. Courses of life-and-death education were instructed in colleges and universities in the 1980s [19]. As Herman Feifel stated in the early days of the death awareness movement that the attitude towards death affects the content and quality of our lives [20]. The quality of death in Western countries is generally better than that in Mainland China. Therefore, how to construct a set of life-and-death education content system integrated with culture, allowing the elderly to face death squarely and build a scientific concept on death has become the key to improving the quality of life and death in contemporary society and medical background.
Life-and-death education offers death information which is helpful for the educated to understand the finiteness of life and the naturalness of death objectively, so as to cherish life and appreciate life [21]. Life-and-death education transmit not only knowledge and skills related to death, broaden the understanding of dying, death, and bereavement, it is also thought provoking. "Acceptance the naturalness of life" and "development of the concept of death" contribute to a better self-realization [10,22]. In addition, life-and-death education can also provide more options for dying and health care.
Life-and-death education highlights the review of the meaning of life and the discussion of death and helps to better promote advance directives or establish a advance care planning, allowing to achieve the goal of palliativeand-hospice care and improve the quality of death [23,24], which is intimately related to palliative-and-hospice care. Based on the terror management theory, life-anddeath education can activate reminders of death or mortality salience effect, trigger a distal defense mechanism to improve the meaning of life [25,26]. As the concept of death influenced by many factors [27,28], especially culture, the development of life-and-death education in accordance with local conditions can benefit the elderly to recognize relevant knowledge and change their negative attitudes towards death escapes or denies. Gradually, they recognize the finiteness of life, and then cherish the moment of life, live more valuable lifestyles, and select optimal medical treatments, thereby living an optimistic life and truly realizing active aging [18].
Life-and-death education should be implemented among groups of all ages, and it is particularly significant for the elderly [14]. However, the elderly has always been one of the groups that is easy to ignore in the research of life-and-death education [14]. Previous life-and-death education researches mostly focused on adolescents [29,30], medical and healthcare professionals [31], and cancer patients [21] to explore the benefits of life-and-death education in reducing negative death emotions, managing the risk of commit suicide, and alleviating alexithymia [32,33]. Most the education contents only focus on the sharing of living wishes and discussion of death recognition, while lacks contents of bereavement, mourning, and death preparations [34], and scientific construction of the life-and-death education contents, especially for the elderly. Therefore, this research attempts to establish expert consensus on a set of scientific and systematic lifeand-death education contents for the elderly by Delphi technique. This project may provide a foundation for better implementation and promotion of life-and-death education among the elderly, and help improve the quality of death and well-being of the elderly.
Methods
This study was approved by the Ethics Committee of the University (NO. 2021.06-03) on June 21, 2021. Delphi method has been widely used in education, health related fields which make full use of the wisdom of experts to construct and improve the new research content. This method is suitable for the purpose of this study. Delphi is a research method that achieves expert consensus through the anonymous and iterative multistage process [35,36]. The trustworthiness of Delphi can be confirmed by credibility, dependability, confirmability, and transferability [37]. The credibility of Delphi can be gained by ongoing iteration and feedback to experts, which can be viewed as member checks [38]. Recruiting authoritative experts with in-depth research and interest in the topic will help increase the content validity and dependability of Delphi method [39]. Confirmability can be assessed through experts' opinions collection, data analysis and detailed description of the consultation process [37]. Transferability of Delphi can be verified by application of the research findings [40].
Expert selection
This study included experts in the fields of palliative-andhospice care, life-and-death education, geriatric nursing, humanities and ethics, and geriatric psychology in the CNKI database, with the following inclusion and exclusion criteria: Inclusion criteria: 1. bachelor's degree or above; 2. professional title qualification of intermediate or above; 3. work experience over 5 years; Exclusion criteria: 1. experts could not participate in this research due to personal reasons; 2. experts whose contact information were not available; 3. experts who had no practical experience of working with the elderly. Experts who meet the inclusion criteria, have conducted in-depth research and published articles in related fields. So the e-mail addresses and some phone numbers could be obtained from the database. Then the experts were invited to participate in the survey by e-mail, phone calls or text message, and they got the information about the content, purpose, and significance of the research in detail. A total of 23 experts agreed to participate in this Delphi study and follow-up questionnaires were sent to them and then returned after fulfillment by emails.
Questionnaire design
The questionnaire for experts mainly consists of three parts: 1. personal sociodemographic information of the experts name, age, professional field, working years, and so on; 2. self-assessment of the familiarity to the questionnaire contents, including the overall familiarity. 3. expert evaluation form for the life-and-death education contents for the elderly. This part was the main body of assessment in the expert questionnaire. Based on retrieval of literature [41][42][43], books [44,45], and websites of hospice [46][47][48] for the elderly, we found that education for the elderly should include physical, psychological, social, spiritual and end-of-life aspects at least. According to the purpose of this study and the previous research, a preliminary draft of the framework of lifeand-death education contents for the elderly was made, and the importance of each part was assessed by experts with scores. A 5-point Likert scale method was employed to measure importance from 5 points to 1 point as "very important"-"very unimportant", in descending order of importance.
Questionnaire procedures
The present study involved three rounds of questionnaires to the expert panel from July to November 2021. Experts did not communicate directly with each other. The researchers sorted out the opinions, suggestions and feedback of each expert and presented them anonymously on the next round of expert correspondence questionnaire. So the experts could get to each other. The first administration of questionnaires to the expert panel was completed in August. This was conducted in the form of emails, and researchers contacted the experts personally, collected their opinions and revised feedback. The first round of survey contained relatively open questionnaire items to seek expert opinions on the lifeand-death education contents for the elderly. Seventeen educational contents were initially constructed. In addition to the scores evaluation, experts could also put forward their own professional opinions and suggestions for modification as well as the addition of other relevant contents to enrich life-and-death education system for the elderly.
According to the results of the first round of inquiries to the expert panel, life-and-death education contents for the elderly were revised and improved following relevant literature retrieval and discussions with professors and research team members. An expert evaluation form for the life-and-death education contents for the elderly was designed, including 4 first-level items, 10 second-level items, and 27 third-level items, and a second round of questionnaire to the expert panel was conducted, and the opinions and feedback from the experts were collected and analyzed.
Based on the opinions of the second-round administration and research purposes, the content system of lifeand-death education for the elderly was modified with some deletion. As a result, an expert evaluation form of a third-round survey was generated containing 4 first-level items, 9 second-level items, and 23 third-level items, and the opinions and feedback from the experts were collected and analyzed.
Data analysis
The returned data were analyzed using Excel and SPSS 24.0. Experts' concern for this study was reflected in the positive coefficient of the expert panel, i.e., the rate of return of a questionnaire [21]. It is generally believed that the positive coefficient above 85% indicates good feedback of the survey from the expert panel. The representativeness of the experts in this research were presented as expert authority coefficient [21]. Generally, an authority coefficient over 0.70 indicates that the expert opinions are reliable and the experts are authoritative. The inclusion criteria for items in this study were the average importance score of each item evaluated by the expert panel > 4.00, the coefficient of variation < 0.20, and the approval rate > 80% [49]. Furthermore, based on some written opinions of the experts with literature retrieval, the proposed contents were discussed among the research team and improved by means of deletion, modification, supplementation, and merger.
Expert sociodemographic information
The present research enrolled a panel of 23 experts at first from five fields of life-and-death education, palliativeand-hospice care, geriatric nursing, geriatric psychology, and humanities and ethics. The working years of experts are between 5 to 40 years, and more than 78% of experts have worked in the professional fields for more than 20 years. The sociodemographic details of the experts were presented as Table 1. And all the experts have experience working with the elderly.
The present study involved three rounds of questionnaires to the expert panel. In the first round of Delphi, 23 experts returned questionnaires in time. In the second round, one expert failed to return the questionnaire due to her busy work, so she had to withdraw from the study, and the assessments from the 22 experts were collected. Also, all 22 experts gave feedback in the third round. In general, the response rate of the experts is more than 95% (> 85%), and the authority coefficient is more than 0.85 (> 0.70), indicating the authority of the experts and the credibility of the Delphi results. Experts were interested in the study through their comments. The main purpose of each round of questionnaires, the number of questionnaire items to be evaluated by experts, item revisions and expert opinions were presented in Table 2.
Evaluation of life-and-death education contents for the elderly
In the first round of questionnaires to the expert panel, the items that did not meet the standards were deleted or modified, seen Table 3 for details. In addition to suggestions for changes in expression, items with overlapping contents were deleted. For example, the "various religions" contains the "Chinese traditional culture". Considering the cognitive acceptance of the elderly and the relevance of education contents for the elderly, the "Life-and-death concepts in traditional Chinese culture" (Confucianism, Taoism, and Buddhism)" were retained ultimately. "Organ transplantation and donation" was also deleted. In addition to the low approval rate by experts, organs of the elderly age naturally, and the age of organ donation generally does not exceed 65 years [50]. It is therefore that this content was not retained. The "Depression and suicide" of the elderly have not yet become the focus of experts' attention, which differs from the western social background [51]. Based on the suggestions of the experts, the contents of life-and-death education was supplemented considering the characteristics of the elderly, and the second round of questionnaires was developed.
In the second round of questionnaires to the expert panel, the answers, modification and explanation of the first-round comments and a total of 41 items at three levels which needed to be evaluated by experts were presented. Then experts put forward some suggestions for revision. For example, "Advance Directives and Wills", both of which in Chinese have the meaning of end-of-life instructions and are likely to be confused by most elderly people who are first exposed to them. But advance directives and wills have different emphases, and it is not necessary to explain at the same time. So, according to experts' advice, wills and advance directives are split into two parts of "Planning before death or dying". The content related to "Euthanasia" ultimately was not included in the education system. As euthanasia remains to be ethically controversial, and it is not permitted by law [52], experts considered that this part of content should be avoided in education for the elderly. "Funeral Ceremony" may originally derive from culture, customs, or religions, and it was integrated into "Chinese funeral Customs" in this study from the perspective of being easy to accept and practical for Chinese elderly. In addition to the deletion of items that did not meet the criteria, duplicate items were removed from the contents as per the proposal of the experts, and partial items were modified as well, as presented in Table 4. After the modification and improvement of the first two rounds of questionnaires and discussion of the research team, the third-round administration was conducted. Experts agreed on the content, and the scores reached consensus standards. And four experts made some additional suggestions on the details under the three-level items. For example, in "Planning before Death or Dying", it is suggested to emphasize enlightening the spiritual needs of the elderly, not only in terms of religion, but also the respect of others, forgiveness, friendship, aesthetics, etc. An expert suggested that palliative-and-hospice care in homes should also be introduced in "Organizations and costs" of "Palliative-and-hospice care". Through the feedback of the third round of expert inquiries, the details of the content of life and death education for the elderly have been enriched and improved.
In the second and third rounds of questionnaires, point-to-point answers, modifications, or explanations to the previous round of expert opinions were presented anonymously. Through multiple rounds of Delphi, an expert consensus on life-and-death education content system for the elderly was finally reached. The content system included 4 first-level items, 9 second-level items, and 23 third-level items as shown in Table 5.
Discussion
Education undertakes the functions of cultural selection, transmission, transformation, criticism, innovation, and remodeling [53]. Life-and-death education can not only convey knowledge of life and death, but also alleviate the negative emotions towards death, help people prepare for death, and improve the quality of death [18]. Based on the social background of aging, this study designed lifeand-death education contents for the elderly to provide a buffer in the process of demise as well as references for other countries.
According to Terror Management Theory, life-anddeath education plays a role as a mortality reminder for the elderly [54]. "Life-and-death literacy promotion in the elderly" gradually transitions the knowledge from life, life cycle and aging that are easily accepted by the elderly to end of life and death. This part aims to reduce the avoidance of death for the elderly through realistic knowledge explanations, and help the elderly face up to the problems of life and death. Proximal defenses may be activated when the elderly receive life-and-death knowledge, that is, the elderly begin to think about death consciously. And the elderly may take effective health behaviors in order to reduce perceived vulnerability and avoid death threats [55]. "Lifeand-death planning of the elderly" provides the elderly with more options for end-of-life care beyond rescues. The purpose of this part is to put autonomy back into the elderly themselves and to let older adults know that they can plan for their own death. When thoughts of death are active but not conscious, distal defenses are activated. The elderly would defend against death by seeking cultural identity, meaning in life [56]. "Lifeand-death concept establishment of the elderly" helps the elderly deepen their understanding of life and death from the perspective of history and culture, and initially form their own views on life and death. Through the introduction of life quality and death quality, the elderly are guided to attach importance to their own death and express their pursuit of good death. "Lifeand-death thoughts of the elderly" enables the elderly to consciously explore their own value and meaning according to their life experiences, religious beliefs or cultural background. This part also discusses the source of happiness for the elderly, with the purpose of enriching the life of the elderly and improving their own wellbeing. The contents of life-and-death education for the elderly constructed in this study include 4 first-level items from the aspects of knowledge, culture and concepts, behavior planning and meaning of life.
From the experts' preference, we found that in the first round of Delphi, the experts scored the highest in "Introduction to palliative-and-hospice care", and the two items with the approval rate reaching 100% were "Introduction to palliative-and-hospice care" and "Lifeand-death concepts in Chinese traditional culture". This shows that "palliative-and-hospice care" is the top priority that experts believe needs to be promoted to the elderly. It may be because palliative-and-hospice care can not only reduce the burden of elderly care, reduce the economic loss of the elderly, but also improve the quality of life-and-death of the elderly [57]. However, it cannot be ignored that the acceptance of palliative-and-hospice care is a medical care decision based on the cognition of life-and-death of the elderly [58]. According to behavioral decision theory, psychology is an important factor affecting people's decision-making [59]. And the influence of life-and-death culture on the psychological phenomenon and psychological process is long-term and difficult to change [60]. Therefore, we designed the concept of lifeand-death from the perspective of traditional Chinese culture by analyzing the origin of the taboo of life-anddeath, the neglected culture of life-and-death, the culture of life-and-death in the new era, and the development of the concept of life-and-death, this prompts the elderly to think about the concept of life and death invisibility. And "Life-and-death concepts in Chinese traditional culture" is also one of the options approved by the experts in their preferences. Culture affects much on the formation of individual life and death attitudes and concepts, and even on the guidance of individual behavior [61]. Therefore, life-anddeath education must take the local social and cultural background and the characteristics of the subjects into consideration. With reference to the contents of lifeand-death education in Taiwan, China, which is welldeveloped [22], this study design combined the basis of traditional Chinese culture and the social reality in mainland China and generated the system of "Life-anddeath concepts in traditional Chinese culture", "Good death in eastern and western cultures", "Funeral culture and bereavement", and "Four ways of emotion expressions (acknowledgment \ apology \ love \ farewell)". It contributed to the elderly to accept and recognize the educational content, and then renew their cognition and concepts. While inheriting the traditional culture, it is also reflecting on the renewal. Under additional cultural backgrounds or in different countries, this part of the content can be referred as per the actual conditions or replaced according to local conditions, thereby minimizing the restructuring work of life-and-death education for the elderly.
Compared with the life-and-death education in the past [62], the content of life-and-death education for the elderly constructed in this study is targeted at the needs of the elderly and their own characteristics. Based on the previous interview and survey needs of the elderly [43], this research integrated professional evaluation of geriatric nursing and geriatric psychologists, and generated the final content system of life-and-death education for the elderly with clear main body and refined contents. Moreover, the contents of "Aging process", "Terminal stage of the elderly", "Physical and mental counseling for the elderly after bereavement" and "Discussion on happiness philosophy of the elderly" were supplemented, which was more object-specific, systematic, and scientific, making up for the content deficiency of previous researches.
As the physical, psychological, and social characteristics of different groups are different, the contents of life-and-death education for the elderly constructed in this study were different from the related education of the rest of groups. Life-and-death education for medical and health workers focuses on palliative-and-hospice care skills [63]. The content of life-and-death education for cancer patients focuses on the acceptance of death by patients and their families and to an optimal choice of palliative-and-hospice care [24]. The contents of life-anddeath education for the elderly constructed in the present research focused highly on the establishment of the death concept of the elderly and the clarification of the meaning of life, so as to help the elderly enrich their lifetime at an old age thereby improving the quality of life and death.
Under the social background of aging population, coupled with the urgent need to improve the death quality of the elderly in China, and palliative-and-hospice care pilot is being fully implemented [64], this research is accurately in line with the older population, and the scientific system of life-and-death education contents is practical and applicable [21]. We believe that life-and-death education is the right of the elderly. We can use this education to mobilize the initiative and enthusiasm of the elderly as a social resource, change the perception that the elderly is a burden on society, give full play to the social value of the elderly, and meet the self-realization needs of the elderly.
Limitations
Limitations of this research include the failure in carrying out life-and-death education practice for the elderly, and a lack of research on the time arrangement and methodology in each part of life-and-death education. Although some of the life-and-death education program parts have not been explored, it is definite that life-and-death education for the elderly should be given in a direct way easy to be accepted and understood. This needs to be further explored in future research.
Conclusions
The expert consensus on the content of life-and-death education for the elderly constructed in this study provides a basis for health professionals to initiate lifeand-death dialogue, death preparation, and end-of-life planning for the elderly. It also plays a positive role in acceptance and implementation of palliative care in older populations. Furthermore, the result can also assist in the planning of hospice care services to improve quality of death of the elderly. This study could enrich life-anddeath education resources for the elderly, and provide theoretical support for relevant research peers and palliative care professionals around the world. | 2022-04-22T13:52:58.379Z | 2022-04-21T00:00:00.000 | {
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4570030 | pes2o/s2orc | v3-fos-license | Malignant Pleural Mesothelioma with Bone Marrow Metastases
A 64-year-old man with the bone marrow metastasis due to malignant pleural mesothelioma (MPM) was diagnosed with anemia, leukoerythroblastosis, thrombocytopenia, and lower back pain. A bone marrow biopsy demonstrated infiltrative malignant mesothelioma lesions in the bone marrow. The patient died within 15 days of the detection of the bone marrow involvement. Physicians should consider performing a bone marrow biopsy to diagnose bone marrow metastasis and treat patients with palliative chemotherapy at an earlier phase of the disease. To our knowledge, this is the first report of an MPM patient having bone marrow metastasis with anemia, leukoerythroblastosis, and thrombocytopenia.
Introduction
Malignant pleural mesothelioma (MPM) is a malignant tumor derived from the mesothelial cells of the serous membranes in the pleura (1)(2)(3). It is classified into the epithelial, biphasic, and sarcomatous types (4). MPM is an aggressive disease with a poor prognosis and limited treatment options; the median survival duration is approximately 1 year (1)(2)(3). Approximately 70% of MPM cases are associated with asbestos exposure, which is known as the primary risk factor for mesothelioma; asbestos is an environmental carcinogen (1)(2)(3)5). The typically long latency period from asbestos exposure to the onset of MPM ranges from approximately 20-50 years (5). MPM generally spreads and invades locally to the chest wall, mediastinum, and diaphragm, and metastasizes to both the hilar and mediastinal lymph nodes. Although distant metastasis to the bone, contralateral lung, peritoneum, or liver is reported to occur in approximately 55% of MPM cases (6)(7)(8), bone marrow metastasis from MPM is extremely rare. Only 3 cases have been reported (9, 10). We herein report the fourth case of an MPM patient with bone marrow infiltration, anemia, leukoerythroblastosis, and thrombocytopenia.
Case Report
A 64-year-old man with MPM was referred to our hospital for chemotherapy. The patient had been immunohistopathologically diagnosed with MPM (epithelial type) at a previous hospital based on a surgical biopsy specimen of the right pleura. He had worked as a drug manufacturer and a manager of drug manufacturing in a pharmaceutical company, and may have experienced low-level exposure to chemical substances without asbestos. A histopathological examination revealed that the tumor cells contained eosinophilic granules and were positive for calretinin, podoplanin (D2-40), Wilms' tumour-1 (WT-1), mesothelin, cytokeratin 5/6 (CK5/6), and thrombomodulin, but negative for carcinoembryonic antigen (CEA), thyroid transcription factor-1 Intern Med 57: 2541-2545, 2018 DOI: 10.2169/internalmedicine.9246-17 Right pleural effusion with thickening in the right front pleura is observed. The increased uptake of FDG in the right front pleura, mediastinal organs, and metastases in the mediastinal lymph nodes was observed.
A B C
(TTF-1), and epithelial cell adhesion molecule (Ber-EP4) ( Fig. 1A-C, and data not shown). Chest X-ray, chest computed tomography (CT), and the 18 F-Fluorodeoxyglucose (FDG) uptake on positron emission tomography (PET)-CT showed the extension of the tumor to the mediastinal organs and metastases in the mediastinal lymph nodes (Fig. 2). The clinical stage was determined to be cT4N3M0 Stage IV (11). After talc pleurodesis, the patient received four cycles of chemotherapy with cisplatin plus pemetrexed, to which he showed a partial response. However, evidence of progressive disease was observed, with a new lesion in the right pleura at three months after his last treatment. Although he was given chemotherapy with the same drug regi-men, he was admitted to our hospital because of general fatigue, dehydration, and right front chest pain after four days of chemotherapy. His general fatigue and dehydration were improved by supportive therapy with fluid replacement, and both nonsteroidal anti-inflammatory drugs (NSAIDs) and morphine reduced the intensity of his pain. He developed myelosuppression, including grade 3 leukopenia, anemia, and thrombocytopenia (Fig. 3). On days 7-11 of hospitalization, filgrastim (recombinant met-human granulocyte-colony stimulating factor) was administered. After the improvement of his myelosuppression, a prolonged fever developed, in spite of the administration of antibiotics. The laboratory data on day 17 of hospitalization showed the following: white
A B
D E C blood cell count, 41,500/mm 3 ; hemoglobin, 7.9 g/dL; alkaline phosphatase, 513 U/L; and lactate dehydrogenase, 1,981 U/L. Moreover, leukoerythroblastic changes were observed in the peripheral blood. A bone marrow biopsy on day 19 revealed the replacement of the marrow by a diffuse neoplastic infiltrate containing eosinophilic granules, granulocytes with an altered nuclear morphology, the absence of megakaryocytes, dyserythropoisis, and poor cellularity ( Fig. 1D and E). The neoplastic cells were accompanied by nuclear deviation and multinuclear cells, and were diffusely positive for cytokeratin (AE1/AE3) and vimentin, focally positive for D2-40, WT-1, and mesothelin, and negative for calretinin, CEA, and TTF-1 (Fig. 1D-F and data not shown). Based on these results, the patient was diagnosed with MPM with bone marrow infiltration. During his prolonged fever, despite the continued administration of NSAIDs and morphine, severe lower back pain also developed on day 14 of hospitalization. Spine magnetic resonance imaging (MRI) on days 16 and 18 of hospitalization revealed metastatic lesions at multiple bone sites, including the thoracic spine, lumbar spine, and sacral vertebra, and spinal cord compression was found in the eleventh and twelfth thoracic vertebra (Th11/Th 12) (Fig. 4). Palliative radiotherapy was administered with irradiation of the thoracic part of the spinal cord (Th11/Th 12) at a dose of 24 Gy divided into six fractions (one fraction per day) on days 22-27 of hospitalization. However, paraplegia of the lower extremities and neurogenic bladder and bowel dysfunction due to metastatic invasion of the spinal cord developed on day 23. On day 31 of hospitalization, the patient developed persistent severe back pain, anemia, and thrombocytopenia, and later died suddenly on day 32 of hospitalization.
Discussion
The postmortem records of 318 patients with MPM demonstrated that extrathoracic metastasis of MPM was observed in the liver (31.9%), peritoneum (24.4%), and bone (13.8%) (8). In these records, metastasis was found in many organs that are not usually regarded as sites of MPM metastasis, including the adrenals (10.2%), spleen (10.8%), and brain (3.0%) (8). There are only 3 reports of MPM patients with bone marrow metastasis (9,10). To the best of our knowledge, this study represents the first report of a MPM patient with bone marrow metastasis and anemia, leukoerythroblastosis, and thrombocytopenia.
Bone metastasis from solid malignant tumors rarely develops into bone marrow metastasis, which is associated with a poor prognosis. Bone marrow metastasis is associated with hematological disorders, including anemia, leukoerythroblastosis, and disseminated intravascular coagulation (DIC). This condition is called disseminated carcinomatosis of the bone marrow (DCBM) (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Previously published reports have shown that DCBM is associated with gastric cancer and includes 3 major symptoms: anemia, lower back pain, and bleeding tendency. The patient in the present case developed anemia, leukoerythroblastosis, thrombocytopenia, and severe lower back pain as characteristics of DCBM, and a bone marrow biopsy revealed infiltrative malignant mesothelioma in the bone marrow. Based on reports of solid malignant tumors, the prognosis of patients with DCBM is considered to be extremely poor (14)(15)(16)(17). These previous reports demonstrated that the median survival of DCBM from gastric cancer after the detection of bone marrow metastases was significantly shorter in patients who were given the best supportive care (BSC) than in the patients who were given palliative chemotherapy (14)(15)(16)(17). The three previous studies by Kim et al., Rhee et al., and Kwon et al. reported that the median overall survival from the time of the detection of bone marrow involvement in BSC was 20 days, 16 days, and 11 days, respectively (14,16,17). Conversely, they showed that the median overall survival in the patients who received palliative chemotherapy was 67 days, 99 days, and 121 days, respectively (14,16,17). Thus, they suggested that palliative chemotherapy should be considered for gastric cancer patients with DCBM (14,16,17). It was also reported that an early diagnosis and prompt chemotherapy could contribute to improved survival of a few months in colon cancer patients with DCBM (22). The patient in the present case was given the best supportive care before the detection of bone marrow metastasis; however, his clinical course was extremely rapid, and he died within 15 days of the detection of bone marrow involvement.
Although the present case showed different pathological findings between the pleura and bone marrow, other malignancies were not detected by PET-CT imaging at the initial presentation or by CT and MRI imaging during treatment. Thus, we determined that the diagnosis in the present case was MPM with bone marrow infiltration. Differences in staining methods and the handling of specimens may have led to the discrepant pathological findings between the pleura and bone marrow. It is acknowledged that the present case was limited by these discrepant findings.
Although mesothelioma is strongly associated with asbestos exposure, approximately 20% of patients with mesothe-lioma have no demonstrable asbestos exposure, even after a detailed assessment (23)(24)(25)(26). It has been reported that other mineral fibers, such as erionite, nanotubes, and irradiation are possible risk factors for the development of mesothelioma (24). This patient worked in a pharmaceutical company and may not have been exposed asbestos or another mineral fiber.
In conclusion, to the best of our knowledge, this is the first report of an MPM patient with bone marrow metastasis, anemia, leukoerythroblastosis, and thrombocytopenia. The prognosis of MPM with bone marrow metastasis with or without palliative chemotherapy is uncertain, because the previous reports only evaluated the prognosis of gastric cancers with DCBM. Although it may be of minimal value to the patients to diagnose such a severe and rapidly progressive case, the accumulation of cases would reveal the true significance of palliative chemotherapy for MPM with bone marrow metastasis. Thus, a bone marrow biopsy should be considered in order to facilitate the diagnosis of MPM and treatment with palliative chemotherapy at an earlier phase of the disease. | 2018-04-26T18:25:47.098Z | 2018-03-30T00:00:00.000 | {
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249240312 | pes2o/s2orc | v3-fos-license | Physics-based neural network for non-invasive control of coherent light in scattering media
Optical imaging through complex media, such as biological tissues or fog, is challenging due to light scattering. In the multiple scattering regime, wavefront shaping provides an effective method to retrieve information; it relies on measuring how the propagation of different optical wavefronts are impacted by scattering. Based on this principle, several wavefront shaping techniques were successfully developed, but most of them are highly invasive and limited to proof-of-principle experiments. Here, we propose to use a neural network approach to non-invasively characterize and control light scattering inside the medium and also to retrieve information of hidden objects buried within it. Unlike most of the recently-proposed approaches, the architecture of our neural network with its layers, connected nodes and activation functions has a true physical meaning as it mimics the propagation of light in our optical system. It is trained with an experimentally-measured input/output dataset built from a series of incident light patterns and corresponding camera snapshots. We apply our physics-based neural network to a fluorescence microscope in epi-configuration and demonstrate its performance through numerical simulations and experiments. This flexible method can include physical priors and we show that it can be applied to other systems as, for example, non-linear or coherent contrast mechanisms.
Introduction
Coherent light is subject to scattering when it propagates through optically heterogeneous samples such as biological tissues. Ballistic light is exponentially attenuated with depth, and the remaining light is scattered giving rise to a complex interference pattern, also called speckle [1]. Most imaging techniques rely on the use of ballistic light and are therefore limited to shallow depths. In the past few years, many advances have been made for imaging at depth, mostly by controlling the phase and/or amplitude of the light being scattered thanks to spatial light modulators (SLMs) [2,3].
The first wavefront shaping experiment consisted in iteratively optimizing the SLM phase pattern using feedback metrics, such as signal strength, to focus the transmitted scattered light onto a diffraction-limited spot [4]. This was a major step forward for optical imaging in turbid medium as scanning the spot across the sample offers an image. Later on, the transmission matrix (TM) that characterizes the linear and deterministic propagation of the optical field through the medium has been experimentally measured [5,6]. Knowing the TM gives more information than optimizing the input field and can also be used for imaging [7]. However in both cases, a feedback signal from the focal plane is needed. A detector (a single pixel detector or a camera) must then have a direct access to the focal plane which is in most scenarios highly invasive. This problem can be overcome by using guidestars, such as nonlinear signals, to provide feedback in a non-invasive way [8,9], but some contrast mechanisms, such as linear fluorescence, are left aside as not easy to use. A few linear fluorescence settings were proposed for imaging but are only able to reconstruct the superficial layers of biological samples with high resolution [10][11][12][13]. Lately, a few approaches have been developed for non-invasive imaging in scattering media with fluorescence feedback, but they are specific and not very flexible [14,15].
Meanwhile, several techniques that utilize emerging computational tools, such as neural networks, have emerged in parallel for controlling light through scattering media such as diffusers and multimode fibres [16,17]. Although these methods offer the advantage of being physicsinformed, they are primarily used in transmission and remain invasive. Moreover, neural networks approaches that are non invasive often include several layers, making it more difficult to interpret the neural network physics and thus add prior physical knowledge [18].
To overcome these issues, we propose a versatile neural network-based TM retrieval method for non-invasive light focusing through scattering media [19]. The approach is based on mapping the input/output information in a microscopy configuration thanks to a 2-layer neural network. It is simple, model-based, and applicable to a variety of imaging scenarios. We present experiments on non-invasive fluorescence imaging and simulations generalizing this concept to a different contrast mechanism (Second Harmonic Generation, SHG).
Principle
In the general context of non-invasive imaging in scattering media, the propagation of light can be decomposed into two steps: the forward path (from the light source to the object of interest, corresponding to the illumination) and the backward path (from the object to the detector, corresponding to the signal re-emitted by the object). Due to scattering, the light waves in both paths are strongly distorted and as such no information can be retrieved directly. Inspired from the classical experiment to measure the TM, our strategy is to introduce an SLM to modulate the incident wavefront, and simply measure the corresponding re-emitted signal from the object with a camera. Most importantly, the latter captures the light that has traveled both the forward and backward paths, unlike in a conventional TM experiment where only the forward path is characterized. As this non-invasive geometry is more complicated to describe in terms of light propagation, it can be seen as a black box describing the relation between the input (on the SLM) and output (on the camera) patterns. It is then possible to make a "digital twin" of the system by describing this black box with a multi-layer neural network with the right constraints [20]. Conventional neural networks are built from an operational layer that involves a trainable matrix-vector multiplication followed by an element-wise nonlinear activation. The weights of the matrix-vector multiplication are adjusted during training in order for the neural network to implement a desired mathematical operation [21]. In the digital twin approach, the operation is linked to the physical properties of the system. By changing the network weights, one can alter the physical transformation performed on the input data in order to learn these parameters according to the physical system. Our approach is based on this representation (see Fig. 1).
We apply this concept to a microscopy imaging system. The physical system and its neural network interpretation are depicted in Fig. 2. The weights of the network connect all inputs to outputs of the system (from SLM to camera) through a fully connected two-layer network. The weights of each layer physically correspond to the coefficients of matrices 1 and 2 as we can see in Fig. 2(b). The first layer connects the input field in to the excitation field exc at the object plane. The weights of this layer correspond to the complex-valued coefficients of the matrix 1 . The size of this matrix is target × SLM , where target is the number of emitters and SLM the dimension of SLM patterns. The weights of the second layer represent 2 , of size CAM × target with CAM the number of camera pixels. According to the physical nature of the process, the forward model can be written as: where 0 = in is the input layer, matrices 1 and 2 constitute the weights of the networks and 1 and 2 are the activation functions, defined according to the imaging process [22]. 1 and 2 are physical priors, they are chosen to mimic our optical system.
In order to find the weights, the network is trained with a dataset composed of input-output pairs { in , out }. The gradient of the function is computed, with respect to the weights of the network (i.e. back propagation [23]). As it is appropriate for regression predictive modeling problems, the loss function we used in the work is simply a 2 -norm loss: After a given number of iteration corresponding to a significant decrease of the loss, the weights of the connection are trained. As a result, these weights correspond to the experimental transmission matrices of the system, 1 and 2 . This result is experimentally validated by focusing on the fluorescence beads using the retrieved 1 or reconstructing the image using 2 . Selective and non-invasive focusing onto beads is done by phase conjugating the retrieved 1 [24,25]. If the focus can be obtained onto most beads, the network learned the physical transform at stake in the system with a given accuracy.
To validate the method, we implement it experimentally in a fluorescence imaging scenario also and also in simulation with a different contrast mechanism (SHG). A different contrast mechanism can be studied by simply modifying the non-linear activation functions of the network, without changing the overall 2-layer architecture. This shows the simplicity of our network and its resulting flexibility since it can be easily applied to other non-invasive model and non-linear imaging settings.
The experimental setup is shown in Fig. 2. A sparse fluorescent sample made of 1 µm beads is placed behind a scattering medium. In order for the 2-layer model to reconstruct the object, all beads are placed in the same plane. An SLM is used to modulate the phase of the incident optical field in order to send different illumination patterns to the sample. Experimentally, random phase masks are displayed on the SLM, denoted in ( ). The phase of the SLM independent pixels is randomly chosen between 0 and 2 , following a uniform distribution. After its propagation through the scattering medium (which corresponds to a first matrix multiplication), a random speckle field exc ( ) is formed and illuminates the fluorescent object. During this first step, the transformation of the optical field between the SLM and the sample plane is linear and deterministic. It is represented by the matrix 1 that maps the SLM input optical field in to the field on the sample in a linear way exc = 1 in . The activation function of the first layer is then 1 = | . | 2 , corresponding to the fact that fluorescence signal is proportional to the intensity of the excitation. In the second step, the fluorescence signal is transmitted through the scattering medium and collected by the camera, a process that can be described by an incoherent and positive matrix 2 : as fluorescence is spatially incoherent, the captured camera image is the sum of the fluorescence speckles emitted by each fluorescent source : out = 2 | exc | 2 . In order to fit our physical system we ensure 2 to be the activation function of the second layer defined by 2 ( ) = ( ), the identity function. We define here eigen patterns, the speckles that are generated by each individual fluorescent emitter (of the focal spot size) on the camera. We made the assumption that the system (scattering medium and fluorescent sample) is static, therefore the eigen patterns are the same over time. Thus, the fluorescence signal out ( ) can be written as: A randomly modulated speckle pattern illuminates a fluorescent object (beads or pollen seeds), hidden behind the scattering medium. exc = 1 in excite the object, which emits a fluorescence signal in return, that is backscattered by the medium and detected in epi-geometry on a camera : out = 2 | exc | 2 . TL : tube lens. (c) Neural network mimicking the experimental setup. The neural network is trained to regenerate fluorescence speckle 2 ≈ out from the input pattern in displayed onto the SLM. 2 is compared with the actual original image out (record in reflection on a camera) through the = || out − 2 || 2 function. Using gradient descent, back-propagation updates the weights ( 1 and 2 ) to minimize the The experiment is completed by two sets of simulations, one to study our linear fluorescence setup, and another to simulate a different contrast mechanisms (SHG) to show how the activation functions and initial conditions on the TMs can be adjusted. Here, a numerical dataset consisting of pairs of input patterns displayed on the SLM, in , and corresponding speckle patterns out = 2 ( 2 1 ( in )) are generated. The values of in are passed to the two fully connected layers which is equivalent to multiplications by the approximate matrices 1 , 2 . An approximate image is then obtained as 2 = 2 ( 2 1 ( 1 0 )). Initially, 1 and 2 are random matrices, complex and real positive-valued, respectively. The derivatives of the function, with respect to the elements of 1 and 2 are computed. A stochastic gradient descent approach (SGD or Adam optimizer in Pytorch) is then applied to gradually update the estimated matrices such that it effectively reduces the overall loss function, and the process is repeated for a fixed number of iterations or epochs, ensuring convergence of the loss function to a minimum value [26]. In this simulation, no noise is added neither on the input nor output data. We also notice that the noise does not significantly impact the reconstruction as long as enough training samples in the dataset can be obtained. (see Supplementary information -Noise Influence to see the impact of noise on the reconstruction).
Discrete object
As a first step, we consider a linear fluorescence forward model based on the two-layer neural network architecture and a discrete object. In machine learning, a test set is typically used to evaluate the fit of a model on a training set [27]. The test set is a part of the original dataset which is set aside and used afterwards to assess the performance of the neural network. We choose a discrete fluorescent object composed of target = 8 targets, and set the dimension of the hidden layer to correspond to the number of targets. The dimensions of input layer and output layer are SLM = 256 and CAM = 256 respectively. Ground truths 1 and 2 are randomly generated following a standard normal distribution, we assume no correlation and no noise is added in the generated ground truth data. We use a training set up to pat =4900 examples to estimate the weights of the matrices 1 and 2 . Then a test set of 500 input examples on the SLM, test (unseen previously by the neural network) is passed to the trained network which generates 500 output images test . Correlations between test and out ; 1 and 1 ; 2 and 2 are plotted with respect to = / pat on Fig. 3, where is the training set size. This procedure is averaged over 10 repetitions for 10 different data sets (i.e different 1 and 2 ). See supplementary information for the choice of the rank, i.e. the size of the hidden layer.
As can be seen in Fig. 3, the loss function is minimized as the number of epochs grows. The correlations between ground truths and neural network guesses of fluorescent output speckle patterns reach a value close to 1 when the training set size increases, meaning that the transmission matrices were well retrieved by the training of the 2-layer neural network. Further, we visualize the reconstruction of both matrices, and observe that the dynamics are different based upon the size of the training set. With small training set sizes, < 500, < 0.09, it seems that the best way to minimize the global loss function is to adjust the coefficients of 2 . When backpropagating the gradient, it rapidly converges towards 2 (correlation > 0.8 for 0.1). At this point ( 0.1), 1 is still nearly completely random: its correlation with 1 is around 0.12. Nevertheless, for larger training set sizes, this correlation significantly increases and reaches almost unity for 0.5. From the comparison between test and out , we show that this method is equivalent to performing the correlations between ground truths and predicted matrices ( 1 and 1 , 2 and 2 ). The use of training and testing sets allows us to implement the two-layer neural network method on experimental data since we can verify the reconstruction of the matrices without knowing the ground truth for 1 and 2 . Correlation between the ground truth out and the neural network guess test , and the ground truth 1 and the neural network guess 1 , and finally between the ground truth 2 and the neural network guess 1 , according to = / pat , with P the training set size. Since the procedure is repeated 10 times for 10 different datasets, the plots represent the mean correlation and its standard deviation in shade. (c) Evolution of the reconstruction of the output image of the test set according to the training set size.
Continuous object
By increasing the dimension of the hidden layer, our physics-based neural network is able to reconstruct more complex continuous objects. We first study the case of continuous objects through simulations. To simulate the ground truth of the fluorescence imaging process, we use the code from [28]. It is simulated by an i.i.d. Gaussian complex with tunable speckle grain size (adjusted in the Fourier plane thanks to a pupil function). In this way, we can control the speckle grain size by varying the pupil size of the pupil function, and we found that the speckle grain size does not have an impact on the TM retrieval. (In the simulation in Figure 4, we show a speckle grain size of 1 pixel and in Fig. 7, we show the case of larger speckle grain of 4 pixels). To construct 1 , is further multiplied element-wise (Hadamard product) with the object vector, so that 1 ∈ C SLM × target . No correlations are embedded in 1 generation. The intensity transmission matrix 2 is simulated in a similar way, but by using real positive coefficients, corresponding to the intensity speckle patterns, 2 ∈ R target × CAM + . Here in our simulation, we chose as fluorescent continuous object a neuron with its connecting dendrites. We study the effect of the rank of the neural network (by changing the dimension of the hidden layer) on the final reconstruction. The training dataset is generated by randomly producing input patterns, and computing the corresponding output speckle images. These speckle images are obtained by propagating the input field with a field transmission matrix, and then multiply it with an intensity transmission matrix to simulate the final set of output fluorescence images. Once the neural network is trained, we phase conjugate 1 and scan through each column to focus the images through the scattering media at all possible locations, and observe the images as they would appear at the object plane. Here we present the original target image without scattering and the focus combined image with phase conjugation 1 , retrieved by the neural network [29]. Figure 4 shows the combined foci with the shape of the targeted neuron. Although the rank (i.e. the dimension of the hidden layer) is a required input parameter, it can be approximated, as it will impact the intensity distribution but not the overall shape. Note that there is no condition on the sparsity of the object in this section.
Discrete object
Experimentally, the measurement was first applied to 8 fluorescent beads of diameter 1 µm, placed on a holographic diffuser (Newport, 10DKIT-C1, 10 as the diffusion angle). A set of known input random patterns ( pat = 15360) are displayed onto the SLM and the corresponding fluorescence speckles are recorded. With this input/output training set, the loss function of the 2-layer neural network is minimized and two transmission matrices 1 and 2 are finally obtained. In order to confirm the quality of this retrieval method, light is focused onto each bead using phase conjugation of 1 [29,30]. When light is successfully focused, it indicates that both transmission matrices were well reconstructed. In Fig. 5, one can see the sum of all the control camera snapshots (placed in transmission) of all the beads foci after phase conjugating 1 , and the non-invasive reconstruction of the object using 2 . From 2 transmission matrix exploitation and the optical memory effect (OME), one can reconstruct the object shape [31][32][33]. The optical memory effect is a type of wave correlation that is observed in coherent fields, allowing control over scattered light through thin and diffusive materials [34]. Here only 16 × 16 macropixels of the SLM are modulated for the input, so our focusing enhancement will be impacted by the limited pixel counts. We expect to have an even higher signal to background ratio (SBR) with an increased number of SLM pixels.
Continuous object
The measurement was also applied to continuous objects like pollen seeds to show that the method can be used with 3D continuous objects and biological ones. The whole approach of acquisition, measurement, and processing is similar to beads samples: random incident wavefronts are produced by the SLM and sent onto the scattering medium, fluorescent speckles are respectively measured on the camera; the neural network is used to retrieve transmission matrices, 1 is used to focus onto the pollen seed and the eigen patterns are used to reconstruct (d) Reconstruction of the object, through scattering medium, from 2 and using the cross-correlation procedure shown in [31]. The white bar corresponds to 10 µm.
the object thanks to OME. The dimension of the hidden layer is increased to match the number of equivalent discrete targets. The latter can be estimated from the minimization of the Frobenius norm of the Non Negative Matrix Factorization (NMF) residual is done [31,32]. To make this reconstruction work, we initialized the weight of the second layer with the result of the NMF algorithm over the output dataset [35], in order to help the training of the neural network. This will help the convergence ratio of the neural network training. Once 1 and 2 are retrieved, the fingerprint of each target is computed by phase conjugation over 1 and a pairwise deconvolution is applied to reconstruct the object non-invasively [32]. Figure 6 shows the ground truth image of a pollen seed and the neural network's reconstruction. The dataset used is the same as in [31].
This shows that the structure of the 2-layer neural network can also be used to retrieve a continuous object hidden in scattering medium in experiments since it can accept some priors such as the NMF over the output data to initialize the second layer.
Other contrast mechanisms
To illustrate the versatility of this neural network approach, simulations for another contrast mechanisms were performed. Here, we study numerically the case of imaging through a scattering medium with contrast from second harmonic generation (SHG). Essentially, this is a coherent phenomenon which can be modelled by a pair of complex valued transmission matrices the same way as before, by simply changing the activation functions to 1 = . 2 and 2 = |.| 2 . The forward model is then: The simulation procedure is the same as before, only the forward model has changed. It is more challenging to retrieve phase information correctly when compared to the fluorescent case. A correlation between the transmission matrices and the corresponding ground truth is shown in Fig. 7. Contrarily to previous results, the correlation does not reach exactly 1 and the training set size needed for high correlation is significantly higher than that in the case of fluorescence. This can be explained by the absence of a phase reference leaving some ambiguity for 2 . Despite this, the retrieved matrices should be sufficient for many applications, such as focusing, as can be seen on the simulation over a continuous object, using the same technique as described in the simulation section by simply changing the forward model over 2 . The focusing is again possible, as shown in Fig. 7. As simulated objects, we chose polystyrene beads and an image of tissue collagen fiber, which are common SHG contrast sources.
Discussion
Our proposed 2-layer physics-based neural network approach enables the efficient retrieval of the two transmission matrices non-invasively, meaning without direct access to the object plane. With phase conjugation over 1 , we are able to focus and thanks to 2 we can successfully reconstruct the object, hidden behind the scattering medium, provided there is some optical memory effect. In order to recover these two TMs, experimentally, for fluorescence, a significant amount of measurements is required: approximately 15000 input/output pairs of experimental data are used for training our network. Through numerical study, we confirmed that it is not necessary to have an accurate estimation of the rank of the object to have two transmission matrices retrieved. In the experiment, the limitation to the reconstruction of these complex objects (for example the pollen seeds) is the contrast of the measured speckle which decreases with a 1/ √︁ target dependency [36]. In terms of transmission matrix reconstruction, our approach is limited by the simple architecture of the network, because of few freedom degrees. Even so, this still allows us to add physical priors, such as changing the initialization and the nature of the components of the two TMs as well as the activation functions. For example, using the result of the non-negative matrix factorization [35] over out to initialize 2 helps find more targets than the previous 2-step approach based on NMF and phase retrieval [31] information -Influence of initialization). Finally, this approach is versatile and applicable to other contrast mechanisms, such as SHG where the positivity constraint on 2 does not hold, but also Raman signal or 2-photons processes. The forward model is simply changed according to the physical phenomenon at stake, as shown in Table 1.
Non-linearity Coherent Non-coherent
Conclusion
Our study presented a physics-based machine learning method for characterizing light propagation through complex samples in order to focus on and reconstruct objects inside scattering media. Compared with other deep learning-based methods, our model is advantageous for data interpretation and incorporation of physical priors. Each node in the network has a physical meaning: it corresponds to the coefficients of the two transmission matrices. In order to test the quality of the neural network reconstruction, training and testing sets were used as is usually done in machine learning approaches. Compared to previous approaches regarding linear fluorescence [31], this new one is more demanding in terms of measurements, because there are no priors on the algorithm. Furthermore, the phase information is not measured. However, the two-layer neural network is more general and less sensitive to noise (see supplementary information -Influence of noise). This approach can also be easily generalized and adapted to other contrast mechanisms such as 2-photon fluorescence or coherent processes where the previous method would not work. Moreover, the physics-based neural network approach is versatile, and additional physical priors may be added in the model to further enhance the capability of the method, such as memory effect [37] information or 3D composition of the sample.
Experimental setup
Full experimental setup with control camera on Fig. 8 Fig. 8. Schematic view of the complete experimental setup. A randomly modulated speckle pattern, generated on the SLM, illuminates a fluorescent object (beads or pollen seeds), hidden behind the scattering medium. The fluorescent speckle is epi-detected on CAM 1. A second camera placed in transmission, CAM 2, monitors the illumination speckle in the plane of the beads and allows register a bright field image of the beads. CAM 2 is only used for passive monitoring. TL: tube lens.
Rank: Hidden layer size
In our reflection geometry, we do not have access to the number of sources non-invasively easily. In order to find this number, we compute the loss function according to the hidden layer size and see which values helps to reach the minimum. As an example, we consider a fluorescent object constituted of N = 8 separate fluorescent targets and see what happens if this real number is under or over estimated. On Fig. 9, one can see the minimum of the according to the rank value. When target < 8 or target > 8, the minimum of the is far above 0. In the case target < 8, the network does not have a sufficient number of degrees of freedom to correctly fit the physical system. This prevents the network to generate patterns that faithfully reproduce the ground truths. When the dimension of the hidden layer is correct, the final loss reaches a minimum. In that case, the number of neurons is high enough to reproduce the physical system with high fidelity. The Loss decreases almost to zero. When target > 8, the is higher but lower than under 8. We think that these additional neurons do not play a major role in the fitting. It may be better to overestimate the dimension of the hidden layer and refine the matrices 1 and 2 of the network afterwards than the opposite. N target Fig. 9. Dimension of the hidden layer. Final Loss value (epoch= 1000) with respect to target . target = 3, ..., 13 whereas the real dimension is target = 8.
Influence of initialization
As said in the main text, the 2-layer neural network is versatile because it is simple to add a prior and give some physical information to it. For the second layer ( 2 ), there is a way to retrieve the weights: the Non-Negative Matrix Factorization (NMF) algorithm, from the speckles recorded on the epi-camera ( out ) [39]. If the result of this algorithm is used as an initialization of the network weights, the reconstruction can be better, and faster because it helps the convergence of the gradient descent. In the Fig. 10, we show on an experimental sample (9 fluorescent beads), how the weight initialization can improve the reconstruction. Using random initialization, it is possible to retrieve four of the nine beads. Initializing the 2-layer neural network with the result of the NMF approach, helps retrieving more beads, even more than the previous approach (8/9 vs 7/9 with the NMF and Phase Retrieval algorithm). (b) Sum of the focus on each bead in the control camera after phase conjugation thanks to 1 found with NMF and Phase retrieval approach ( [31]). 7/9 beads found. Only the ones with an SNR higher than 20 are added to the sum. (c) Sum of the foci with 1 from 2-layer neural network optimization with random initialization of the weights. 4/9 beads found. Only the ones with an SNR higher than 20 are added to the sum. (d) Sum of the foci with 1 from 2-layer neural network optimization with NMF initialization of the weights of the second layer. 8/9 beads found. | 2022-06-02T06:47:58.896Z | 2022-06-01T00:00:00.000 | {
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203604657 | pes2o/s2orc | v3-fos-license | Efficacy of Cotesia flavipes (Hymenoptera: Braconidae) in Reducing Diatraea tabernella (Lepidoptera: Crambidae) Injury in Sugar Cane
Abstract Sugar cane stem borers, Diatraea spp. (Lepidoptera: Crambidae), are the most important pests affecting sugar cane in Colombia. To date, the use of egg parasitoids such as Trichogramma exiguum Pinto & Platner (Hymenoptera: Trichogrammatidae), and larval parasitoids such as Billaea claripalpis Wulp and Lydella minense Townsend (Diptera: Tachinidae) have been the principal biological control approaches for pest management. However, a pest outbreak of Diatraea tabernella Dyar in the northern Cauca River Valley demonstrated that conventional control measures are insufficient, and that new pest control methods must be sought. Field evaluations were made using 2 sources of Cotesia flavipes Cameron (Hymenoptera: Braconidae): a commercial colony maintained in the laboratory, and a colony recovered from previous field releases (field-refreshed). Three releases of the parasitoid were made, each release consisting of 4 g of C. flavipes cocoons (about 4,000 wasps) per ha. The results from both sources of C. flavipes were compared with check plots where no releases were made. Larvae of D. tabernella were collected 2 different times (45 d and 75 d after the first release) and observed in laboratory. The proportion of larval parasitism ranged between 0.32 and 0.55, with no significant differences between sources of C. flavipes. Parasitism differed significantly from the check plots, where the proportion of larval parasitism was less than 0.1. Our results indicate that wasps from the check plots experienced an increase in the number of cocoons and wasps per parasitized larva between the first and the second larval collection. The high levels of parasitism in fields where C. flavipes was released resulted in a reduction of up to 65% in the percentage of bored internodes, demonstrating the potential of this natural enemy to effectively control D. tabernella. Changes in the number of progeny per parasitized larvae (cocoons and wasps) in the check plots can be explained as the increase of parasitoids in an area under the influence of nearby releases, and the subsequent effects of multiple parasitism. In addition, comparisons between the 2 parasitoid sources indicate higher biological efficiency in the field-refreshed plots expressed in an increase in adult longevity between the first and second collection times.
Palabras Claves: control biológico; barrenadores del tallo; Lydella minense Colombia offers perhaps one of the best examples of effective biological control of the sugarcane stem borers, Diatraea spp. (Lepidoptera: Crambidae), major pests of sugar cane in the country (Vélez 1997). The newly hatched larvae of this pest feed on leaf parenchyma and migrate toward the cane stem, which they penetrate after the second larval instar, feeding on plant conducting tissues, building tunnels, and finally leaving the stalk as moths (Vargas et al. 2015a). Sugarcane stem borers can reduce crop performance significantly by decreasing stalk weight, and in some cases causing plant death. Losses due to stem borer damage have been estimated at around 0.83% of reduced cane weight, and an additional 0.26% yield at milling per ha for each percentage unit of internodes bored (Vargas et al. 2015a). To control this pest, biological control using egg or larval parasitoids has proved effective not only in Colombia, but elsewhere in the Americas (Vargas et al. 2015a).
In 2012, Diatraea tabernella Dyar was detected attacking sugar cane crops in the Cauca River Valley, between Viterbo (Department of Caldas) and La Unión (Department of Valle del Cauca). It was also the predominant and most widely distributed species in cane fields of the Risaralda Sugarcane Mill (Vargas et al. 2013), surpassing in number and aggressiveness Diatraea saccharalis F. and Diatraea indigenella Dyar & Heinrich, species traditionally found in the region. As early as 1914, this insect had been reported in Colombia in the Department of Chocó (Box 1931), but it was only in 2012 that its presence was recorded in commercial sugar cane fields in the Cauca River Valley (Vargas et al. 2013).
Diatraea tabernella also is considered the most important and widely distributed pest of sugar cane in Panama and Costa Rica, and has been observed to attack from germination through harvest in Costa Rica (Valverde et al. 1991). Based on the observed impact of this pest in Central American countries, its potential to limit sugar cane production, not only in the Cauca River Valley but elsewhere in Colombia, should not be overlooked.
According to Guagliumi (1962), an alternative to effectively reduce sugarcane borer populations could be the use of the wasp Cotesia flavipes Cameron (Hymenoptera: Braconidae). The biological characteristics of this natural enemy, such as its short life cycle and high population growth rate, are fundamental elements of effective biological control (Bellows & van Driesche 1999). Cotesia flavipes is a larval endoparasitoid native to Southeast Asia, first found parasitizing larvae of the genus Chilo (Lepidoptera: Crambidae) (Mohyuddin 1971). A large number of host species of the families Pyralidae and Noctuidae have been reported for this species (Li et al. 2005). The life cycle of C. flavipes consists of 4 development stages: egg, 3 larval instars, pupa (cocoon), and free-living winged adult (Hernández 2010). These parasitoids take 13 to 14 d to complete larval development within the host, after which they exit the Diatraea larvae to build a silk cocoon in which they pupate.
Cotesia flavipes has been introduced as a biocontrol agent of the sugarcane borer in several countries, including Brazil, Peru, Madagascar, Indonesia, and the US (David & Easwaramoorthy 1991;Fernández et al. 2006). However, in Colombia, and especially in the Cauca River Valley, this parasitoid has not been used because of its history of low adaptation in this region (Aya et al. 2017), explaining its absence from Diatraea hosts in the region thus far (Vargas et al. 2013). The objectives of this study were to evaluate the effect of releasing C. flavipes in areas highly infested by D. tabernella in the northern Cauca River Valley where they were absent at the time of this study, and then to analyze its adaptation to both the host and the study area. We also sought to determine whether continuous rearing of C. flavipes under laboratory conditions could decrease the adaptation potential of the released parasitoid as compared to its offspring obtained from material recovered in the field (field-refreshed).
Materials and Methods
The study was carried out in 9 commercial sugar cane fields located in the municipality of Toro, Department of Valle del Cauca (4.6028°N, 76.0355°W), planted with the sugar cane variety CC 85-92, and having similar agronomic practices. To verify the absence of C. flavipes in the test area, on 9 Sep 2014 an initial larval collection was made of 22 larvae, which produced 21 D. tabernella moths and only 1 D. saccharalis moth, demonstrating, as expected, that the parasitoid was rare or absent in the study area. In each commercial field, a plot measuring 100 m × 100 m (1 ha) was established, and separated from all other plots by at least 100 m.
The commercial insectary Laboratorios Biocol S.A.S. supplied the biological material used in the study. The C. flavipes specimens were from 2 different sources: a commercial source permanently maintained in the laboratory, and a field refreshed source. The latter consisted of individuals from a colony obtained from recovered parasitized individuals from fields in different sugar cane farms where previous releases were made (Vargas et al. 2015b).
Treatments used were as follows: release of individuals from the commercial source, release of individuals from a field refreshed source, and plots with no releases of the parasitoid. Each treatment had 3 replicates (plots) randomly assigned to each treatment. Three releases of C. flavipes, at 15 d intervals, were made in a 2 mo-old crop on 9 Oct, 23 Oct, and 6 Nov 2014. Each release consisted of 4 g parasitoid cocoons per ha, totaling 12 g per ha in the 3 releases, which is equivalent to approximately 12,000 wasps per ha, with a 1.5:1.0 female:male ratio.
An initial collection of Diatraea larvae was conducted 15 d after the last release of the parasitoid (i.e., 45 d after the first release, on 24 Oct 2014, from this point forward referred to as the first collection), and the level of parasitism by C. flavipes in the borers was evaluated. A second collection was carried out 1 mo after the first collection (i.e., 75 d after the first release, 19 Nov 2014, and from this point forward referred to as the second collection) to determine the establishment potential of the parasitoid in the field. Both collections were made with a sampling effort of 2 man-h per plot.
Collected larvae were tagged and placed individually in plastic Petri dishes for identification of the stem borer species at Laboratorios Biocol S.A.S., located in La Victoria, also in the department of Valle del Cauca. Larvae were fed slices of fresh corn cobs, according to the methodology of Rodríguez et al. (2004), and were maintained at temperatures of 24 ± 2 °C and 65% RH, with a photoperiod of 12:12 h (L:D). The following variables were recorded: total number of captured larvae, proportion of borers parasitized, number of C. flavipes cocoons formed per parasitized larva, number of wasps emerged per parasitized larva, female:male ratio of parasitoids obtained, time from cocoon formation to adult parasitoid emergence, and adult longevity.
To assess the effect of the releases of natural enemies on the reduction of pest injury, at 6 mo of crop age the percent of bored internodes was estimated. A randomly distributed sample of 20 stalks was observed within each plot to determine the level of boring.
A 3 × 2 completely randomized factorial design was used for the field experiment, with 3 treatments and 2 collection times. A binomial-type distribution was assumed for proportion variables. Data were analyzed using the Glimmix procedure, and multiple comparisons between treatments were performed based on the Tukey-Kramer test (α = 0.05) using SAS 9.3 (SAS Institute 2011). The GLM procedure was used to analyze the percentage of bored internodes, whereas multiple tests were performed using the Tukey test (α = 0.05), and using SAS 9.3 (SAS Institute 2011).
Results
Despite the decrease in the average number of D. tabernella larvae captured between the first (38 larvae per plot in 2 man-h) and second collection times (23 larvae per plot in 2 man-h), no effect could be attributed to the treatments. The average proportion of parasitism by C. flavipes on D. tabernella ranged from 0.32 to 0.55 among the 2 C. flavipes sources and the 2 collection times. Although no difference in parasitism was observed between the 2 sources of C. flavipes (Fig. 1), statistical differences were found between the commercial source and the check for both collection times (F = 12.21; df = 2, 12; P = 0.001). Parasitism tended to increase between the first and second collection, but this was not a statistically significant difference (F = 3.23; df = 1, 12; P = 0.097). Also, there was no interaction between treatments and collection times (F = 0.06; df = 2, 12; P = 0.941). The proportion of parasitism observed in the check plots ranged from 0.04 and 0.09 for the 2 collection times, indicating that check plots were probably colonized by parasitoids released in nearby plots, assuming there were no previously parasitized larvae in the field. On the other hand, a natural proportion of parasitism by L. minense was observed in larvae collected in the different plots, and ranged from 0.07 to 0.16 between treatments and collection times.
Statistical differences found in the duration of the cocoon stage of C. flavipes were attributable to the treatment (F = 5.21; df = 2, 6; P = 0.048) and collection times (F = 14.57; df = 1, 116; P < 0.001). A significant interaction was observed between treatment and collection times (F = 11.9; df = 1, 116; P < 0.001), where C. flavipes wasps in the check treatments displayed a shorter development time (up to 1 d) in the first collection time (Fig. 2).
With respect to the number of cocoons formed per parasitized larva (Fig. 3A), the maximum average number was 62.2 cocoons per larva from the refreshed source of C. flavipes, and the minimum average was 26.5 cocoons per larva from check plots. However, no statistically significant differences were found among treatments (F = 1.43; df = 2, 5; P = 0.320). The number of cocoons per larva increased between the first and the second collection times, especially in the check and the refreshed source of C. flavipes treatments (F = 14.36; df = 1, 146; P < 0.001). In addition, a significant interaction was found between treatments and collection times (F = 7.59; df = 2, 143; P < 0.001). The same pattern was observed when considering the number of wasps emerging per larva, with a tendency for fewer wasps per larva in check plots, but there was not a statistical difference among treatments (F = 4.71; df = 2, 5; P = 0.059). However, statistically significant differences were found between the 2 collection times (F = 26.11; df = 1, 145; P < 0.001). Fewer wasps were found per larva from check plots on the first collection time, and a significant interaction was found between treatments and collection times (F = 14.54; df = 1, 145; P < 0.001) (Fig. 3B). As in the case of the number of cocoons per larva, the number of wasps per larva tended to increase between the first and second collection times for the refreshed source of C. flavipes and colonizing wasps from the check plots. However, there was a statistically significant difference only in the case of the check treatment, where the number of wasps produced per larva increased up to 3 times from the first to second collection times (Fig. 3).
No statistically significant differences were found in offspring sex ratio at the treatment level (F = 2.63; df = 2, 5; P = 0.165) or due to collection time (F = 0.01; df = 1, 135; P = 0.915), nor the interaction between treatment and collection time (F = 1.12; df = 1, 135; P = 0.327). However, the first collection time revealed a higher female:male ratio in check plots (2.6:1) and in the refreshed source of C. flavipes (1.8:1) compared to the commercial source of C. flavipes (1.3:1). On the other hand, in the second collection, all treatments presented a female:male ratio of 1.8:1, which corresponds to a proportion of 0.64 females.
The proportion of adults emerging fluctuated between 0.51 for check plots in the first collection time and 0.94 for the same treatment in the second collection; however, due to large variations in data, no differences were found among treatments (F = 0.52; df = 2, 5; P = 0.625). In the case of sources of C. flavipes, the proportion of adult emergence ranged from 0.83 (commercial source in the second collection time) to 0.87 (check in the first collection time). On the other Observations were made at a first collection time (45 d after the initial release) and a second collection time (75 d after the initial release). Bars with the same capital letter do not differ between treatments within the same collection time. Bars with the same lowercase letter do not differ within each treatment between collection times (Tukey-Kramer, α < 0.05). hand, significant differences were observed due to collection time (F = 6.8; df = 1, 139; P = 0.010). There also was a significant interaction between treatment and collection time (F = 5.47; df = 1, 139; P = 0.005), specifically involving check plots, which recorded a 50% increase in adult emergence from the first to second collection times.
Adult longevity ranged from 2.0 d (check plots at first collection time) to 3.7 d (refreshed source at second collection time), but no statistically significant differences were found between treatments (F = 1.28; df = 2, 6; P = 0.345). On the other hand, longevity increased 1 d between the first and the second collection times in the refreshed source of C. flavipes (F = 4.24; df = 1, 145; P = 0.040). Likewise, a significant interaction was found between the treatment and collection time (F = 3.82; df = 1, 145; P = 0.024) (Fig. 4).
With respect to the effect of releases of C. flavipes on pest damage, a decrease up to 65% in the percentage of bored internodes was observed at 6 mo of crop age in plots where releases of C. flavipes were made (commercial and refreshed) as compared with check plots (F = 5.68; df = 2; P = 0.004). Post-hoc tests do not reveal differences between the 2 sources of the parasitoid (Tukey, α < 0.05; Fig. 5).
Discussion
In the study conducted in the Cauca River Valley, the proportion of parasitism was as high as 0.53 in plots where releases were made, clearly demonstrating the suitability of C. flavipes for parasitism of D. tabernella under field conditions. The occurrence of parasitism in check plots also confirmed that the parasitoid adapted well to the per Diatraea tabernella larva after 3 releases of 4 g of cocoons per ha each, from 2 sources of C. flavipes (field refreshed and commercial), compared to a check with no releases. Observations were made at a first collection time (45 d after the initial release) and a second collection time (75 d after the initial release). Bars with the same capital letter do not differ between treatments within the same collection time. Bars with the same lowercase letter do not differ within each treatment between collection times (Tukey-Kramer, α < 0.05).
Fig. 4. Average adult longevity (d) of
Cotesia flavipes wasps obtained on Diatraea tabernella larvae after 3 releases of 4 g of cocoons per ha each, from 2 sources of C. flavipes (field refreshed and commercial), compared to a check with no releases. Observations were made at a first collection time (45 d after the initial release) and a second collection time (75 d after the initial release). Bars with the same capital letter do not differ between treatments within the same collection time. Bars with the same lowercase letter do not differ within each treatment between collection times (Tukey-Kramer, α < 0.05).
Fig. 5.
Average percentage of bored internodes (± SE) evaluated in 6-mo-old sugar cane plots (20 stalks evaluated per plot) after 3 releases of 4 g of cocoons per ha each, from 2 sources of C. flavipes (field refreshed and commercial), compared to a check with no releases. Bars with the same letter do not differ statistically (Tukey, α < 0.05). study area, considering the low level of natural parasitoid dispersal (Sallam et al. 2001). Badilla (2002) reported similar levels of parasitism (50%) in Costa Rica and concluded that the introduction of C. flavipes had proved successful in managing the pest, parasitizing the 3 borer species found in that country (D. tabernella, D. saccharalis, D. guatemalella Schaus). According to the same author, C. flavipes also was found to adapt well to the different ecological regions where sugar cane is planted in Costa Rica.
According to Sallam et al. (2001) a female C. flavipes can travel up to 64 m during her lifespan of 2 to 3 d, and although plots in this study were at least 100 m apart, the time between each of the 3 releases (15 d) and the 2 collection times (30 d) totaled 75 d between the first release of the parasitoid and the second collection time. The generational time of C. flavipes is around 20 d (Hernández 2010). Therefore, 2 generations could have passed when the first collection was made 15 d after the 3 releases had been completed, and at least 3 generations by the second collection, 45 d after the 3 releases had been made. During the multiple generations, individuals could have travelled between the plots where the releases were made and the check plots. This also could affect the ability to contrast the different sources of C. flavipes.
It is important to mention that L. minense has been widely released as a biocontrol agent of Diatraea spp. in the study area; however, there is no evidence that releases of C. flavipes triggered a decrease in the activity of these tachinid flies during the study period. Although the work carried out by Weir and Sagarzazu (1998) showed that flies were more effective than wasps in inoculations on D. saccharalis larvae in the laboratory, Rossi and Fowler (2004) observed a marked tendency for populations of the tachinid flies L. minense and B. claripalpis to decrease at sites in Brazil where C. flavipes had been released. However, these studies in the Cauca River Valley are, to the best of our knowledge, the first in Colombia, and additional studies should be conducted on future releases of C. flavipes in relation to population densities of L. minense.
An interaction was found between treatments and collection times with respect to the number of cocoons and adults formed by larvae, indicating that in check plots there was an increase in the number of cocoons and wasps per larva in relation to collection time. This might be explained by the fact that there were no parasitoid releases in these plots, therefore fewer ovipositing adults and perhaps less multiple parasitism, leading to fewer cocoons and wasps per parasitized larva in the first larvae collection. The increase in parasitism in the check plots between collection times can, therefore, be explained by the additional releases and time for dispersal from the refreshed and commercial plots, and as Potting et al. (1997) suggest, a lack of discrimination between already parasitized hosts and non-parasitized ones, mostly in non-experienced females.
Although wasp size was not measured in the present study, fewer parasitoids per larva resulted in a shorter cocoon stage in the case of the checks, which is consistent with a tradeoff between favorable conditions during development (e.g., number of individuals per larva) and reduced development time, and possibly, larger-sized adults. In fact, in the context of life history evolution, it is assumed that there is a tradeoff between the time an individual takes to develop and its size as a sexually matured adult (Stearns 1992). Larger adult size generally is understood to translate into higher fecundity in insects (Honěk 1993), and in parasitoid wasps in particular (Charnov et al. 1981).
In this regard Goubault et al. (2007) pointed out that the optimal oviposition per host would not only be affected by future reproduction expectancy, but also would be flexible and affected by intergenerational biological efficacy, where larger individuals (the result of less sibling competition) would translate into more competitive adults. Mayhew and Glaizot (2001) in turn pointed out that one of the great-est tradeoffs in the life history of parasitoid insects is between clutch size and resulting size of offspring; however, in the particular case of C. flavipes the equation also needs to consider fitness consequences of superparasitism under a very common low rate of host encounter (Potting et al. 1997).
Studies carried out on C. flavipes in Kenya corroborate that the parasitoid tends to produce a higher proportion of females, even when exposed to different hosts such as Sesamia calamistis Hampson (Lepidoptera: Noctuidae) (0.55 females) and Chilo partellus (Swuinhoe) (Lepidoptera: Crambidae) (0.68 females) (Obonyo et al. 2008). Similarly, Wiedenmann et al. (1992) found high female:male ratios in the lab when studying C. flavipes parasitism on D. saccharalis. Studies carried out in sugar cane fields in Cuba by Barroso et al. (2003) also demonstrated a higher proportion of females (0.58 females) in C. flavipes during the acclimatization and colonization processes. The observed tendency of a higher proportion of females in the first evaluation of both the refreshed source and check suggests that the female:male encounter rate was high, and attributable to a balanced sex ratio of released insects. It also suggests good parasitoid adaptation (Fernandez & Sharkey 2006).
Both rate of host encounters and adult longevity notably affect the reproductive success of parasitoid insects in the field, indicating that these insects generally are affected by time constraints (Bezemer & Mills 2003). The significant interaction between treatment and collection time for wasp longevity indicates that the refreshed source was able to increase its longevity by 1 d between the first and second collection times, which translates into more opportunities for wasps to search for hosts, and in turn increases parasitic potential, as illustrated by the greater number of cocoons per parasitized larvae, especially during the second evaluation 45 d after release. Factors such as nutritional quality, host size, temperature, and relative humidity are known to affect the longevity and fecundity of C. flavipes females (Charnov et al. 1981;Jervis & Copland 1996;Emana 2007). In this case, we argue that factors associated with increased longevity in the refreshed source would be related to a higher biological efficiency mediated by selection in the field to tolerate greater environmental stress (van Lenteren 2003).
The reduction of pest injury observed in sugar cane plots where the wasp was released, as compared with check plots, is consistent with the parasitism observed, and confirms the parasitoid's potential to adapt to the environmental conditions of the northern Cauca River Valley, as well as to the host D. tabernella. The reduction in the level of pest injury is similar to that reported by Vargas et al. (2015a), when estimating the effect of releases of the tachinid L. minense on reducing the damage caused by Diatraea spp. in sugar cane fields.
This study presents useful biological observations on the parasitoid's functionality when introduced into a new environment. Comparisons between a source previously exposed to field conditions and those maintained in the lab suggest differences at the level of biological efficiency by an increase in adult longevity between the first and second collection times in the refreshed source, which translates into more opportunities for wasps to search for hosts, and in turn an increase in parasitic potential. More recent observations in the Cauca River Valley have proven its potential for adaptation, persistence, and effectiveness as a biological control agent under field conditions, as C. flavipes has been expanding its distribution range in the region (Aya et al. 2017;Vargas et al. 2018). Therefore, C. flavipes is currently included in the biological control programs, not only in the northern Cauca River Valley, but across the region. It should be highlighted that Cotesia also could prove useful in the management of other Diatraea species; however, additional studies are needed to explore the potential of this natural enemy to manage different species of the Diatraea complex. | 2019-10-01T13:03:53.092Z | 2019-09-30T00:00:00.000 | {
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19672126 | pes2o/s2orc | v3-fos-license | Imaging Appearance of Dextranomer/Hyaluronic Acid Copolymer Implant Injections for Treatment of Velopharyngeal Insufficiency
BACKGROUND AND PURPOSE: Dextranomer/hyaluronic acid copolymer implants are used in treating velopharyngeal insufficiency. These posterior nasopharyngeal implants can be mistaken for pathologic conditions such as retropharyngeal abscess on imaging. We studied the imaging appearance of dextranomer/hyaluronic acid copolymer implants in patients treated for velopharyngeal insufficiency. MATERIALS AND METHODS: A consecutive series of patients with velopharyngeal insufficiency treated with dextranomer/hyaluronic acid copolymer were included in this study. Data on patient characteristics and volume of dextranomer/hyaluronic acid copolymer injected were obtained. Postoperative imaging characteristics on plain radiography, CT, and MR imaging were assessed. The imaging appearance of postoperative complications was determined. RESULTS: Sixteen patients were included in this study. Seven patients underwent postoperative plain radiographs, 5 patients underwent CT, and 9 patients underwent MR imaging. Plain radiographs demonstrated soft-tissue swelling in the retropharyngeal space, which resolved at 1 month. On CT, dextranomer/hyaluronic acid copolymer implants appeared as bilateral nasopharyngeal soft-tissue masses isoattenuated to hypoattenuated relative to muscle in 80% (4/5) of patients. On MR imaging, dextranomer/hyaluronic acid copolymer implants appeared as bilateral nasopharyngeal soft-tissue masses that were isointense to muscle on T1 (8/9, 88.9%) and hyperintense to muscle on T2 (8/9, 88.9%) and demonstrated no restricted diffusion (4/4, 100.0%) or peripheral enhancement (7/7, 100.0%). CONCLUSIONS: The normal postoperative findings of posterior nasopharyngeal dextranomer/hyaluronic acid copolymer injection on MR imaging is characterized by the presence of bilateral nasopharyngeal soft-tissue masses that are isointense to muscle on T1 and hyperintense on T2, with no restricted diffusion or peripheral enhancement. Velopharyngeal dextranomer/hyaluronic acid copolymer implants are iso- to hypoattenuated to muscle on CT and are not visible radiographically once associated implantation-related swelling has resolved.
P osterior pharyngeal wall augmentation is a promising technique for the treatment of velopharyngeal insufficiency (VPI). 1 By augmenting the posterior pharyngeal wall so it extends more anteriorly, an easily reached contact point for the soft palate is created, allowing velopharyngeal closure. Numerous implant materials have been used, including cartilage, fat, paraffin, and calcium hydroxyapatite. [1][2][3][4][5][6][7] Injection of dextranomer/hyaluronic acid has emerged as an effective technique for treating vocal cord dysfunction and laryngeal insufficiency, and augmenting the pos-terior pharyngeal wall in the treatment of VPI. [8][9][10][11][12] Because many patients receiving these implants will undergo imaging studies unrelated to VPI, characterization of the normal imaging appearance of hyaluronic acid implants in the posterior pharyngeal wall is important to avoid confusion with pathologic processes.
Patient Population
After institutional review board approval, a retrospective series of patients who received posterior pharyngeal wall augmentation with hyaluronic implants (dextranomer/hyaluronic acid copolymer or hyaluronic acid) between April 1, 2010, and August 31, 2012, and who underwent follow-up imaging was reviewed. For all patients, data on age, sex, underlying disease, and volume of polymer injected were collected.
Procedural Details
All surgical procedures were performed by an otorhinolaryngologist. Preoperative nasoendoscopy was used to determine the level of velopharyngeal closure. Patients were treated under general endotracheal anesthesia. A catheter was inserted into each nostril and withdrawn through the oral cavity and clamped to itself to retract the palate. Mirror visualization of the nasopharynx was correlated with the findings of the nasoendoscopy. Following identification of the level of velopharyngeal closure, dextranomer/hyaluronic acid copolymer or hyaluronic acid solution was injected to augment the posterior nasopharyngeal wall. Careful aspiration before the injection was performed to avoid placement within a vessel. Irrigation was then performed, and all hardware was removed from the patient's mouth. Procedural success was generally evaluated with a postoperative endoscopic evaluation and/or a video swallow study.
Imaging Characteristics
Neck MR imaging was generally performed on 1.5T scanners. Fast spin-echo T1 and T2 images were obtained. Imaging parameters for T2 sequences were the following: TR ϭ 3000 ms, TE ϭ 100 ms, section thickness ϭ 4 -5 mm, matrix size ϭ 256 ϫ 256 pixels. Imaging parameters for T1 sequences were the following: TR ϭ 467 ms, TE ϭ 10 ms. Postgadolinium imaging was generally performed by using fat-saturated echo-spoiled gradient-echo imaging (TR ϭ 170 ms, TE ϭ 3.2 ms) or fat-saturated spin-echo sequences (TR ϭ 483 ms, TE ϭ 20 ms). Diffusing-weighted imaging was performed by using TR ϭ 10,050 ms and TE ϭ 72 ms. Section thickness was 5 mm for axial images and 4 mm for sagittal images. Matrix size was 256 ϫ 256 pixels. In general, images were obtained from the orbits to the superior mediastinum. For CT neck imaging, the parameters were the following: kVP ϭ 120, mA ϭ 400, kernel ϭ H41s, section thickness ϭ 2 mm. Image matrix size was 512 ϫ 512 pixels, and FOV was 250 ϫ 250 mm.
In addition to imaging patients following injection of hyaluronic acid, we imaged a vial of dextranomer/hyaluronic acid copolymer adjacent to a vial of water as a reference standard with both T1-and T2-weighted sequences. T2-weighted image parameters were the following: TR ϭ 4000 ms, TE ϭ 110 ms, section thickness ϭ 4 mm, matrix size ϭ 256 ϫ 256 pixels. Imaging parameters for the T1 images were the following: TR ϭ 500 ms, TE ϭ 11 ms, section thickness ϭ 4 mm, matrix size ϭ 256 ϫ 256 pixels.
All imaging was reviewed by a board-certified neuroradiologist with 22 years' experience (J.I.L.). Imaging characteristics studied included postoperative x-ray findings, postoperative CT findings, and postoperative MR imaging findings. For patients receiving postoperative CT studies, data were obtained on the attenuation of the implant relative to surrounding muscle and the appearance of the posterior pharyngeal wall in cases in which contrast was used. For patients undergoing postoperative MR imaging studies, data were obtained on the imaging appearance of the dextranomer/hyaluronic acid copolymer implants relative to surrounding muscle on T1-weighted sequences, T2-weighted sequences, diffusionweighted sequences, and postcontrast T1-weighted sequences. Data on time to imaging were also obtained.
For patients who experienced complications related to dextranomer/hyaluronic acid copolymer injection (ie, retropharyngeal abscess), the appearance of these complications on both CT and MR imaging was studied and compared with the normal imaging appearance of dextranomer/hyaluronic acid copolymer implants.
Patient Population
Fifteen patients were included in our study. The median age of these patients was 10 years (minimum age ϭ 3 years, maximum age ϭ 68 years). Five patients were adults (33.3%), and 10 patients (66.6%) were children. Eight patients were female (53.3%), and 7 patients were male (46.7%). Among the patients included in our study, the most common underlying causes of VPI were postsurgical complications after treatment of a malignancy (3/15, 20.0%), neurofibromatosis 1 (3/15, 20.0%), and velocardiofacial syndrome (3/15, 20.0%). The mean volume of hyaluronic acid injected was 3.2 Ϯ 1.2 mL (minimum ϭ 1 mL, maximum ϭ 6 mL). Fourteen patients were injected with dextranomer/hyaluronic acid copolymer, and 1 patient was injected with hyaluronic acid. The patient injected with hyaluronic acid alone underwent only CT. Clinical data for the patients included in our study are summarized in the Table.
Imaging Appearance of Hyaluronic Acid Implants
Plain Radiographs. Seven patients in this study (46.7%) were studied with plain radiographs at some point in their postoperative period (range ϭ 2 days to 15 months). Four patients had plain radiographs within 1 month of the procedure. Of these, 3 patients (75.0%) had prevertebral soft-tissue swelling. Three patients (20%) had plain radiographs after 1 month, and the plain radiograph findings were normal in all 3 cases. The dextranomer/hyaluronic acid copolymer implants were not specifically visible in any patients on plain radiography. The postoperative radiographic appearance of patients receiving these implants is shown in Fig 1. CT Appearance. Five patients (33.3%) underwent CT during the postoperative period (minimum ϭ 2 months, maximum ϭ 9 months). Four of the patients received dextranomer/hyaluronic acid copolymer injection, and 1 patient received hyaluronic acid implants. On CT, both hyaluronic acid copolymer and hyaluronic acid implants appeared as bilateral nasopharyngeal soft-tissue masses that were isoattenuated to hypoattenuated relative to muscle in 80% (4/5) of patients. The CT appearance of these implants is demonstrated in Fig 2. One patient presented with a ring-enhancing retropharyngeal mass with central low attenuation consistent with a large retropharyngeal abscess at 8 months after the procedure. The CT imaging appearance of this retropharyngeal abscess is demonstrated in Fig 3. The abscess was surgically aspirated and grew Grampositive anaerobic bacilli.
No peripheral enhancement was observed in patients who received gadolinium (7/7, 100.0%). The MR imaging appearance remained stable with time. The MR imaging appearance of these dextranomer/hyaluronic acid copolymer implants is demonstrated in Fig 4. One patient developed a retropharyngeal abscess at the injection site, which was characterized by T2 hyperintensity, mild T1 hyperintensity, restricted diffusion, and peripheral enhancement. This is demonstrated along with the CT appearance in Fig 3. MR imaging of the ex vivo dextranomer/hyaluronic acid copolymer vial by using T2 imaging parameters demonstrated that the dextranomer/hyaluronic acid copolymer was hyperintense with a signal intensity similar to that of water. Using T1 imaging parameters, the ex vivo dextranomer/hyaluronic acid copolymer was hyperintense with a higher signal intensity than that of water. These findings are demonstrated in Fig 5.
DISCUSSION
We have demonstrated that on MR imaging, posterior nasopharyngeal hyaluronic acid injection is characterized by the presence of bilateral nasopharyngeal soft-tissue masses that are isointense to muscle on T1 and hyperintense on T2, with no restricted diffusion or peripheral enhancement. On CT, hyaluronic acid injections are generally iso-to hypoattenuated to adjacent muscle. On plain radiographs, the normal imaging appearance is softtissue fullness within 1 month of the procedure, which normalizes thereafter. Dextranomer/hyaluronic acid copolymer implants are not radiodense on plain radiography or CT.
These findings are important to recognize to distinguish the normal postoperative imaging appearance of dextranomer/hyaluronic acid copolymer injections from nasopharyngeal pathology. Because VPI is associated with so many diseases in which imaging is involved in the diagnosis and follow-up, it is highly likely that patients with VPI will undergo imaging in which the posterior nasopharynx is included. In our series of patients with VPI who underwent imaging, 3 had undergone surgical treatment for a head and neck/brain malignancy, 3 had neurofibromatosis type 1, and 3 had velocardiofacial syndrome. Several previous studies have demonstrated a strong association between neurofibromatosis type 1, velocardiofacial syndrome and postradiation, and surgical treatment for head and neck malignancies and VPI. [13][14][15][16][17] In our series, 1 patient developed a retropharyngeal abscess at the injection site. The imaging appearance of this abscess is clearly distinct from the normal postoperative imaging of hyaluronic acid implants by its increased size, restricted diffusion, and peripheral enhancement. It is important for clinicians and radiologists to be able to distinguish the appearance of a retropharyngeal abscess from the normal appearance of hyaluronic acid to avoid misdiagnosis and unnecessary intervention.
To our knowledge, no studies currently exist on the postoperative imaging findings of patients undergoing posterior pharyngeal wall augmentation with dextranomer/hyaluronic acid copolymer implants. However, the imaging appearance of dextranomer/hyaluronic acid copolymer implants in patients treated for vesicoureteral reflux has been previously described. Similar to our study, hyaluronic acid implants have been shown to have a CT and radiographic attenuation comparable with that of soft tissue. 18,19 Some studies have demonstrated that these implants can calcify after 24 months. 18,19 In our study, no evidence of calcification of these implants was seen, but no patients received follow-up radiography or CT after 24 months. On MR imaging, these implants have been shown to be hyperintense on T2-weighted imaging and isointense on T1-weighted imaging. No contrast enhancement is seen. 18,20 The diffusion characteristics of these implants have not been previously characterized.
Limitations
There are a number of limitations to our study. Because this was a retrospective study, the use of the various imaging modalities could not be standardized. There is a wide range of follow-up times for postoperative imaging, which limits our ability to determine any temporal changes to the imaging appearance of hyaluronic acid implants. Furthermore, no patients who received dextranomer/hyaluronic acid copolymer implants for VPI have yet been followed beyond 2 years, the period beyond which calcifications have been reported in the urologic literature.
CONCLUSIONS
The normal postoperative findings of posterior nasopharyngeal dextranomer/hyaluronic acid copolymer injection are characterized by the presence of bilateral nasopharyngeal soft-tissue masses that are isointense to muscle on T1 and hyperintense on T2, with no restricted diffusion or peripheral enhancement on MR imaging and are iso-to hypoattenuated with adjacent muscle on CT. Familiarity with this appearance is critical in distinguishing the normal postoperative state from nasopharyngeal pathology. | 2017-06-22T07:21:56.011Z | 2015-06-01T00:00:00.000 | {
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234850264 | pes2o/s2orc | v3-fos-license | Multi-Scale Evaluation on Two Locations and Digital Fruit Imaging Highlight Morpho-Agronomic Performances and Antioxidant Properties in Chilli Pepper Hybrids
: Chilli pepper is a vegetable crop widely consumed mostly as fresh food or dried as a spice. The nutritional contribution due to the presence of beneficial healthy-related compounds and the versatility of uses have increased its cultivation over the last decade. In Southern European coun-tries chilli production uses established cultivars and/or landraces that are well adapted to specific environments but do not often meet the requirements of the industry, particularly for packaging and processing. In this study, 10 commercial hybrids were evaluated in two diverse environment sites for their productivity and the content of phytochemicals including, carotenoids, capsaicinoids, ascorbic acid and tocopherols. Fruits were assessed using automated tools for the analysis of size, shape and colour parameters. The pepper materials were promising in terms of productivity, whereas a lower level of capsaicinoids and ascorbic acid were detected. Genotype by environment analysis indicated minimal environmental influence on yield, fruit shape, and capsaicinoids. The integration of different sources of phenomics data demonstrates how breeding activities of hybrids have focused on yield and morphology rather than quality linked to phytochemicals content. cultivars [12] cultivars, of assessment the same
Introduction
Chilli pepper is among the most fascinating and consumed spice foods, largely appreciated for its high nutritional and health contribution to human diets. Today, the crop is cultivated worldwide covering a surface of about four million hectares and a production over 40 million tonnes [1].
Chilli pepper cultivars are highly variable regarding morphological characteristics and levels of bioactive compounds, especially capsaicinoids which contribute to their typical flavour and pungent taste that are unique of this species. These characteristics make chilli fruits a multidisciplinary item, as food in fresh, dried or in paste form (e.g., sauce, cream) or for industrial use, such as colouring additive or agent for cosmetics [2]. The broad diversity of chilli pepper includes both cultivated (Capsicum annuum) and other domesticated species, that includes accessions such as the habanero (C. chinense), tabasco (C. frutescens), rocoto (C. pubescens) and aji types (C. baccatum). Most of these are widely grown in the Caribbean, South America, and various Asian areas, where a combination of culinary and cultural factors has favoured consumption of very spicy types [3]. The cultivated C. annuum spicy types, on the other hand, have a more global market and are the most consumed in the Europe where mild spicy varieties are preferred according to the needs of consumers [4]. Europe is the second major importer of chillies after Asia and is also an exporter of dried and packaged products. Considering the increasing trend of spicy foods, it is necessary to develop and cultivate new cultivars suitable to meet both producers and consumer expected needs [4].
To date, diverse local varieties selected by farmers are grown especially in small to medium sized farms. These materials although well adapted to specific habitats, could still hold some intrinsic genetic variability since they are mostly open-pollinated varieties with off-types from high genetic diversity. These constraints are not desired for packaging and processing where the production chain requirements are stricter [5,6]. In previous works, we characterized panels of chilli genotypes including open-pollinated cultivars and landraces for their agronomic performance and nutritional characters, as well as for the effect of genotype by environment interaction on traits [6,7]. The growing interest of seed companies in the development of new chilli cultivars has led to the release of improved uniform varieties with high yield, such as F 1 hybrids.
This research aims to broaden the information on these types of varieties, in order to: (a) understand the characteristics on which selection has been mostly focused, (b) determine whether improved cultivars are more stable than the landraces by estimating the G × E effect, (c) investigate the possible existing gap between the performance of improved and unimproved cultivars, and d) define further objectives to be pursued in chilli breeding.
Thus, ten outstanding hybrids (representing cherry, horn, and jalapeno morphotypes) were grown at two environmentally different locations. Phenotyping was carried out for 18 agronomic and biochemical traits, as well as for over 40 morphometric and colour fruit parameters. Multi-trait analysis provides novel knowledge for the exploitation of chilli cultivars in further breeding programs.
Plant Material and Field Trials
Plant material consisted of ten commercial hybrids of cultivated pepper selected for fresh and dried consumption. Hybrids included cherry ('Bomber' and 'Topik') and jalapeno ('Jalapride' and 'Newpark') morpho-types with round and ovoid shape, respectively, whereas the remaining selection had a horn shape ('Anastar', 'Eris', 'Haruba', 'PH11421', 'Vulcan', 'Zigano'). Seeds were obtained from various companies (Sativa, Esasem, United Genetics). Plants were grown at two locations: Battipaglia (BP) in the Sele Valley of Campania Region (40 • 37 N; 14 • 58 E, 65 m a.s.l.) and Montanaso Lombardo (ML) in the Po Valley of Lombardia Region) (45 • 20 N; 9 • 26 E, 80 m a.s.l). The two sites are located at over 800 km of distance apart and are characterized by very different pedoclimatic conditions (Table 1). Cultivation conditions have been performed equally in the two considered locations. Seeds were sown in April and seedlings transplanted in May in a randomized block experimental design with three replicates. Ten plants were grown for each genotype in each experimental unit at a density of 2.7 plants/m 2 . Fruits were harvested at commercial ripening, from August to October, according to the different varieties' characteristics. Fields were managed according to standard agronomic practices. For plant fertilization, 120 kg ha −1 of N, 100 kg ha −1 of P 2 O 5 and 80 kg ha −1 of K 2 O were applied. Based on crop evapotranspiration, plants were irrigated throughout the entire cultivation period using a drip irrigation system. Effective control of pests and diseases was pursued with the aim of having healthy plants until the end of the cycle. Data from each variety were grouped and presented as the average of the three typologies 'cherry', 'jalapeno' and 'horn' shape.
Morpho-Agronomic Characterizations
Agronomic traits scored at both locations included total yield (grams) [TY] of fully ripe fruits assessed as: the total weight of fruits taken from each plant at full ripening stage; average fruit weight (FW) (in grams) obtained by dividing the total yield by the number of fruits harvested; fruit length (FL) and fruit width (FD) (in centimeters) measured by using manual calliper on ten fruits; and fruit shape index (FS) as length/width ratio.
Chemical and Biochemical Characterization
Chemical and biochemical assessment was conducted on fruits from each replication in both fields. The plant material for the analyses was established after selecting fresh peppers without apparent defects. Peduncles were removed from selected fruits and then cut along the longitudinal axes according to common practices and carefully dried until constant weight in a forced-air oven at 45 • C for 48 h. The dried material was powdered by a waring blender (Waring Commercial, Stamford, CT, USA) at 4 • C and stored in dark bottles at −20 • C until analyses. Chemical traits were measured using a supernatant solution obtained after suspending 2 g of powder in 25 mL of deionized water, subsequent stirring (15 min), and decantation. The total soluble solid content (SSC), expressed in • Brix on 100 g on dried weight ( • Bx dw), was measured using a Multi-Scale refractometer RFM 91 (Bellingham-Stanley Ltd., Kent, UK). The pH and the titratable acidity (AC), expressed as mEq% dw, were determined using a titroprocessor mod 682 equipped with a Dosimat 665 apparatus (Metrohm, Herisau, Switzerland). Biochemical traits measurements included: (a) total carotenoids (TC) and their red (CR) and yellow (CY) fraction; (b) ascorbic acid (AsA); (c) capsaicin (CAPS), dihydro-capsaicin (DHC), nordihydro-capsaicin (NDHC); (d) Scoville units (SHU); and (e) gamma-tocopherol (γtoc), alpha-tocopherol (α-toc). Carotenoids were measured using spectrophotometric methodology, whereas the remaining traits were analysed by High Performance Liquid Chromatography (HPLC). Details of analytical protocols used can be found in previous works [6,7].
Digital Fruit Analysis
A representative bulk of fifteen ripe fruits for each accession and from the BP site were cut longitudinally and scanned with CanoScan Lide 200 (Canon, Italy) at 300 dpi resolution in a dark room using a black background in order to avoid any bias due to light. Resulting images were analysed using the software Tomato Analyzer 3.0 (TA) [8]. Thirty-eight quantitative fruit size and shape traits were evaluated. Fruit color was assessed by handheld colorimeter (Minolta Chroma Meter CR-210; Minolta Corp., Osaka, Japan) to obtain CIELab (L*, a*, b*) coordinates along with Chroma [(a*) 2 + (b*) 2 ] 0.5 and Hue angle (arctan b*/a*) [9].
Data Analysis
All traits were subjected to a two-way ANOVA to analyse the main effects of genotype (G), environment (E), and their interaction (G × E) by using JMP v7.0 software package (SAS Institute, 2007, Cary, NC, USA). Means were compared by using Tukey HSD (honest significant difference) test (p < 0.05). Coefficient of variation (CV) in percentage was expressed as the ratio of the standard deviation to the mean value multiplied by 100. Correlation test was used to compare the distribution of biochemical compounds and morpho-agronomic traits. Pearson analysis was carried out testing at p < 0.01. Correlogram was constructed and visualized using the Corrplot package implemented in R version 3.0.2 (R Development Core Team) [10]. Principal component analysis (PCA) was performed using the computer package XLSTAT 2012.1.
Phenotypic Variability
A variable level of diversity was found for the assessed traits. Considering the three typologies studied, differences (p < 0.001) were found for 10 out of 18 agronomic and biochemical traits ( Table 2). Only pH did not differ among cultivar groups considering the two sites. 4.9 ± 1.7 a 4.77 ± 1.14 a 3.5 ± 1.5 a 5.22 ± 1.25 a 3.51 ± 1.2 b 3.67 ± 1.04 b 5.76 *** § Acronym's description is in the method section. TY is expressed in g per plant; FW is expressed in g per fruit; FL and FD are expressed in cm; SSC is expressed as • Bx dw; AC is expressed as mEq% dw; TC, CR, CY, CAPS, DHC, NDHC are expressed as mg/kg dw; AsA, γ-toc and α-toc are expressed as mg/100 g dw. # * Indicate significance at p < 0.05, ** indicate significance at p < 0.01, and *** indicate significance at p < 0.001; ns = not significant.
Horn types were higher yielding with greater fruit weight and shape parameters at both sites, with 'Zigano' having outstanding values (Table S1). The evaluated accessions had similar productivity between locations except for 'Jalapride' that had low yield at BP.
Bioactive compounds content differed according to the chilli pepper type. Higher levels of Vitamic C (AsA) were found in horn types at both sites with 'Vulcan' having the greatest amount (Table S1). In comparison, high levels of carotenoids were found in both cherry and horn types ( Table 2). Capsaicinoids and related Scoville scale units were more genotype-dependent rather than based on cultivar group, with high levels of pungency found in both jalapeno types as well in 'PH11421' (Table S1). Cherry types exhibited the highest levels of γ-tocopherols with 'Topik' having high amounts at both locations, whereas α-tocopherols primarily accumulated in horn types with 'PH11421' having the highest amount compared to the other cultivars across both locations (Table S1).
Genotype by Environment Interaction
The results of combined analysis of variance for agronomic and biochemical traits are reported in Table 3. A strong effect of the genotype (G) with high level of significance (p < 0.001) was detected in 16 out of the 18 assessed traits. The main source of variation was due to G, which accounted on average for 70% of total variation, expressed by TSS%. The strongest effect was found for morpho-agronomic traits fruit shape and fruit length exhibiting TSS value over 97%. Among biochemical traits, the highest values were found for the minor components of capsaicinoids (DHC and NDHC) and tocopherols (γ-toc). The environmental (E) factor, as well as the G × E interaction, represented 12% and 18% of the total variation, respectively. Several traits were not influenced by location differences or G × E interaction (Table 3), among them, SSC did not show any effect due to either E or G × E, whereas all capsaicinoids did not differ between environmental locations. Table 3. Analysis of variance and significant levels for the genotypic (G) and environmental effects due to grow sites (E) for ten chilli pepper cultivars, and the combined effects (G × E) for the traits evaluated.
Digital Fruit Analysis
The digital assessment involved the measurement of morphometric parameters by fruit scans and automatic inspection of surface colour through CIELab coordinates. For each accession, a total of 30 image sections were analysed for 38 size and shape parameters. The ANOVA detected highly significant differences (p < 0.001) for all size traits, whereas, among shape attributes, five parameters showed lower (Proximal Indentation Area and H. Asymmetry.ob) or null (Proximal Angle Micro, Proximal and Distal Eccentricity) significance (Table 4). 04 § significant at p < 0.05, ** significant at p < 0.01, *** significant at p < 0.001, and ns: not significant.
The largest variation was found for fruit size and shape traits, and well as Lobedness Degree and Circular Homogeneity, the latter explaining most of the variance with the highest F-ratio. Fruit size-related traits were highly variable between accessions, having a relatively high F-ratio, except for Maximum width and Height Mid-width ( Table S2 reports the results of tomato analyzer descriptors for each accession. The cultivar 'Anastar' had the highest average values for the fruit size traits apart from Width Mid-height and Maximum Width which were higher in 'Bomber' and 'Zigano', respectively. Additionally, 'Topik' showed the lowest average value for these traits except for Width Mid-height and Maximum Width which was lower in 'Eris' and 'Newpark', respectively. CIELab coordinates (Table 5) (Table S2) with a mean of 33.26. Jalapeno types had the lower mean value for the CIELab colour coordinates, resulting in fruits with less intense red colour. The highest values were instead found in 'Eris', a horn-shape type. Among colour coordinates, b* was the variable that exhibited a CV higher than L* and a*. On the contrary, L* and Hue had the lower coefficient of variability indicating low level of variation among genotypes. Overall, fruits were almost stable in terms of colour parameters in all genotypes as indicated by reported standard deviation values (Table S2).
Trait Correlation and Multivariate Analysis
For each growing site, correlograms between pairs of variables were generated considering a p threshold of 0.01 ( Figure 1). As expected, high correlations mainly occurred among the same trait's categories. Specifically, fruit weight (FW) and soluble solids (SSC) were positively correlated to carotenoids and negatively correlated to capsaicinoids in both BP and ML (Figure 1a,b), showing significative positive values only at BP. At both locations, negative correlations were found between carotenoids and capsaicinoids (maximum value-0.49 for CY vs. DHC in Figure 1a, and-0.53 for CY vs. NDHC in Figure 1b) with tighter values at BP (Figure 1a). Negative significant correlations were evidenced for morphological traits (coefficient of −0.76 for FS and FD at both sites) and for fruit length and γ-toc (values of-0.53 and-0.50 for BP and ML, respectively), whereas positive correlations were found for α-toc and capsaicinoids (0.40 vs. DHC in BP and 0.41 vs. SHU in ML). Considering the single growing sites, a larger number of significant correlations were found at BP than ML (81 vs. 60), although the trend was similar at the two locations. Correlations with opposite signs and different significance between BP and ML were found for FW vs. pH, FS vs. SSC, CR and TC vs. α-toc. Furthermore, divergent correlations were found in the two grown sites for AsA vs. γ-toc as well as for AsA vs. CAPS.
The principal component analysis that considered the entire dataset of 62 traits scored, explained 61.18% of the variation in the first two dimensions (Figure 2). Traits and genotypes were evenly distributed in the two axes of the biplot. Most of the variation (46.03%) was explained by the first PC which separated cherry and jalapeno types from horn types, positioned in the negative and positive part of the axis (PC 1 ), respectively. The PCA highlighted major traits of the studied genotypes which were those related to yield, fruit weight, and size. Horn types were discriminated in positive axis of both PC 1 and PC 2 , whereas, among biochemical traits, the main discriminators were related to SSC and carotenoids both located in positive axis values of PC 1 and PC 2 . Instead, on the opposite side, those related to pungency clearly discriminated the genotypes positioned in the region of negative values of both PC 1 and PC 2 ( Figure 2, Table S3). It is interesting to note that these latter observations are in full accordance with the correlation data presented in Figure 1.
Comparison of Yield and Ascorbic Acid Content with Local Varieties
To determine the potentiality of F 1 hybrids and to investigate any gaps with the current chilli local varieties, we compared our results with previous studies performed on pungent landraces in Mediterranean environments. As a reference, we considered studies assessing typical Italian chilli cultivars [6,7,11], Balkan pungent lines [12], and a set of Spanish pepper landraces [13].
Five landraces having cherry ('Calabrese Ciliegino' and 'Cerasiforme'), jalapeno ('Naso di Cane') and horn ('Piccante di Cayenna' and 'Sigaretta calabrese') shape, were evaluated in diverse trials during seasons 2014-2016 [6,7,11]. Considering the average performance, hybrids produced two to three times higher with reference to the cherry and horn cultivar groups, whereas the jalapeno F 1 types were slightly higher in terms of fruit weight and total yield respect the reference 'Naso di Cane'. The same trend was observed for the Balcan hot varieties that showed substantial lower productivity than commercial hybrids (Figure 3). Although low levels of ascorbic acid were detected in the commercial hybrids, contents in local cultivars were higher and up to 10 times more in some instances.
Comparison of Yield and Ascorbic Acid Content with Local Varieties
To determine the potentiality of F1 hybrids and to investigate any gaps with the current chilli local varieties, we compared our results with previous studies performed on pungent landraces in Mediterranean environments. As a reference, we considered studies assessing typical Italian chilli cultivars [6,7,11], Balkan pungent lines [12], and a set of Spanish pepper landraces [13].
Five landraces having cherry ('Calabrese Ciliegino' and 'Cerasiforme'), jalapeno ('Naso di Cane') and horn ('Piccante di Cayenna' and 'Sigaretta calabrese') shape, were evaluated in diverse trials during seasons 2014-2016 [6,7,11]. Considering the average performance, hybrids produced two to three times higher with reference to the cherry and horn cultivar groups, whereas the jalapeno F1 types were slightly higher in terms of fruit weight and total yield respect the reference 'Naso di Cane'. The same trend was observed for the Balcan hot varieties that showed substantial lower productivity than commercial hybrids (Figure 3). Although low levels of ascorbic acid were detected in the commercial hybrids, contents in local cultivars were higher and up to 10 times more in some instances. [6,7,11] were considered. For the pungent cultivars from the Balkans, only the production data reported in [12] and related to the 2018-2019 seasons were considered. For Spanish cultivars, we considered the average ascorbic acid levels described in Rodríguez-Burruezo et al. [13]. All studies used the same method of assessment reporting values in the same scale.
Discussion
Chilli pepper is one of the most popular spices with multi-consumption uses as fresh food, dried in powder, processed as sauce or cream. This versatility has leads to an increase in the cultivation of types addressed for different market segments. Previous studies in chillies reported the assessment of different germplasm material including local [6,7,11] were considered. For the pungent cultivars from the Balkans, only the production data reported in [12] and related to the 2018-2019 seasons were considered. For Spanish cultivars, we considered the average ascorbic acid levels described in Rodríguez-Burruezo et al. [13]. All studies used the same method of assessment reporting values in the same scale.
Discussion
Chilli pepper is one of the most popular spices with multi-consumption uses as fresh food, dried in powder, processed as sauce or cream. This versatility has leads to an increase in the cultivation of types addressed for different market segments. Previous studies in chillies reported the assessment of different germplasm material including local landraces, breeding lines and established cultivars [6,7,[11][12][13]. The need to standardize the production chain has led to the development of commercial hybrid cultivars aimed to have better performance. Thus, ten hybrids were selected and cultivated at Italian farms with two contrasting environments to assessing a broad range of agronomic and biochemical traits for investigating their performance, as well as variation due to environmental factors. We further refined the characterization of the cultivars based on a detailed assessment of fruit morphological features and colour. This approach was pursued to highlight the level of variability among the pepper genetic materials using established phenotyping tools [14,15].
The accessions differed (p < 0.05) in fruit size and fruit shape, being divided into two primary roundish and horn groups, the former including circular and oval types known as cherry and jalapeno. The traits leading to the selection of fruit morphology characters were identified. In fact, most of the variation was related to fruit size and shape index traits. Less variation among accessions was instead for fruit colour coordinates, which did not allow accessions to form defined groups. Although digital measurements were carried out on fruits from a single location, we can consider the obtained information reliable, considering the low environmental influence on fruit morphological traits obtained across the two environmental locations. In fact, all fruit shape traits showed a strong genotypic effect, with no G × E detected for FD or FS, and only a low significance for FL. Thus, the environment had little influence on manipulating these fruit traits. Considering possible bias in manual measurements due to the curvature of fruits in the longitudinal section (in particular hornshaped types) we implemented a more precise method for shape traits based on fruit scans. The observations based on the fruit scans provided more detailed information allowing the measurement of more traits than those visually recorded and manually collected. This confirms the potentiality of scans for fruit phenotyping in chilli pepper. We found similar trait correlations across sites, although a greater number was found at BP. This may be due to the diverse pedoclimatic conditions of the cultivation sites, and/or the quantitative nature of the traits measured which are affected by environmental changes.
In comparison with data from previous studies [6,7,11,12], we observed an increase in productivity of hybrids with respect to cultivars and/or landraces cultivated in the same field sites. Instead, minor content of ascorbic acid has been observed with respect of what usually reported in local varieties of chillies [6,7,11,13]. Anyhow, the levels of AsA founded are in line with the previous essays on F 1 hybrids [16]. Interestingly, we found a low influence of the environmental changes on ascorbic acid contents in fruits to what was previously observed [6,7]. Although there are not many data of AsA content in chilli hybrids, we suggest our results are due to a possible heterotic effect resulting from the hybrid combination, but also the lesser effect of the environment on low vitamin C genotypes. On the contrary, carotenoids and α-tocopherol were significantly influenced by the environment which agrees with previous studies [6,7,12]. Wall and colleagues [17], although not describing the G × E interaction, report a variable level of carotenoids across two years of evaluation in different chillies. In addition, the total carotenoid content found in the hybrids is lower in respect to those reported in other C. annuum hot cultivars including serrano, cayenne [17], and jalapeño [18].
These findings highlight how the breeding activities for commercial varieties could have focused on high-yielding and morphologically stable genotypes rather than highquality ones. This seems to be confirmed by the observed strong genotypic effect over the environment for agronomic traits. The similar trend observed for the values of capsaicinoids across locations suggests pungency should be added as an additional primary objective in the breeding of chilli pepper for the market toward the development of slightly spicy cultivars. In fact, the SHU levels encountered in this study were 30-50-fold lower than the typical hot pepper such as habanero types (about 300,000 SHU). This reflects the culinary and cultural uses of the Mediterranean diet. The high productivity combined with greater fruit weight certainly represents an advantage in manual harvesting, which is still very common in chillies, with a better final product yield for both fresh and processed market.
Conclusions
This report has shown the potential of chilli hybrids regarding their agronomic and qualitative performance. By deep phenotyping, we determined the characteristics of different types of chilli pepper highlighting how morphology, yield and capsaicinoids are the main drivers of selection toward the development of high yielding and mild-pungent varieties. Instead, additional efforts are needed for the improvement of quality-linked traits. Further studies involving sensory analyses and panel tests could give additional insight into the preference of consumers for the different existing genotypes of chillies.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/agronomy11040805/s1, Table S1: Mean Values, standard deviation, and results of post-hoc Tukey's for morpho-agronomic and biochemical traits in ten chilli cultivars evaluated at Battipaglia (BP) and Montanaso lombardo (ML); Table S2: Mean values, standard deviation, and results of post-hoc Tukey's for Tomato Analyzer attributes and CIELab colour traits in ten chilli cultivars; Table S3: Variable contribution in % for the traits in the first two PC.
Data Availability Statement:
The data presented in this study are available on request from the corresponding author. | 2021-05-21T16:56:35.894Z | 2021-04-19T00:00:00.000 | {
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251272449 | pes2o/s2orc | v3-fos-license | Biologically Derived Gold Nanoparticles and Their Applications
Nanotechnology is a rapidly evolving discipline as it has a wide variety of applications in several fields. They have been synthesized in a variety of ways. Traditional processes such as chemical and physical synthesis have limits, whether in the form of chemical contamination during synthesis operations or in subsequent applications and usage of more energy. Over the last decade, research has focused on establishing easy, nontoxic, clean, cost-effective, and environmentally friendly techniques for nanoparticle production. To achieve this goal, biological synthesis was created to close this gap. Biosynthesis of nanoparticles is a one-step process, and it is ecofriendly in nature. The metabolic activities of biological agents convert dissolved metal ions into nanometals. For biosynthesis of metal nanoparticles, various biological agents like plants, fungus, and bacteria are utilized. In this review paper, the aim is to provide a summary of contemporary research on the biosynthesis of gold nanoparticles and their applications in various domains have been discussed.
Introduction
Nanotechnology is an evolving area due to its wider range of applications in a variety of disciplines [1,2]. Optics, electronics, catalysis, biomedicine, magnetics, mechanics, and energy research are some of the fields where nanotechnology is applied [3]. Nanobiotechnology is a collaborative area that entails technology research and development in a variety of domains such as nanotechnology, biotechnology, chemistry, material science, and physics [4]. It is about the biofabrication of nano-objects or bifunctional macromolecules that may be utilized to produce or modify nano-objects [5]. Nanoparticles are metallic units that come in a variety of forms, including spherical, triangular, and rod shaped [6]. Nanoparticles have distinct features (chemical, physical, optical, and so on) as compared to bulk material [7]. Currently, research on nanoparticles production is one of the hot topics.
One of most well-defined noble metals is gold. It is utilized in automobiles as a heat insulator and as a reflective coating on some high-end CDs [8]. Gold nanoparticles (GNPs) are being researched for usage in ultrasensitive chemicals, optoelectronic devices, or biological sensors, or as catalysts [9]. Among all types of nanomaterials, metallic nanoparticles are the most promising because of their excellent antibacterial characteristics due to their huge surface area to volume ratio. Researchers are interested in the antibacterial effect of metallic nanoparticles because of the rising microbial resistance to antibiotics or the creation of resistant strains. Silver, platinum, gold, titanium, iron, palladium, aluminum, and copper [10] are some of the metallic nanoparticles that have received a lot of attention recently owing to their critical value. Gold has been used in medicine in various forms throughout the history of civilization. Rheumatic illnesses, such as discoid lupus erythematosus and restorative dentistry, and different skin inflammation conditions, such as urticaria, pemphigus, and psoriasis, have been treated with gold and gold compounds [11].
Biological agents such as plant tissues, bacteria, fungi, actinomycetes, and other molecules have been used for synthesis of gold nanoparticles. e extracellular synthesis of gold nanoparticles has attracted a lot of interest because it avoids many stages of the synthesis process. In general, there are two techniques for nanoparticle synthesis, that is, a 'topbottom' and a 'bottom-top' strategy. Nanoparticles could be synthesized by chemical (chemical reduction) or biological (uses of plant, microbes, etc.) processes through self-assembly of atom new nuclei that develop into nanoscale particles in the bottom-top approach [12]; however, in the top-bottom method, appropriate bulk materials are reduced into small particles using different lithographic processes. Physical and chemical processes for nanoparticle synthesis are not environmentally friendly due to the usage of toxic substances that pose a variety of biological dangers and are costly [13]. is review provides a summary of contemporary research on biosynthesis of gold nanoparticles and their applications in various domains.
General Chemistries of Gold
ere are six possible oxidation states for gold, ranging between −1 and +5, due to its comparatively high electronegativity. Auric (Au (I)) or auric (Au (III)) are two of the main oxidation states for gold complexes [14]. To dissolve gold in aqueous solution, the oxidation and complexation processes work together. Au (I) or Au (III) could form a stable complex in the presence of complex ligands, or, in solution, these could be reduced to metals of gold. Stabilities of gold's complex is governed not just by complex ligand's property, but also by the donor atom of ligands which is directly attached to gold atoms [15]. e first rule, according to research, is that stability of gold's complex reduces as the electronegativity of the donor atom rises. In solution, the stability of the gold halide complex, for example, follows the I-> Br-> Cl-> F patterns [16]. e second rule is that Au (III) is preferred to Au (I) in harsh ligands, whereas Au (I) is preferred to Au (III) in gentle ligands (III). Preferred coordination numbers of Au (I) are 2, which results in a linear complex, whereas Au (III) has a preferred coordination number of 4, which results in a square planar complex. Two precursor uses in the production of GNPs are the gold (III) chloride complexes or the gold thiosulfate (I). In most GNP biosynthesis techniques, the gold (III) chloride complexes are extensively employed as precursors.
Green Synthesis of Gold Nanoparticles
One of the basic and technical concerns is the production of nanoscale gold within the regulated phase or shapes. Michael Faraday described production of gold colloids, now known as GNPs, nearly 150 years ago using phosphorous to decrease AuCl4 ions. A variety of biological, physical, or chemical methods have been explored in the past years in order to create GNPs for usage in electrical, biotechnological, industrial, pharmaceutical, agricultural, or medical sectors [17]. ese methods are used to manufacture gold nanostructures with well-defined compositions, such as colloids, clusters, wires, powders, tubes, rods, and thin films [18]. Physical and chemical approaches to make GNPs have been used in the past, as shown in Figure 1.
ese approaches have yielded GNPs within size ranging from 1 to 100 nm or varieties of morphology. ese synthesis processes have certain limitations despite their considerable research, such as the use of harsh chemicals, rigorous synthesis conditions, energy or capital demands [19], or lower productivities [20]. Currently, mix-shaped nanoparticles (NPs), produced by synthetic methods, need highcost, low-yield purification processes such as differential centrifugation [6]. Furthermore, these processes create more sludge and pose environmental risks due to harmful solvents or additives. As a result, there is a growing need to create clean, nontoxic, ecologically friendly, and long-term synthesis methods. A key issue is the development of high-yield, low-cost NPs production technologies. Because of their wide range of applications, researchers in nanoparticles synthesis had turned to a biological system.
Biosynthesis has been shown to be a viable method for producing tiny particles on a wide scale [21] (Figure 2). It is worth noting that biologically produced NPs have higher stability [23] and better morphological control. Biological systems that create NPs include bacteria, fungus, actinomycetes, and plants [24,25]. Microbes create NPs intracellularly and/or extracellularly due to their inherent potential [26]. However, due to the further processing procedures, such as ultrasonication or treatments with appropriate detergents, extracting NP generated via intracellular biosynthesis is often challenging [27]. As a result, bacteria that produce NP extracellularly must be thoroughly screened [28]. Microorganisms as potential biofactories for GNP production is a promising new field of study. Additionally, it can easily be scaled up for larger-scale production and is economical, time-saving, and ecologically friendly [29]. Next sections go through the various microbial synthesis techniques for GNPs in further depth.
Bacteria.
Prokaryotes have gained a lot of interest in the field of GNP synthesis among microorganisms. For the first time, bacterial generation of GNP in Bacillus subtilis 168 was described, indicating the presence of 10-35 nm octahedral NPs in the cell wall [30]. Rhodopseudomonas capsulata generated spherical GNPGNP within diameters of 10-20 nm at a lower concentration [31] or nanowires within networks at high concentrations [32]. In a study, GNP synthesis has been reported in six cyanobacteria which include Plectonema sp., Calothrix sp., Anabaena sp., and Leptolyngbya sp. GNP [33]. Govindaraju et al. [34] reported synthesis of GNPs from single celled protein, that is, Spirulina platensis GNP. Table 1 summarizes the synthesis of bacterial GNP.
Ahmad et al. [44] demonstrate microbial generation of monodispersed GNPs from an extremophilic ermomonas sp. A study reported that after 48 hours of incubation with aqueous chloroauric acid (HAuCl 4 ) solution at pH ranges of 4.0-7.0, bacterium Rhodopseudomonas capsulata produces spherical GNPs in 15-25 nm ranges [31]. Furthermore, pH of the solution is an important factor that influences types of biogenic AuNPs or location of gold deposition in the cell. Due to metal ion reduction by enzyme present in cell walls or on the cytoplasmic membrane but not in the cytosols, alkalotolerant Rhodococcus sp. formed more intracellular monodispersed GNPs on the cytoplasmic membrane than on the cell walls. Pseudomonas aeruginosa cell supernatant was used for the reduction of gold ions and extracellular production of GNPs [36]. Heterotrophic sulphate-reducing bacteria were employed in the bacterial cell membrane to decrease gold (I) thiosulfate complexes Au (S 2 O 3 ) 2 to elementals golds of 10 nm sizes, resulting in H2S as a metabolic end product [48]. E. coli DH5 biologically reduces chloroauric acid to Au0, leading to the synthesis of nanoparticles on the cell surface, which were mostly spherical but also included some triangles or quasihexagons. is cell-bounded nanoparticles might be useful in hemoglobin or protein electrochemistry [49]. Rhodobacter capsulatus, a photosynthetic bacterium with a larger biosorption capacities for HAuCl 4 , have also been found to bioreduce trivalent aurum. Fungi are believed to be more advantageous for GNP synthesis than other bacteria because fungal-mycelial meshes, unlike bacteria, could withstand flow pressure, agitations, or other bioreactor conditions. ey are easy to culture and manage. ey produce more reductive protein extracellular secretions and are more easily processed downstream [51]. Fungus Trichothecium sp. was reported to produce GNPs both extracellularly and intracellularly [52]. Under stationary conditions, gold ion interacting within Trichothecium sp. fungal biomass results in rapid extracellular formations of GNPs with spherical rod-like and triangular shapes, whereas in case of shaking condition it resulted in intracellular formation of the GNPs. A study reported that whenever gold ions are exposed to extremophilic actinomycete ermomonospora sp., it reduces metal ions extracellularly [53]. Table 2 provides an overview of fungal derived GNP production.
Plant.
One of the most significant methods for biosynthesis of nanoparticles was the use of plant extracts (Figure 3). In a study, Azadirachta indica leaf extract showed bioreduction of Au 3+ or Ag + ion [69]. Aloe vera leaf extract was used to make gold nanotriangle and spherical silver nanoparticles [70]. Some of the ecological benefits of processing plants or their extracts in producing GNPs include uses of nontoxic biocomponents to cap or reduce GNP, limiting waste generation, eliminating the need for further purification methods or ease of availability. Flavonoids, phytosterols, quinones, and other plant biocomponents contribute to the formation of GNPs because they include functional groups that aid in the reduction and stability of GNPs. To create specified shapes and sizes of GNPs, technique requires the combination of gold salt with plant extracts for a certain period of time under various reaction variables such as pH, incubation duration, and temperature.
Song et al. [71] reported GNPs synthesis from leaf extract of two plants, that is, Magnolia kobus and Diospyros kaki. GNPs were synthesied by using a plant extract mixed with an aqueous HAuCl4 solution. At a reaction temperature of 95°C, more than 90% of the GNPs were recovered in just a few minutes. Emblica officinalis fruit extract was also used as a reducing agent in extracellular synthesis of extremely stables Ag nanoparticles [72]. In a study, Cinnamomum camphora leaf extract was used to make gold nanoparticles [73]. Further information on the plants that have been used for synthesis of GNPs has been provided in Table 3.
Algae.
Algae is one of the potential biological agents which can be utilized for the synthesis of different types of nanoparticles. ere is a current interest in the study of algal mediated synthesis of metal nanoparticles, with a focus on the evaluation of the effect of reaction conditions, such as pH, temperature, and stirring rate, upon the final nanoparticles with respect to size, morphology, stability, and so forth [102]. A study employed algal system to explore procedure of gold's reduction by Chlorella vulgaris biomass from gold (III) chloride solution [103]. XAS data show that Au (III) was significantly reduced to Au (I), also Au (I) is coordinated along sulfur atom from free-sulfhydryl residue or lighter-atoms elements, most likely nitrogen. Another study reported that elemental gold was largely precipitated on cell wall of Sargassum natans biomass [104]. According to a study, hydroxyl groups of saccharide or carboxylates anions of amino acid residues from peptidoglycans layers on cell walls seemed to be the gold binding site [105]. A marine alga Sargassum wightii was also used for production of GNPs [106]. After 12 hours of reaction, stable GNPs in the size range of 8-12 nm was produced through reducing aq. AuCl4-ions within extracts of marine alga, with 95% of golds recovered. Some other algae which are used for the synthesis of gold nanoparticles include Acanthophora spicifera, Kappaphycus alvarezii, Chlorella pyrenoidosa, Sargassum myriocystum, Stoechospermum marginatum, Sargassum wightii, and Laminaria japonica [107]. Arockiya Aarthi Rajathi et al. [108] reported synthesis of gold nanoparticles using Stoechospermum marginatum and the synthesized nanoparticles were 18.7-93.7 nm in size. Another study reported synthesis of gold nanoparticles using Tetraselmis kochinensis with 5-35 nm in size [109]. Abdel-Raouf et al. [110] reported synthesis of gold nanoparticles using Galaxaura elongata with the size range of 3.85-77.13 nm. Sargassum cymosum synthesized gold nanoparticles were reported by Costa et al. [111] with the size range of 7-20 nm.
Biomolecules.
Biomolecules are molecules that are produced by living organisms to help the body's biological processes. Some of the biomolecules include amino acids, nucleic acids, carbohydrates, and lipids. Carbonyl and hydroxyl groups of biomolecules convert Au 3+ ion to Au 0 atom. After that, Au 0 is capped, yielding stable GNPs. e biosafety of the reactants employed in the manufacture of GNP's may be addressed using this technique. Table 4 depicts the various biomolecule-mediated GNP production methods. List of different biomolecules which were utilized for gold nanoparticles synthesis has been reported in Table 4.
Advantages of Biologically Synthesized Gold Nanoparticles
Biogenic gold nanoparticles are free from hazardous byproducts which are generally found in case of chemical synthesis, that resulted in minimising usage in different applications [128]. To be used in biomedical applications, gold nanoparticles must be biocompatible. Biological production of gold nanoparticles had various advantages, including its simplicity, one-step nature, environmental friendliness, cost effectiveness, and biocompatibility [129]. Furthermore, no external stabilising agents are required since biogenic components of plants and microorganisms serve as stabilising or capping agents. Biosynthesis of gold nanoparticles requires less time than chemical one. Another advantage of biological synthesis is that it could decrease numbers of chemical synthesis steps required, such as adding functional groups to surfaces of gold nanoparticles to make them physiologically active [129].
Applications of GNPs
e productions of inorganic or metal-based nanomaterials had encouraged establishment of newer industry which brings together experts from several sectors to hunt for new type of nanoparticles having distinct property. Developing or designing creative or cost-effective processes for scaling up the nanomaterial manufacturing has not only given an intriguing topic of research but will also address future human needs such as health, safety, and environmental concerns. Nanomaterials are rapidly being used in industry, also these would soon replace hazardous or toxic chemical usage. e utilization of nanoparticles or nanocomposites is relatively safer option, opening up new areas for antibacterial research. Different ancient cultures (India, China, or Egypt) employed gold to heal diseases like smallpox, syphilis, skin ulcers, or measles [130].
Medical Application.
Gold nanoparticles are versatile materials with several uses in a wide range of industries. Gold particles were coated with DNA and inserted into plant embryos or plant cells by researchers. is ensures that some genetic material enters and transforms the cells. is technique improves plant plastids. Because GNPs may be detected using a number of methods, including optical absorptions fluorescent or electrical conductivities, they had been mainly used in biosensor labelling and bioimaging applications [131]. GNPs were focused and amplified in regions of interests, giving contrasts for observations or visualisations. Light energy causes free electrons in GNPs to form collective oscillations k/a, a surface plasmon, which has the property of considerably absorbing and scattering visible light. e excited electron plasma thermally relaxes by the transfer of energies to gold lattices, causing GNPs to heat up as a result of light absorption. e interaction of GNPs with light can assist in optical microscopy, fluorescent microscopy, photothermal imaging, or photoacoustic imaging. In addition, transmission-electrons microscopy [132] may be used to investigate interactions of GNPs with electrons wave or X-ray. Gold nanoparticles have long been used to carry therapeutic compounds into cells [133]. Before being administered to cells by gene guns or particle ingestion, the chemicals are adsorbed on the surface of GNPs.
Anticancer Activity.
GNPs are used to treat cancer because of their biocompatibility. GNPs might be utilized to treat epithelial ovarian cancer. ey have the capacity to suppress the evolution of ovarian cancer and metastasis [134]. Growth factors VEGF (vascular-endothelial growth factors) are involved in the development of ovarian cancer and tumour growth. GNPs have also been demonstrated to inhibit activity of VEGF, which promotes cell proliferation, in multiple myeloma (MM), a plasma cell cancer. As a result of VEGF inhibition, cell-cycle inhibitor proteins such as p21 or p27, that limit proliferations, are upregulated [135]. Chronic lymphocytic leukaemia (CLL) is a kind of leukaemia characterised by an excess of lymphocytes that originate in bone marrow but could spread to different organs. GNPs have been found to inhibit the action of factor produced by CLL cells or to promote apoptosis [136] because they have the potential to impair the function of heparinbased growth factors.
Tumour Detection.
Newly developed functionalized GNPs (dendrimers) have been developed to target and destroy tumours and combat cancer [137]. GNPs are intended not only to recognise, target, and destroy tumours, but also to transport an additional chemical that can delay or kill cancerous cells. Dendrimers function as arm for GNPs, allowing other molecules to be attached to the arms. Laser and infrared light heat gold's particle, prompting dendrimer in releasing chemicals that kill tumours. e Mie equations suggest that the surface plasmon resonance scattering of GNPs will increase as the nanoparticle size grows. By conjugating GNP to anti-EGFR antibodies, using stronger scattering images of GNP coupled to antibodies which just adhere to cancerous cells and not to noncancerous cells, researchers were able to distinguish between cancerous and noncancerous cell [138]. A basic optical microscope is used to view the scattering. ey obtain 500% greatest bindings ratios to sick cells compared to nonmalignant cells, allowing cancerous cells to be spotted using a dark field microscope to examine scattered light. Because of their higher X-ray's absorption coefficient, simplicity of synthetics modification, nontoxicity, surfaces functionalities for colloidal stabilities, targets distribution, GNPs had received most attention as an X-ray's contrasting agent. Low-molecular-weight vascular contrasting age agents, like iodinates compounds, are common. ese iodinated aromatics have a high-water solubility, indicating minimal toxicity. However, the period of blood circulation is brief, and waste is quickly removed by the kidneys. As a result, a limited imaging windows might necessitate numerous injections, increasing risks of thyroidgland dysfunctions.
Antibacterial Activity.
Gold nanoparticles are able to inhibit bacterial growth by conferring themselves onto the bacterial cell surface due to their surface changes. Alteration of surface releases reactive oxygen species which causes protein denaturation, DNA destruction, and mitochondrial disfunction and finally leads to cell death [139]. A study reported synthesis of gold nanoparticles using Mentha piperita and evaluated its antibacterial effect against E. coli and S. aureus and found that gold nanoparticles showed antibacterial activity against E. coli only [140]. Another study reported synthesis of gold nanoparticles using Commelina nudiflora and found that it was effective against Salmonella typhi and Enterococcus faecalis [141]. Abdel-Raouf et al. [110] reported synthesis of gold nanoparticles using Galaxaura elongate and evaluated its antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa and MRSA.
Removal of Pollutants.
GNP-based technologies are being developed now for pollution control and water purification in the environment. Bimetallic gold-palladium nanoparticles have been found to be a potent catalyst for degrading trichloroethene (TCE), which is one of the primary contaminants in groundwater, into a nontoxic form [142]. GNPs in water purifications system have been shown to effectively gather and eliminate halocarbon-based pollutants from drinking water [143] and improve mercury oxidation from coal-fired power plants [144].
Ornamental Applications.
GNPs were developed to selectively oxidize biomass-derived chemicals such as furfurals or hydroxymethyl furfurals to produce methyl-esters, carbon monoxide (CO), or trimethylamine. ese chemicals are used in polymers and industrial solvents, as well as in flavour and fragrance applications [145]. A range of gases, including carbon monoxide (CO) and nitrogen oxides, have been detected using Au nanoparticle-based gas sensors (NOx) [146].
Removal of Inorganic
Compounds. Green GNPs are widely recognised for their catalytic activity, particularly their catalytic reduction abilities. Although gold is not commonly used as a catalyst, gold nanoparticles have been reported to decrease ferrocyanide (III), nitroarenes, cyanosilylation of aldehydes, and deoxygenation of epoxides into alkenes. For example, p-nitrophenols, which are common by-products of the production of herbicides, pesticides, and synthetic dyes and are known to be environmentally poisonous and inhibitory in nature, have been successfully reduced to p-amino phenols by green synthesized GNPs, which would otherwise be incapable of being converted to its neutral and nontoxic form even by the strongest reducing agent [147][148][149].
Future Perspective
Nanotechnology is a rapidly expanding area with several applications in various fields. Gold nanoparticles are synthesized using a variety of processes because of their vast range of uses. Traditional chemical procedures have limitations, either in the form of chemical contamination during the synthesis process or in future applications. Chemical reduction of gold uses a variety of chemicals (reducing agents) that are usually hazardous and difficult to dispose of owing to environmental concerns. Synthesis is also carried out at higher temperatures in a variety of different situations, which generate a lot of heat and are highly expensive. Biological synthesis of gold nanoparticles has sparked considerable attention because it is a quick, ecofriendly, nonpathogenic, and cost-effective method that can be completed in a single step at room temperature and pressure. Compared to standard physical and chemical techniques, biological synthesis is an environmentally friendly approach that uses a diverse variety of resources such as plants, bacteria, actinomycetes, yeast, and fungi. e biosynthesis of gold nanoparticles is still in its early stages of investigation. ere are a few issues that must be addressed. Several studies are still required to better understand the impacts of time, temperature, light, and other elements on the formation of gold nanoparticles, as well as the control of the nanoparticle size and shape. Furthermore, researchers face challenges due to a lack of knowledge of the chemical components and mechanisms involved in the reduction and stability of biosynthesized gold nanoparticles. As a consequence, more studies are recommended on the mechanism of gold nanoparticle synthesis and its influence on the shape and size of gold nanoparticles for various applications.
Conclusion
Biological approach for nanoparticle synthesis is an important alternative in the development of clean, nontoxic, cost-effective, and environmentally friendly technologies for the synthesis of GNPs, with substantial advantages over previous approaches. Many biological agents have the capacity to synthesise GNPs both inside and outside the cell. Research into the production of GNPs is still in its early stages. For widespread usage of GNPs in commercial applications, more research is required on biosynthesis processes and with well-defined size and shape. e capacity to vary the features of GNPs simply by changing their size or shape is intriguing, and it will be employed in unique applications in the future. GNPs are employed in a wide range of applications, including electronics and catalysis, as well as biology, medicine, and medical diagnostics and therapy. However, further research into the mechanics and kinetics of GNP production is required, since this might lead to process optimization, eventually leading to GNP synthesis with strict control over size, shape, and large-scale manufacturing.
Data Availability
All data used to support the findings of this study are included within the article.
Conflicts of Interest
e authors declare no conflicts of interest. | 2022-08-03T15:24:43.903Z | 2022-08-01T00:00:00.000 | {
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30123571 | pes2o/s2orc | v3-fos-license | Inhibitory Effect of Propolis on Platelet Aggregation In Vitro
Platelet hyperactivity plays an important role in arterial thrombosis and atherosclerosis. The present study was aimed to investigate the effects of different extracts of propolis and components of flavonoids on platelet aggregation. Platelet-rich plasma was prepared and incubated in vitro with different concentrations of the tested extracts and components of flavonoids. Platelets aggregation was induced by different agonists including adenosine diphosphate (ADP, 10 μM), thrombin receptor activator peptide (TRAP, 50 μM), and collagen (5 μg/mL). At 25 mg/L to 300 mg/mL, the water extract propolis (WEP) inhibited three agonists-induced platelet aggregations in a dose-dependent manner. The flavonoids isolated from the propolis also showed markedly inhibited platelet aggregation induced by collagen, ADP, and TRAP, respectively. The components including caffeic acid phenethyl ester (CAPE), galangin, apigenin, quercetin, kaempferol, ferulic acid, rutin, chrysin, pinostrobin, and pinocembrin and their abilities of inhibiting platelet aggregation were studied. It was concluded that propolis had an antiplatelet action in which flavonoids were mainly implicated.
Introduction
Nowadays, cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide [1][2][3] and bring a huge burden to the world economic development and people's living standard. It has been widely known that platelets play important roles in both hemostasis and pathogenesis of CVD such as acute coronary syndrome [4]. Platelet inhibition has shown improved short-and long-term clinical outcomes for CVD patients. However, increased bleeding risk and the high rates of recurrent ischemic events could not be ignored [5]. Furthermore, the activation of platelets was also related to circulation and vascular damage in patients with hypertension and diabetes [6]. Antiplatelet drugs used clinically to treat and prevent coronary syndromes and stroke are accompanied by a variety of side effects such as thrombocytopenia, hemorrhage, gastric ulcers, and therapeutic resistance [7,8]. More safe and effective antiplatelet drugs would be urgently needed based on the current situation.
In recent, natural products and alternative medicine are now getting significant attention. Propolis, a complex mixture containing various compounds, such as flavonoids, terpenes, β-steroids, aromatic aldehydes, and alcohols, is a plant-derived substance collected from plant materials by honeybees [9,10]. Propolis is extensively used in food and beverages to improve health because of its unique pharmacological activities including antimicrobial, antioxidant, immunomodulatory, hepatoprotective, antitumor, and cardioprotective effects [11][12][13][14][15][16][17][18][19][20][21]. Recently, Liu and his colleagues put forward that chrysin in propolis performs antiplatelet activity via inhibiting platelet alphaIIbbeta3-mediated signaling pathway [22]. In addition, evidence data showed that propolis coated Co-Cr could significantly reduce adhesion of platelets [23]. Many studies demonstrated that the propolis components were varied in different geographic and climatic zones [24]. Thus, the aim of the present study was to examine the effects of propolis extracts on human platelet aggregation in vitro and to identify the nature of the compounds responsible for the antiplatelet activity.
Preparation of Water Extract of Propolis (WEP) and
Flavonoid. According to previous research [25], a certain amount of dried propolis sample frozen at −20°C was dissolved in 30 mL of distilled water at 60°C for 7 h. The crude extract was filtered, then centrifuged at 28000 rpm for 30 min, and the supernatants were concentrated under reduced pressure to produce the WEP. Flavonoid was obtained from another 10.0 g dried propolis dissolved in 300 mL 70% ethanol 7 h at room temperature using the same method [26].
Component Analysis of WEP and Flavonoid.
For the components of WEP and flavonoid, a ZQ-4000 HPLC apparatus (Waters, USA) was used. The chromatographic separation was achieved using a Waters Symmetry C18 column (4.6 mm I.D. × 150 mm, 3.5 μm), with the column oven temperature maintained at 25°C. The mobile phase consisted of acetonitrile and 2% acetic acid. The mobile phase flow rate was 0.2 mL/min, and UV absorbance was monitored at 280 nm. Diluted standard solutions of galangin, CAPE, apigenin, quercetin, kaempferol, ferulic acid, rutin, chrysin, pinostrobin, and pinocembrin were analyzed in the same HPLC conditions, and furthermore, the calibration of the detector response was done. Quantification of the main bioactive compounds from WEP and flavonoid was based on the calibration curves. The results were confirmed by the Department of Agriculture Bee Products Quality Supervision and Inspection Center (Beijing).
The Platelet Aggregation Tests.
Human platelet suspensions were prepared as previously described [27]. In this study, human volunteers had been given informed consent. In brief, blood was collected from healthy human volunteers who had taken no medicine during the preceding 2 weeks and was mixed with 3.4% Na-citrate (9 : 1, vol/vol). The blood was centrifugated at 1100 rpm for 15 min at 20°C. Then, platelet-rich plasma (PRP) was collected with pipette and stored at room temperature. The tubes containing PRP were centrifuged at 3000 rpm for 10 min at 20°C to obtain the platelet-pool plasma (PPP). After a platelet count (Sysmex SE-9000, Kobe, Japan), a standard concentration of 200 × 109/L was obtained by diluting PRP with PPP.
The turbidimetric method was applied to measure platelet aggregation stimulated by the various kinds of agonists using an aggregometer [28]. Previous study mentioned different flavonoid components present in propolis (CAPE, galangin, acacetin, and pinostrobin) were able to inhibit platelet aggregation, so CAPE, galangin, acacetin, and pinostrobin were tested in our study [29]. All aggregation tests were performed within 2 h after isolation and carried out in triplicate. Each person had his/her own 0% PRP and 100% PPP aggregation calibration. Before aggregation was started, standard platelet concentration was incubated in solutions of different concen-
Statistical Analysis.
The results were shown as mean ± standard deviations, and each experiment was performed in triplicate. Statistical analysis was carried out using SPSS19.0, and differences were considered to be significant at a level of P < 0 05.
Results and Discussion
3.1. Chemical Components of Propolis. Flavonoids are the major constituents of propolis and contribute greatly to the pharmacological activities of propolis. Usually, the quality of propolis was based on flavonoids quality [30]. The contents of flavonoids depend on the harvest region because the characteristics of propolis are influenced by the local plant varieties and weather [31]. Report of component analysis of WEP and flavonoids was shown in Table 1. The contents of CAPE were the highest in the WEP, followed by galangin, ferulic acid, quercetin, kaempferol, and apigenin. Pinostrobin was the most abundant chemical compound in flavonoids, followed by galangin, chrysin, CAPE, rutin, kaempferol, pinocembrin, apigenin, quercetin, and ferulic acid. Figures 1 and 2.
The results showed that the crude WEP inhibited platelet aggregation in a dose-dependent manner, which might be stimulated with different agonists including ADP, collagen, and TRAP. For instance, 300 mg/L WEP significantly decreased platelet aggregation induced by ADP, collagen, and TRAP to 35.00 ± 5.34%, 35.00 ± 6.61%, and 42.33 ± 13.24% (n = 3; P < 0 01), respectively, indicating that propolis contains compounds having antiplatelet aggregation activity. This effect might be attributed to polar compounds such as flavonoids and polyphenols. Flavonoids inhibited the three agonist-induced platelet aggregations in a dose-dependent manner, and the percent of inhibition were 53.11 ± 1.90%, 51.12 ± 9.30%, and 51.98 ± 22.20% at concentrations of 400 mg/L, respectively. This finding was consistent with previous studies showing the antiplatelet aggregation activity of flavonoids in vitro [32] and in vivo [33]. We also test four purified components, including CAPE, galangin, acacetin, and pinocembrin. All test dose CAPE added to the PRP platelet aggregation induced by collagen and TRAP significantly decreased (P < 0 05), and the most antiplatelet aggregation effect occurred in the collagen-included platelet aggregation test group with an inhibition of 58.82 ± 4.51% at 176 μM. Significant inhibition was not detected in the ADP-included platelet aggregation test group when adding 6 μM CAPE.
When 46 μM or more galangin was added to PRP, significant inhibition was detected in the collagen-included platelet aggregation test group in a dose-dependent manner. Galangin was not able to inhibit ADP-induced platelet aggregation until 370 μM galangin had been added. Acacetin could significantly inhibit platelet aggregation induced by ADP, collagen, and TRAP at concentrations of 44, 176, and 352 μM in the concentration-dependent manner (P < 0 05). The antiplatelet aggregation activity of pinostrobin isolated from propolis (23 μM, 92 μM, and 370 μM) was evaluated upon ADP-, collagen-, and TRAP-induced aggregation. As shown in Figures 1(f) and 2(f), the platelet aggregation could be suppressed by pinostrobin at concentrations of 23-370 μM (P < 0 005). CAPE appeared to be more effective inhibitors than galangin and acacetin. This difference in potency could be explained by their chemical structures [34]. The isolated flavonoids markedly inhibited platelet aggregation induced by ADP, TRAP, and collagen. The inhibition might be introduced by holding back fibrinogen binding to its platelet membrane receptor (glycoprotein (GP) IIb-IIIa), which is a final and common pathway of platelet aggregation. It was reported that the flavonoid phloretin diminished ADP and TRAP-stimulated expression of the activated form of the GPIIb/IIIa complex and reduced platelet aggregation stimulated by ADP [30]. Several other studies had shown that flavonoids inhibited platelet function through a multitude of mechanisms including decreasing phospholipase C activity [31], scavenging of reactive oxygen species such as superoxide anion, and inhibiting cyclic nucleotide phosphodiesterase and thromboxane A2 synthesis [32].
Conclusion
The main components of propolis were galangin, CAPE, apigenin, quercetin, kaempferol, ferulic acid, rutin, chrysin, pinostrobin, and pinocembrin. The WEP and flavonoids extracted from propolis exhibited dose-dependent inhibitory effects on platelet aggregation induced by different agonists including ADP, collagen, and TRAP, respectively. CAPE, galangin, and pinostrobin might be mainly implicated. Further studies are necessary to clarify the mechanism of platelet inhibition.
Conflicts of Interest
The authors indicated no potential competing interests. | 2018-04-03T01:34:58.303Z | 2017-10-10T00:00:00.000 | {
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52902121 | pes2o/s2orc | v3-fos-license | Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo
The chemotherapeutic agent temozolomide (TMZ) kills tumor cells preferentially via alkylation of the O6-position of guanine. However, cells that express the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), or harbor deficient DNA mismatch repair (MMR) function, are profoundly resistant to this drug. TMZ is in clinical use for melanoma, but objective response rates are low, even when TMZ is combined with O6-benzylguanine (O6BG), a potent MGMT inhibitor. We used in vitro and in vivo models of melanoma to characterize the early events leading to cellular TMZ resistance. Melanoma cell lines were exposed to a single treatment with TMZ, at physiologically relevant concentrations, in the absence or presence of O6BG. Surviving clones and mass cultures were analyzed by Western blot, colony formation assays, and DNA methylation studies. Mice with melanoma xenografts received TMZ treatment, and tumor tissue was analyzed by immunohistochemistry. We found that MGMT-negative melanoma cell cultures, before any drug treatment, already harbored a small fraction of MGMT-positive cells, which survived TMZ treatment and promptly became the dominant cell type within the surviving population. The MGMT-negative status in individual cells was not stable, as clonal selection of MGMT-negative cells again resulted in a mixed population harboring MGMT-positive, TMZ-resistant cells. Blocking the survival advantage of MGMT via the addition of O6BG still resulted in surviving clones, although at much lower frequency and independent of MGMT, and the resistance mechanism of these clones was based on a common lack of expression of MSH6, a key MMR enzyme. TMZ treatment of mice implanted with MGMT-negative melanoma cells resulted in effective tumor growth delay, but eventually tumor growth resumed, with tumor tissue having become MGMT positive. Altogether, these data reveal stochastic expression of MGMT as a pre-existing, key determinant of TMZ resistance in melanoma cell lines. Although MGMT activity can effectively be eliminated by pharmacologic intervention with O6BG, additional layers of TMZ resistance, although considerably rarer, are present as well and minimize the cytotoxic impact of TMZ/O6BG combination treatment. Our results provide rational explanations regarding clinical observations, where the TMZ/O6BG regimen has yielded mostly disappointing outcomes in melanoma patients.
recovery. After reaching near-confluency in the 10-cm plate, cells were harvested, lysed, and analyzed by Western blot.
Treatment with 5-Azacytidine
A2058 cells were repeatedly exposed to 50 µM 5-AzaC for 48-72 h. After each treatment, the drug-laced medium was removed and the cells were allowed to recover in drug-free medium. After approximately one week of recovery, the cell culture was transferred onto 2 plates. Cells on one of these plates were harvested for WB analysis, whereas cells on the other plate received the next dose of 5-AzaC. This cycle was repeated 4 times, thus generating cell lysates from cells that had received 1, 2, 3 or 4 rounds of drug treatment.
Immunoblots
Total cell lysates were analyzed by Western blot analysis as described earlier [41]. We used the following primary antibodies. For the detection of MGMT, we used a polyclonal antibody (#2739) from Cell Signaling Technology (Beverly, MA, USA) and a monoclonal antibody (ab39253) from Abcam (Cambridge, MA, USA). Anti-MSH2 (#2017) and anti-MSH6 (#5424) antibodies were from Cell Signaling. Human TRA-1-85 antibody (MAB3195) was from R&D Systems (Minneapolis, MN, USA) and beta-actin antibody (sc-47778) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-antibody conjugates (i.e., secondary antibodies) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). All antibodies were used according to the suppliers' recommendations. All immunoblots were repeated at least once to confirm the results. Band intensities of MGMT were quantified using ImageJ, normalized relative to the respective actin control bands, and expressed in relation to the strongest reference band.
MethyLight Analysis
MGMT promoter methylation status was analyzed by standard MethyLight assay [42], using genomic DNA extracted from A2058 cells and derived clones. The procedure was performed by the USC Pathology Core lab. The primer/probe combination to determine methylated MGMT promoter sequences was the same as the one that is commonly used to analyze MGMT methylation status in glioblastoma patients [43].
Animal Model
All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of USC on 27th July 2016, #20038, and all rules and regulations were followed during experimentation on animals. Athymic mice (Harlan, Inc., Indianapolis, IN, USA) were implanted with 4 × 10 6 cells into the right flank. About two weeks later, once palpable tumors had developed, animals were assigned to different treatment groups. The control group received vehicle only (45% glycerol, 45% ethanol, 10% DMSO), whereas the treatment group received TMZ dissolved in vehicle. Administration was by gavage.
Tissue Analysis
Upon euthanasia of animals, tumor tissues were collected and stored frozen or fixed in formalin. For the detection of MGMT protein by immunohistochemistry, tumor tissues were processed as described earlier [44]. The primary antibody was mouse monoclonal anti-human MGMT from Abcam (cat #ab39253). The secondary antibody was biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA), followed by incubation with the Vectastain Elite avidin-biotin-peroxidase complex kit (Vector Laboratories). Positive staining was visualized with amino-ethylcarbazole substrate (red). Hematoxylin was used as the counterstain to mark nuclei (blue).
Statistical Analysis
All parametric data were analyzed using the Student t-test to calculate the significance values. A probability value (p) < 0.05 was considered statistically significant.
Sensitivity to TMZ Correlates with MGMT Protein Levels
For our study, we chose two MGMT-positive (A375 and CaCl 74-36) and two MGMT-negative lines (A2058 and M24) ( Figure 1A). As expected, MGMT expression levels in these lines were closely aligned with sensitivity to TMZ, when tested in colony formation assays (CFAs): the IC50 (concentration of drug that reduces colony formation by 50%) was in the range of 10-20 µM for MGMT-negative cells, and well above 100 µM for MGMT-positive cells. Exemplary results are shown in Figure 1B (and similar results were obtained for CaCl 74-36 and M24). To put these data in perspective: achievable peak plasma concentrations of TMZ in patients are 40-50 µM [45,46]. Thus, IC50 values that are greater than 100 µM, as observed for our MGMT-positive cells, clearly indicate profound TMZ resistance. The molecular basis for this resistance could be validated with O6BG, because inclusion of this MGMT inhibitor exquisitely sensitized MGMT-positive A375 cells to TMZ, but had no effect in MGMT-negative A2058 cells ( Figure 1B). Essentially identical results were obtained with CaCl 74-36 and M24 cells (not included in the figure).
Statistical Analysis
All parametric data were analyzed using the Student t-test to calculate the significance values. A probability value (p) < 0.05 was considered statistically significant.
Sensitivity to TMZ Correlates with MGMT Protein Levels
For our study, we chose two MGMT-positive (A375 and CaCl 74-36) and two MGMT-negative lines (A2058 and M24) ( Figure 1A). As expected, MGMT expression levels in these lines were closely aligned with sensitivity to TMZ, when tested in colony formation assays (CFAs): the IC50 (concentration of drug that reduces colony formation by 50%) was in the range of 10-20 µM for MGMT-negative cells, and well above 100 µM for MGMT-positive cells. Exemplary results are shown in Figure 1B (and similar results were obtained for CaCl 74-36 and M24). To put these data in perspective: achievable peak plasma concentrations of TMZ in patients are 40-50 µM [45,46]. Thus, IC50 values that are greater than 100 µM, as observed for our MGMT-positive cells, clearly indicate profound TMZ resistance. The molecular basis for this resistance could be validated with O6BG, because inclusion of this MGMT inhibitor exquisitely sensitized MGMT-positive A375 cells to TMZ, but had no effect in MGMT-negative A2058 cells ( Figure 1B). Essentially identical results were obtained with CaCl 74-36 and M24 cells (not included in the figure).
MGMT-Negative, but Not MGMT-Positive, Cell Populations Adjust to TMZ Treatment
It is generally observed that increasing drug concentrations lead to correspondingly decreasing numbers of emerging colonies in CFAs. However, it is oftentimes unclear why some cells withstand much higher drug concentrations and continue to proliferate. We therefore selected cells that had survived a single round of high-dose drug treatment and investigated the potential basis for their survival. This was done both with MGMT-positive and with MGMT-negative cells.
In the case of MGMT-positive A375 cells, the IC50 of TMZ treatment was about 300 µM, yet even at higher concentrations, there were small numbers of survivors. We therefore treated these cells with a single dose of 700 µM TMZ, representing IC99.9. From about 100,000 treated cells, 100 colonies emerged, and 12 individual clones were isolated for further analysis (Figure 2A). We performed CFAs for all 12 of these clones and determined that their IC50s were in a fairly narrow range around 300 µM, i.e., there was no significant change as compared to the non-drug treated parental A375 cells ( Figure 2B). Also, when MGMT protein levels were analyzed, no difference was observed between parental cells and individual clones ( Figure 2C). Several of these clones were subjected to treatment with TMZ in the presence of O6BG, which caused sensitization to TMZ, confirming that their resistance mechanism was based on MGMT, similar to the parental cells (see Figure 1B). Thus, even though these clones represented the 0.1% fraction of A375 cells able to survive high-dose (700 µM)
MGMT-Negative, but Not MGMT-Positive, Cell Populations Adjust to TMZ Treatment
It is generally observed that increasing drug concentrations lead to correspondingly decreasing numbers of emerging colonies in CFAs. However, it is oftentimes unclear why some cells withstand much higher drug concentrations and continue to proliferate. We therefore selected cells that had survived a single round of high-dose drug treatment and investigated the potential basis for their survival. This was done both with MGMT-positive and with MGMT-negative cells.
In the case of MGMT-positive A375 cells, the IC50 of TMZ treatment was about 300 µM, yet even at higher concentrations, there were small numbers of survivors. We therefore treated these cells with a single dose of 700 µM TMZ, representing IC99.9. From about 100,000 treated cells, 100 colonies emerged, and 12 individual clones were isolated for further analysis (Figure 2A). We performed CFAs for all 12 of these clones and determined that their IC50s were in a fairly narrow range around 300 µM, i.e., there was no significant change as compared to the non-drug treated parental A375 cells ( Figure 2B). Also, when MGMT protein levels were analyzed, no difference was observed between parental cells and individual clones ( Figure 2C). Several of these clones were subjected to treatment with TMZ in the presence of O6BG, which caused sensitization to TMZ, confirming that their resistance mechanism was based on MGMT, similar to the parental cells (see Figure 1B). Thus, even though these clones A similar approach was performed with MGMT-negative A2058 cells. Ten thousand cells were treated with 200 µM TMZ, which killed about 98%. From the emerging survivors, 12 individual clones were isolated ( Figure 3A). CFAs were performed for half of these clones, revealing significantly increased IC50s as compared to the IC50 of parental A2058 cells ( Figure 3B). Intriguingly, the elevated IC50s of these clones were around 300 µM, and thus very similar to the IC50 of MGMT-positive A375 cells (and other MGMT-positive cells). Western blot analysis of 11 of these clones revealed the prominent appearance of MGMT protein in all of them ( Figure 3C). We also compared expression levels of MSH6 and MSH2, two proteins of the MMR pathway, and detected highly variable expression of these two proteins. To differentiate the potential contribution of MGMT, MSH6, and MSH2 to TMZ resistance in these clones, we performed CFAs in the presence of O6BG in clones 21, 23, 28, and 30. In these cases, inclusion of O6BG restored full sensitivity to TMZ, i.e., the IC50 was reduced from about 300 µM to ~25 µM, which is the IC50 of the MGMT-negative A2058 parental population. We therefore concluded that rapid emergence of drug resistance after single TMZ treatment was based on the presence of MGMT in these clones.
It is noteworthy that several clones, in particular clones 28 and 31, displayed prominent upregulation of MSH2 and MSH6 expression. This was interesting because increased expression of MSH6 has recently been shown to be associated with TMZ resistance [47]. However, in the case of Clone 28, our above analysis did not support a critical role for MSH6, because the addition of O6BG A similar approach was performed with MGMT-negative A2058 cells. Ten thousand cells were treated with 200 µM TMZ, which killed about 98%. From the emerging survivors, 12 individual clones were isolated ( Figure 3A). CFAs were performed for half of these clones, revealing significantly increased IC50s as compared to the IC50 of parental A2058 cells ( Figure 3B). Intriguingly, the elevated IC50s of these clones were around 300 µM, and thus very similar to the IC50 of MGMT-positive A375 cells (and other MGMT-positive cells). Western blot analysis of 11 of these clones revealed the prominent appearance of MGMT protein in all of them ( Figure 3C). We also compared expression levels of MSH6 and MSH2, two proteins of the MMR pathway, and detected highly variable expression of these two proteins. To differentiate the potential contribution of MGMT, MSH6, and MSH2 to TMZ resistance in these clones, we performed CFAs in the presence of O6BG in clones 21, 23, 28, and 30. In these cases, inclusion of O6BG restored full sensitivity to TMZ, i.e., the IC50 was reduced from about 300 µM to~25 µM, which is the IC50 of the MGMT-negative A2058 parental population. We therefore concluded that rapid emergence of drug resistance after single TMZ treatment was based on the presence of MGMT in these clones.
It is noteworthy that several clones, in particular clones 28 and 31, displayed prominent upregulation of MSH2 and MSH6 expression. This was interesting because increased expression of MSH6 has recently been shown to be associated with TMZ resistance [47]. However, in the case of Clone 28, our above analysis did not support a critical role for MSH6, because the addition of O6BG (i.e., inhibition of MGMT) restored full sensitivity to TMZ, assigning the drug-resistance mechanism in this clone entirely to MGMT. Nonetheless, since we did not investigate all of the other clones, there remains a possibility that alterations in MSH2 or MSH6 might contribute to the development of TMZ resistance as well.
Cancers 2018, 10, x 7 of 23 (i.e., inhibition of MGMT) restored full sensitivity to TMZ, assigning the drug-resistance mechanism in this clone entirely to MGMT. Nonetheless, since we did not investigate all of the other clones, there remains a possibility that alterations in MSH2 or MSH6 might contribute to the development of TMZ resistance as well.
TMZ Resistance Emerges after Single Drug Treatment Even at Low Concentration
To complement our analysis of individual surviving clones, we next studied mass cultures of surviving cells after single TMZ treatment. MGMT-negative A2058 or M24 cells were exposed to TMZ at increasing concentrations from 10 to 500 µM ( Figure 4A). After this one-time drug exposure, cells were allowed to recover. Surviving cells received fresh medium on a regular basis, until the entire population reached confluency and could be harvested for Western blot analysis. This process took longer for cell populations exposed to higher TMZ dosages, because of more extensive cell death following drug treatment. As shown in Figure 4B,C, all drug-treated cell populations had converted to MGMT-positive status. While 10 µM TMZ exerted weak effects, cells treated with only 25 µM TMZ displayed pronounced MGMT positivity that was only slightly further increased at much higher
TMZ Resistance Emerges after Single Drug Treatment Even at Low Concentration
To complement our analysis of individual surviving clones, we next studied mass cultures of surviving cells after single TMZ treatment. MGMT-negative A2058 or M24 cells were exposed to TMZ at increasing concentrations from 10 to 500 µM ( Figure 4A). After this one-time drug exposure, cells were allowed to recover. Surviving cells received fresh medium on a regular basis, until the entire population reached confluency and could be harvested for Western blot analysis. This process took longer for cell populations exposed to higher TMZ dosages, because of more extensive cell death following drug treatment. As shown in Figure 4B,C, all drug-treated cell populations had converted to MGMT-positive status. While 10 µM TMZ exerted weak effects, cells treated with only 25 µM TMZ displayed pronounced MGMT positivity that was only slightly further increased at much higher TMZ concentrations. Together, these results demonstrate rapid emergence of MGMT-positive cell populations following a single TMZ treatment, including at low, physiologically relevant concentrations.
MGMT-Positive Cells Exist within an MGMT-Negative Population
We hypothesized that rapid appearance of MGMT positivity might be due to the presence of a small number of MGMT-positive cells among the otherwise MGMT-negative population of cells. To investigate this model, we randomly isolated 12 clones of individual A2058 cells in the absence of any drug treatment ( Figure 5A). Western blot analysis of these cells demonstrated prominent expression of MGMT protein in one of these 12 clones (7 of the 12 clones are shown in Figure 5B). We then performed CFA with the one positive clone (Clone 43) in comparison to one of the other negative clones (Clone 41). As shown in Figure 5C, Clone 43 was highly TMZ-resistant, and inclusion of O6BG restored its drug sensitivity. In comparison, Clone 41 was TMZ sensitive (similar to the parental A2058 cell line), and inclusion of O6BG did not result in an obvious difference. Thus, this approach indicated the presence of MGMT-positive, TMZ-resistant cells among the greater MGMT-negative overall population, even before any drug treatment was applied.
MGMT-Positive Cells Exist within an MGMT-Negative Population
We hypothesized that rapid appearance of MGMT positivity might be due to the presence of a small number of MGMT-positive cells among the otherwise MGMT-negative population of cells. To investigate this model, we randomly isolated 12 clones of individual A2058 cells in the absence of any drug treatment ( Figure 5A). Western blot analysis of these cells demonstrated prominent expression of MGMT protein in one of these 12 clones (7 of the 12 clones are shown in Figure 5B). We then performed CFA with the one positive clone (Clone 43) in comparison to one of the other negative clones (Clone 41). As shown in Figure 5C, Clone 43 was highly TMZ-resistant, and inclusion of O6BG restored its drug sensitivity. In comparison, Clone 41 was TMZ sensitive (similar to the parental A2058 cell line), and inclusion of O6BG did not result in an obvious difference. Thus, this approach indicated the presence of MGMT-positive, TMZ-resistant cells among the greater MGMT-negative overall population, even before any drug treatment was applied. We next investigated the issue of stability of a given (MGMT-negative) phenotype of individual cells within the entire population, i.e., do MGMT-negative cells maintain their negative phenotype, or is there potential conversion from negative to positive status, which of course would greatly bolster the development of TMZ resistance. To address this question, we selected three of the abovedescribed MGMT-negative clones (41, 42 and 46) and exposed each one to a single dose of TMZ at different concentrations. Cells were allowed to recover and proliferate until sufficient cells were available for harvest ( Figure 6A). Western blot analysis revealed that these TMZ-treated cell populations had become MGMT positive ( Figure 6B), clearly demonstrating that MGMT positivity can rapidly emerge from a cell population derived from an initially MGMT-negative clone. We next investigated the issue of stability of a given (MGMT-negative) phenotype of individual cells within the entire population, i.e., do MGMT-negative cells maintain their negative phenotype, or is there potential conversion from negative to positive status, which of course would greatly bolster the development of TMZ resistance. To address this question, we selected three of the above-described MGMT-negative clones (41, 42 and 46) and exposed each one to a single dose of TMZ at different concentrations. Cells were allowed to recover and proliferate until sufficient cells were available for harvest ( Figure 6A). Western blot analysis revealed that these TMZ-treated cell populations had become MGMT positive ( Figure 6B), clearly demonstrating that MGMT positivity can rapidly emerge from a cell population derived from an initially MGMT-negative clone. Cancers 2018, 10, x 10 of 23
MGMT Expression Is Not Regulated by Promoter Methylation
To gain insight into the mechanism by which these melanoma cells might switch their MGMT status from negative to positive, we investigated control of promoter activity by methylation. It is well recognized that cells are able to silence MGMT expression through methylation of their MGMT promoter [18]. In fact, in the case of glioblastoma (GBM) patients, standard of care generally involves analysis of MGMT promoter methylation status in resected tumor tissue as the key predictive factor of TMZ efficacy [48]. We used MGMT-negative A2058 and Clone 41 cells, and MGMT-positive Clone 28 and Clone 43 cells, extracted DNA, and analyzed MGMT promoter methylation by MethyLight assay. However, no promoter methylation could be detected in any of the four samples ( Figure 7A), indicating that differential MGMT expression in these cells was not due to differences in promoter methylation. As this finding was somewhat unexpected, it was validated by treating A2058 cells with 5-aza-deoxycytidine (5AzadC). 5AzadC is an epigenetic modifier that can be used to remove methyl groups from DNA, resulting in re-activation of promoters silenced by methylation [49]. We exposed A2058 cells to several rounds of treatment with 5AzadC, followed by Western blot analysis of MGMT expression. As shown in Figure 7B, there was no emergence of MGMT protein expression in response to extensive 5AzadC treatment. Combined, these results exclude promoter methylation as a decisive factor of differential MGMT expression in the analyzed cell lines.
MGMT Expression Is Not Regulated by Promoter Methylation
To gain insight into the mechanism by which these melanoma cells might switch their MGMT status from negative to positive, we investigated control of promoter activity by methylation. It is well recognized that cells are able to silence MGMT expression through methylation of their MGMT promoter [18]. In fact, in the case of glioblastoma (GBM) patients, standard of care generally involves analysis of MGMT promoter methylation status in resected tumor tissue as the key predictive factor of TMZ efficacy [48]. We used MGMT-negative A2058 and Clone 41 cells, and MGMT-positive Clone 28 and Clone 43 cells, extracted DNA, and analyzed MGMT promoter methylation by MethyLight assay. However, no promoter methylation could be detected in any of the four samples ( Figure 7A), indicating that differential MGMT expression in these cells was not due to differences in promoter methylation. As this finding was somewhat unexpected, it was validated by treating A2058 cells with 5-aza-deoxycytidine (5AzadC). 5AzadC is an epigenetic modifier that can be used to remove methyl groups from DNA, resulting in re-activation of promoters silenced by methylation [49]. We exposed A2058 cells to several rounds of treatment with 5AzadC, followed by Western blot analysis of MGMT expression. As shown in Figure 7B, there was no emergence of MGMT protein expression in response to extensive 5AzadC treatment. Combined, these results exclude promoter methylation as a decisive factor of differential MGMT expression in the analyzed cell lines.
Other Resistance Mechanisms Are Present, but Are Considerably Rarer
While the above results characterized MGMT as a prominent contributor to TMZ resistance of these melanoma cell lines, we next asked whether other mechanisms of resistance were present as well. To investigate this aspect, we used O6BG to block any MGMT activity, thus allowing potential other elements of resistance to emerge. However, when we seeded 100,000 A2058 cells and treated them with 100 µM TMZ in the presence of O6BG, we obtained no survivors-as compared to more than a thousand surviving colonies after treatment with TMZ in the absence of O6BG (see also Figure 3). This outcome indicated that resistance based on MGMT was by far the most prevalent mechanism of resistance in these cells, and any other mechanism, if present, would have a frequency below 10 −5 . It therefore appeared necessary to investigate a larger number of cells. We seeded 3 × 10 6 A2058 cells, followed by a one-time treatment with 100 µM TMZ in the continuous presence of O6BG ( Figure 8A). After 6 weeks, a single colony of slow-growing cells emerged (Clone 61), which was expanded for further characterization. Western blot analysis revealed that these cells did not express MGMT protein, similar to the parental A2058 culture. However, unlike the A2058 culture, Clone 61 had lost expression of MSH6 ( Figure 8B), and loss of this key MMR protein provided a reasonable explanation for their MGMT-independent resistance to TMZ.
Clone 61 was also used in CFAs with increasing concentrations of TMZ in the presence or absence of O6BG. As expected, the cells were highly resistant to TMZ, and inclusion of O6BG made no difference ( Figure 8C), confirming that MGMT did not contribute to these cells' resistance phenotype. Furthermore, similar to the treatment approach presented in Figure 4, we seeded 100,000 Clone 61 cells per well and treated these populations with increasing concentrations of TMZ, followed by Western blot analysis after cells had recovered from drug treatment. Unlike the results obtained with parental A2058 cells (Figure 4) and other selected clones (Figure 6), TMZ treatment of Clone 61 did not result in the emergence of MGMT-positive populations ( Figure 8D). On one hand, this outcome suggested that loss of MSH6 was sufficient for drug resistance, and MGMT was not required. On the other hand, in view of the very rapid emergence of MGMT positivity after treatment of A2058 or M24 cells with TMZ (Figure 4), it also diminished the possibility that TMZ in general
Other Resistance Mechanisms Are Present, but Are Considerably Rarer
While the above results characterized MGMT as a prominent contributor to TMZ resistance of these melanoma cell lines, we next asked whether other mechanisms of resistance were present as well. To investigate this aspect, we used O6BG to block any MGMT activity, thus allowing potential other elements of resistance to emerge. However, when we seeded 100,000 A2058 cells and treated them with 100 µM TMZ in the presence of O6BG, we obtained no survivors-as compared to more than a thousand surviving colonies after treatment with TMZ in the absence of O6BG (see also Figure 3). This outcome indicated that resistance based on MGMT was by far the most prevalent mechanism of resistance in these cells, and any other mechanism, if present, would have a frequency below 10 −5 . It therefore appeared necessary to investigate a larger number of cells. We seeded 3 × 10 6 A2058 cells, followed by a one-time treatment with 100 µM TMZ in the continuous presence of O6BG ( Figure 8A). After 6 weeks, a single colony of slow-growing cells emerged (Clone 61), which was expanded for further characterization. Western blot analysis revealed that these cells did not express MGMT protein, similar to the parental A2058 culture. However, unlike the A2058 culture, Clone 61 had lost expression of MSH6 ( Figure 8B), and loss of this key MMR protein provided a reasonable explanation for their MGMT-independent resistance to TMZ.
Clone 61 was also used in CFAs with increasing concentrations of TMZ in the presence or absence of O6BG. As expected, the cells were highly resistant to TMZ, and inclusion of O6BG made no difference ( Figure 8C), confirming that MGMT did not contribute to these cells' resistance phenotype. Furthermore, similar to the treatment approach presented in Figure 4, we seeded 100,000 Clone 61 cells per well and treated these populations with increasing concentrations of TMZ, followed by Western blot analysis after cells had recovered from drug treatment. Unlike the results obtained with parental A2058 cells (Figure 4) and other selected clones (Figure 6), TMZ treatment of Clone 61 did not result in the emergence of MGMT-positive populations ( Figure 8D). On one hand, this outcome suggested that loss of MSH6 was sufficient for drug resistance, and MGMT was not required. On the other hand, in view of the very rapid emergence of MGMT positivity after treatment of A2058 or M24 cells with TMZ (Figure 4), it also diminished the possibility that TMZ in general might trigger stable MGMT expression (as opposed to the selection of pre-existing MGMT-positive cells). Figure 4). After complete recovery, mass cultures were analyzed for MGMT protein levels by WB analysis with actin as the loading control.
Repeated TMZ Treatment Accelerates the Emergence of Resistance
In all of the above experiments, TMZ was applied as a single treatment. In clinical use, however, it is given repeatedly. For instance, during the adjuvant phase after completion of the radiation schedule, it usually is given for 5 consecutive days every 4 weeks [50]. We therefore pursued the question as to whether repeated drug treatments at physiologically relevant concentrations would increase the likelihood of resistance development. Because the pre-existing presence of MGMTpositive cells was expected to quickly dominate the cell population in response to TMZ treatment, we included O6BG in these experiments in order to eliminate protection by MGMT. Three million cells were seeded and treated with 10 µM TMZ in the continuous presence of O6BG once daily for 5 consecutive days. This resulted in about 250 colonies of surviving cells ( Figure 9A).
We isolated 12 of these clones and performed Western blot analysis, which revealed loss of MSH6 in all cases ( Figure 9B). Intriguingly, two of the clones (77 and 79) also were MGMT positive. Figure 4). After complete recovery, mass cultures were analyzed for MGMT protein levels by WB analysis with actin as the loading control.
Repeated TMZ Treatment Accelerates the Emergence of Resistance
In all of the above experiments, TMZ was applied as a single treatment. In clinical use, however, it is given repeatedly. For instance, during the adjuvant phase after completion of the radiation schedule, it usually is given for 5 consecutive days every 4 weeks [50]. We therefore pursued the question as to whether repeated drug treatments at physiologically relevant concentrations would increase the likelihood of resistance development. Because the pre-existing presence of MGMT-positive cells was expected to quickly dominate the cell population in response to TMZ treatment, we included O6BG in these experiments in order to eliminate protection by MGMT. Three million cells were seeded and treated with 10 µM TMZ in the continuous presence of O6BG once daily for 5 consecutive days. This resulted in about 250 colonies of surviving cells ( Figure 9A).
We isolated 12 of these clones and performed Western blot analysis, which revealed loss of MSH6 in all cases ( Figure 9B). Intriguingly, two of the clones (77 and 79) also were MGMT positive. CFAs of several of these clones confirmed that they were highly TMZ-resistant, and inclusion of O6BG had no effect (see representative examples in Figure 9C). It is noteworthy that O6BG also had no effect in those 2 MGMT-positive clones, revealing that their resistance status was not determined by MGMT, but that the presence of MGMT was probably of stochastic origin, as described above. Altogether, these results present loss of MSH6 expression as a protective backup mechanism of tumor cells under conditions of inactivated MGMT. CFAs of several of these clones confirmed that they were highly TMZ-resistant, and inclusion of O6BG had no effect (see representative examples in Figure 9C). It is noteworthy that O6BG also had no effect in those 2 MGMT-positive clones, revealing that their resistance status was not determined by MGMT, but that the presence of MGMT was probably of stochastic origin, as described above. Altogether, these results present loss of MSH6 expression as a protective backup mechanism of tumor cells under conditions of inactivated MGMT.
Emergence of MGMT-Driven TMZ Resistance Occurs In Vivo
Finally, we investigated MGMT expression and its relation to TMZ resistance under in vivo conditions. Mice were implanted with MGMT-positive A375 or MGMT-negative A2058 cells. In the absence of any drug treatment, well-grown tumors were collected after three weeks and subjected to immunohistochemistry with a human-specific MGMT antibody. As shown in Figure 10A, tissue grown from A375 cells stained positive for MGMT, as expected. In comparison, tissue derived from Figure 9. Drug resistance after repeated low-dose TMZ plus O6BG treatment. (A) Treatment schedule was as follows: 3 × 10 6 A2058 cells were seeded into a 25-cm plate and treated with 10 µM TMZ in the presence of O6BG once daily over 5 days. After about 15 days, there were approximately 250 colonies, which were pooled and expanded. After sufficient proliferation, 3 × 10 6 of these cells were seeded again into a 25-cm plate and exposed to a single treatment of 100 µM TMZ in the presence of O6BG. Another 15 days later, approximately 2000 colonies emerged, which were sub-cultured to allow for the isolation of 12 individual clones (numbered 71 through 82), which were analyzed by WB and CFA. (B) Cell lysates were prepared from all clones and analyzed by WB for MGMT and MSH6 protein levels. Lysates from A375 and A2058 cells were included for comparison. (Clones 81 and 82 are not included in this figure; clone 76 was lost.) Numbers under the blot indicate quantification of MGMT bands, with reference to the actin signal, and relative to Clone 79 (set at 1.0); <0.1 indicates below detection limit. (C) To confirm drug sensitivity in correlation with MGMT or MMR deficiency, clones 71 and 79 were treated with increasing concentrations of TMZ in the presence or absence of O6BG, and CFA was performed 12 days later (n = 3, ±SE).
Emergence of MGMT-Driven TMZ Resistance Occurs In Vivo
Finally, we investigated MGMT expression and its relation to TMZ resistance under in vivo conditions. Mice were implanted with MGMT-positive A375 or MGMT-negative A2058 cells. In the absence of any drug treatment, well-grown tumors were collected after three weeks and subjected to immunohistochemistry with a human-specific MGMT antibody. As shown in Figure 10A, tissue grown from A375 cells stained positive for MGMT, as expected. In comparison, tissue derived from A2058 cells appeared negative, although a small number of positive cells could clearly be recognized, consistent with our in vitro finding that the A2058 cell population harbors a minority of MGMT-positive members.
developed, half the mice received daily TMZ treatment for 2 weeks, while the other half received vehicle only. At the end of the treatment period, vehicle-treated animals presented with large tumors, which necessitated euthanasia, along with collection of tumor tissue. At this time point, TMZ-treated animals had only very small tumors, indicating clear therapeutic activity of TMZ. These latter animals were not euthanized, but were kept for another 3 weeks without any further TMZ treatment. During these 3 weeks, tumor growth resumed, and the tissues were collected at the end of this time period. Figure 10B presents Western blot analysis of tumor tissues from this experiment. A1-A4 represent tumor tissues derived from A2058 cells, whereas M1-M3 are from M24 tissues. As shown, tumor tissues from vehicle-treated animals (A1, A2, M1, M2) are MGMT-negative, just like their in vitro-cultured counterparts (shown on the left side of the blot). In contrast, both A2058 tumors from TMZ-treated animals (A3, A4) were strongly MGMT-positive. In comparison, the one tumor obtained from M24 cells in TMZ-treated animals was MGMT-negative. In conclusion, in the case of A2058, we could establish proof-of-principle that TMZ treatment triggered the selection of MGMT-positive cells, which were derived from a pre-existing minority of cells that survived and eventually dominated the tumor mass. , using a human-specific MGMT antibody. A1, A2, M1 and M2 are tumors from mice that only received vehicle treatment (control); A3, A4 and M3 are tumors from mice that received TMZ therapy (M4 animal was lost). To confirm the presence of human tumor cells in tissues harvested from mice-especially in those lanes where the MGMT signal was negative-we used an antibody against TRA-1-85/CD147, an epitope that is present only on human cells, but not mouse cells. Please note that specificity of this antibody was confirmed by lack of reactivity with the lane containing lysate from mouse embryo fibroblasts (MEF). Actin was used as the loading control. Numbers under the blot indicate quantification of MGMT bands, with reference to the actin signal, and relative to A4 sample (set at 1.0); <0.1 indicates below detection limit; only lanes with tumor tissue samples were measured.
We then asked the question whether in vivo treatment with TMZ would trigger the selection and accumulation of MGMT-positive cells, as it did in vitro. To establish proof-of-principle of this process, we implanted A2058 and M24 cells into the flanks of mice. Once palpable tumors had developed, half the mice received daily TMZ treatment for 2 weeks, while the other half received vehicle only. At the end of the treatment period, vehicle-treated animals presented with large tumors, which necessitated euthanasia, along with collection of tumor tissue. At this time point, TMZ-treated animals had only very small tumors, indicating clear therapeutic activity of TMZ. These latter animals were not euthanized, but were kept for another 3 weeks without any further TMZ treatment. During these 3 weeks, tumor growth resumed, and the tissues were collected at the end of this time period. Figure 10B presents Western blot analysis of tumor tissues from this experiment. A1-A4 represent tumor tissues derived from A2058 cells, whereas M1-M3 are from M24 tissues. As shown, tumor tissues from vehicle-treated animals (A1, A2, M1, M2) are MGMT-negative, just like their in vitro-cultured counterparts (shown on the left side of the blot). In contrast, both A2058 tumors from TMZ-treated animals (A3, A4) were strongly MGMT-positive. In comparison, the one tumor obtained from M24 cells in TMZ-treated animals was MGMT-negative. In conclusion, in the case of A2058, we could establish proof-of-principle that TMZ treatment triggered the selection of MGMT-positive cells, which were derived from a pre-existing minority of cells that survived and eventually dominated the tumor mass.
Discussion
Methylation of the O6-position of guanine constitutes the key toxic lesion placed onto the DNA strand by TMZ. In cells expressing MGMT, the DNA repair mechanism of this protein removes this foreign methyl group, thus eliminating the toxic threat and protecting survival of the cells. However, in the absence of MGMT, O6-methylated guanine mispairs with thymidine, triggering futile cycles of attempted DNA repair by the MMR system, resulting in DNA strand breaks and apoptosis. Intriguingly, if the MMR system is defective, as is the case when key MMR proteins are absent, O6-methylguanine: thymidine mispairs do not trigger cell death; rather, the cells survive at the expense of an increased rate of mutations, in particular G:C to A:T transitions. For this reason, MMR-deficient cells, for example those lacking expression of the MMR protein MSH6, are profoundly resistant to TMZ [12,[51][52][53].
The key findings of our study are as follows: (i) two established melanoma cell lines, which appear to lack MGMT expression, in fact harbor a small percentage of MGMT-positive cells. (ii) MGMT expression is stochastic, and isolation of individual, MGMT-negative clones again results in a population with mixed MGMT status. (iii) A one-time exposure to TMZ, at physiologically relevant concentrations, effectively depletes the MGMT-negative population and promptly enriches for MGMT-positive, TMZ-resistant survivors. (iv) The combination of TMZ with O6BG prevents the emergence of MGMT-based TMZ resistance; however, an alternative resistance mechanism, loss of the MMR protein MSH6, appears (although at much lower frequency).
Our study focused on TMZ concentrations that are physiologically relevant. Peak concentrations of TMZ measured in the plasma of melanoma patients are about 50 µM [45,46]. Based on these levels, we considered cells that survived treatment with >50 µM TMZ as unresponsive to TMZ. Intriguingly, while the IC50s of our two MGMT-negative cell lines were in the range of 10-20 µM, thus marking them as TMZ sensitive, we nonetheless obtained a few surviving colonies even at 100 µM TMZ (and above). In the case of A2058, this TMZ-resistant fraction was 1-3%, whereas with M24 cells it was 0.5-1.0%. The addition of O6BG substantially reduced colony survival at these elevated TMZ concentrations, providing an early indication that MGMT was involved in conferring drug resistance (Figure 1).
To further investigate the mechanisms of TMZ resistance in these cells, we applied an in vitro strategy that used only a single, one-time drug exposure. We hypothesized that any survival of cells under such conditions must be due to a pre-existing resistance mechanism, quite likely based on the involvement of MGMT. The veracity of this model was supported by the following findings: (i) When MGMT-negative A2058 or M24 cells were exposed to TMZ, the resulting surviving cell population had become strongly MGMT positive (Figure 4). Because only a single exposure to TMZ had been applied, the resistance mechanism must have been already present at the time of addition of the drug. (ii) When we isolated 12 clones, picked at random from A2058 cells in the absence of any drug treatment, one of them turned out to be strongly MGMT-positive (Clone 43) ( Figure 5), indicating that rare MGMT-positive cells were present among the general MGMT-negative cell population, even before any TMZ treatment was applied. This was further substantiated by an independent experiment, where two of 12 individually isolated clones after treatment of cells with TMZ + O6BG turned out to be strongly MGMT-positive, even though MGMT was not involved in the drug resistance mechanism of these (MSH6-deficient) cells (Figure 9).
On one hand, it makes sense that the presence of even a few MGMT-positive cells among an otherwise MGMT-negative population will result in rapid expansion of this minority fraction upon TMZ treatment. On the other hand, it was unclear whether MGMT status within such a population was stable or fluid. We therefore addressed the question whether MGMT-negative cells among a mixed population would retain their negative status, or whether they would be able to convert and produce progeny with mixed MGMT status. Two experimental results indicated that the latter model applied: (i) when individual A2058 cells were isolated and cloned, the majority of them presented as MGMT-negative in Western blot analysis ( Figure 5); however, upon single treatment with TMZ, all these cell cultures promptly converted to strong MGMT-positive status (Figure 6), in a fashion similar to the parental A2058 population ( Figure 4). (ii) However, extensive TMZ treatment of Clone 61, which represents A2058 cells with TMZ resistance based on MMR deficiency, did not result in increased MGMT expression (Figure 8). This outcome constituted an important control, because it excluded the possibility that mere treatment with TMZ might have been the trigger for increased MGMT expression in MGMT-negative clones. Lack of MGMT stimulation by TMZ is further supported by a study showing that TMZ exposure of melanoma cells in vitro does not alter MGMT mRNA expression [54].
Combined, the above results favor a model where MGMT expression stochastically appears in a small fraction of the melanoma cell population, thus providing the seeds of TMZ resistance even before the commencement of any TMZ treatment. The underlying mechanism of this switch between different MGMT statuses remains to be established, although we excluded differential methylation of the MGMT promoter as an epigenetic control of this event (Figure 7). We focused on promoter methylation, because in glioblastoma this modification has been recognized as a predictive and prognostic factor for therapy with methylating agents, such as TMZ, and determination of MGMT promoter methylation status is standard practice for this tumor type [55,56]. In melanoma, however, the role of this epigenetic modification is less clear. The percentage of tumors with methylated MGMT is lower than in glioblastoma, and clinical studies with melanoma patients concluded that MGMT promoter methylation status did not correlate with clinical outcome after TMZ [57,58]. The results shown in Figure 7 are in agreement with the view that promoter methylation does not appear to play a critical role in melanoma. Our analysis of A2058 cells and derived Clone 41 reveals that their lack of MGMT protein expression ( Figure 5) is not due to silencing of the promoter by methylation (Figure 7); as a corollary, the rare emergence of MGMT-positive cells, such as Clone 43 ( Figure 5), from an otherwise MGMT-negative cell population cannot be caused by promoter demethylation. In any case, irrespective of the underlying control mechanism, our study provides proof of principle of stochastic MGMT expression in vitro, and there are indications that similar events might take place in patient tissue. For example, a few studies have documented intratumoral heterogeneity with regard to MGMT status in patient samples derived from melanoma [59,60], as well as glioblastoma [61].
In the clinical setting, efforts were undertaken to overwhelm the well-recognized MGMT obstacle via addition of MGMT inhibitors to TMZ therapy. However, despite the potent activity of such combinations in pre-clinical models [23,35,36], neither glioblastoma nor melanoma patients experienced significant benefit over TMZ alone [33,34,62,63] and the underlying reasons have remained unclear. Consistent with earlier in vitro studies, we found that TMZ combined with O6BG was hugely potent in all our melanoma cell lines, irrespective of their MGMT status. For example, in MGMT-negative A20158 cells, where a single treatment with 100 µM TMZ killed 98% of the cells, inclusion of O6BG increased efficacy by 10,000-fold to 99.9999% (Figure 8). Similarly, in M24 cells, O6BG increased TMZ efficacy by a factor of >2000. In the face of such enormous enhancements in vitro, it is puzzling why they cannot be translated into the clinic-although our analysis of surviving clones might provide some basis for rational speculation (below).
In comparison to single treatment with 100 µM TMZ + O6BG, where we obtained only 1 surviving clone (Figure 8), we obtained >100-fold more survivors when we applied conditions that were more clinically relevant, namely a 5-day cycle of 10 µM TMZ + O6BG. Overall cell killing was still very high at about 99.99% ( Figure 9). As expected, MGMT did not play a role in TMZ resistance of these survivors; instead, 12 out of 12 analyzed clones revealed prominent down-regulation, if not loss, of MSH6 expression, a key component of the MMR pathway. Loss of function of MSH6 (or of other MMR proteins) has been shown to confer profound resistance to TMZ in a variety of tumor cell lines [52]. In glioblastoma, MSH6 mutations or loss of expression have been associated with tumor progression during TMZ treatment and ensuing TMZ resistance [12,13,64], but the role of MSH6 in clinical melanoma is less well characterized [65].
Contrary to the loss of MSH6, a recent study in glioblastoma found increased expression of MSH6, which was associated with TMZ resistance [47]. The authors proposed that aberrantly increased MSH6 might disrupt fine-tuned cooperation among MMR proteins, resulting in incompetence of DNA repair and tolerance of TMZ-induced lesions. This tentative model is intriguing in view of our findings that several clones of TMZ-resistant A2058 cells displayed increased expression of MSH2 and MSH6 proteins ( Figure 3C). Although among the clones we analyzed, the primary resistance mechanisms was based on MGMT, it is conceivable that in some of the other clones elevated expression of these MMR proteins might contribute to the development of TMZ resistance.
It remains to be determined how MSH6 is upregulated in some cells (Figure 3; reference [47]), but downregulated in others (Figures 8 and 9). It is possible that TMZ-induced mutations may play a role. This might be particularly relevant in response to repeated drug administration, as is the standard approach in the clinic [50] and in many pre-clinical studies aimed at generating drug-resistant variants [8][9][10][11]47]. Also, our MSH6-deficient clones shown in Figure 9 were derived after 5-time TMZ treatment. On the other hand, our MSH6-deficient Clone 61 originated from a single TMZ exposure of the parental cells, and so did the MSH6-overexpressing clones shown in Figure 3, tentatively minimizing a role for mutations, but favoring more immediate drug impact or selection of pre-existing cells with aberrant MSH6 expression. In any case, irrespective of the underlying molecular mechanism, our finding that a single, clinically relevant cycle of TMZ/O6BG results in survivors with a consistent MSH6-deficient phenotype demonstrates the emergence of a secondary resistance mechanism beyond MGMT, and provides a reasonable explanation for why tumor cells might escape TMZ/O6BG treatment in the clinic.
Despite the emergence of secondary resistant cells, treatment with TMZ/O6BG in vitro still achieves 99.99% cell killing, which was observed in different preclinical studies and has provided the rationale to test this drug combination in patients. So why is a corresponding positive effect not observed in the clinic? Naturally, the preclinical conditions are different from the situation in a patient, which complicates direct comparisons and invites reasonable speculations: Our observation that TMZ/O6BG-treated cells rapidly produce an MMR-deficient phenotype is intriguing in the context of other studies showing that exposure to alkylating agents like TMZ can foster a hypermutational phenotype [12][13][14]. This model implies that, in addition to conferring TMZ resistance, impaired MMR leads to many additional mutations that provide a growth advantage and accelerate tumor progression and cross-resistance to additional drugs with different targets [7,[12][13][14]66,67]. While this scenario seems detrimental for the patient, it may open new avenues for therapeutic intervention with checkpoint inhibitors, which appear to have increased efficacy when applied to genetically unstable and hypermutated tumors [68,69]. In fact, pembrolizumab, an antibody inhibitor of programmed cell death 1 (PD-1) receptor on T cells, which initially was approved to treat advanced melanoma, received additional approval for MMR-deficient solid cancers, representing the first time the FDA approved a cancer drug based on tumor genetics rather than tumor site [70,71]. Thus, while TMZ combined with O6BG does not appear to yield impressive long-term results, its short-term benefits, in addition to buying melanoma patients some extra time, could potentially prime the recurrent tumor tissue to greater sensitivity to checkpoint inhibitors, such as pembrolizumab. While this model is speculative, it is testable and should provide impetus for further studies.
Conclusions
Our study reveals rare expression of MGMT as a stochastically pre-existing, key determinant of TMZ resistance in melanoma cell lines ( Figure 11). Upon treatment with TMZ at physiologically relevant concentrations, MGMT-negative cells are eliminated, but the tiny fraction of MGMT-positive cells survives and becomes the dominant, drug-resistant population. Combining TMZ with the MGMT inhibitor O6BG suppresses survival of MGMT-positive cells, but drug-resistant survivors still emerge, due to the presence of even rarer cells that harbor defective MMR (i.e., impaired MSH6 expression) that conveys TMZ resistance. These findings might explain, at least in part, why TMZ-based treatment regimens, with or without added O6BG, do not achieve impressive clinical outcomes in melanoma patients.
Of note, MMR-deficient cells, which escape TMZ + O6BG combination treatment, are prone to develop a hypermutation phenotype, which results in increased display of neoantigens as potential targets for the immune system. It is thus conceivable that melanoma patients recurring after TMZ + O6BG might respond favorably to interventions that administer immune checkpoint inhibitors ( Figure 11).
Conclusions
Our study reveals rare expression of MGMT as a stochastically pre-existing, key determinant of TMZ resistance in melanoma cell lines ( Figure 11). Upon treatment with TMZ at physiologically relevant concentrations, MGMT-negative cells are eliminated, but the tiny fraction of MGMT-positive cells survives and becomes the dominant, drug-resistant population. Combining TMZ with the MGMT inhibitor O6BG suppresses survival of MGMT-positive cells, but drug-resistant survivors still emerge, due to the presence of even rarer cells that harbor defective MMR (i.e., impaired MSH6 expression) that conveys TMZ resistance. These findings might explain, at least in part, why TMZbased treatment regimens, with or without added O6BG, do not achieve impressive clinical outcomes in melanoma patients.
Of note, MMR-deficient cells, which escape TMZ + O6BG combination treatment, are prone to develop a hypermutation phenotype, which results in increased display of neoantigens as potential targets for the immune system. It is thus conceivable that melanoma patients recurring after TMZ + O6BG might respond favorably to interventions that administer immune checkpoint inhibitors ( Figure 11). | 2018-10-14T17:56:42.165Z | 2018-09-28T00:00:00.000 | {
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216212933 | pes2o/s2orc | v3-fos-license | ARTIFICIAL INTELLIGENCE TECHNIQUES-BASED LOW VOLTAGE RIDE THROUGH ENHANCEMENT OF DOUBLY FED INDUCTION WIND GENERATOR
Wind energy is increasingly used as renewable energy worldwide. According to grid codes, wind turbines (WT) should essentially be coupled to grid throughout as well as following fault and source reactive power toward the grid with an objective of maintaining grid voltage. Doubly fed induction generator (DFIG), a wind turbine type enabling speed adjustment, is getting established currently in wind industry. Many DFIGs employ crowbar-based system to shelter the converter at the rotor side during disturbed and/or distorted grid voltage circumstances. Although it helps in protecting the generator, it does not warrant an appropriate grid support. This shortcoming led to designing anew coordinated controller that excludes or even cancels the need of a crowbar. This paper proposes fault confrontation controller (FCC) design to augment the feature -of low voltage ride through (LVRT) in this turbine. Considering the system’s nonlinear nature, an attractive FCC was constructed using computational intelligence (CI) techniques, namely fuzzy logic, back propagation network (BPN) and adaptive neuron fuzzy inference system (ANFIS).The simulation study demonstrates that the ANFIS system gives the best results for the proposed system .
I. Introduction
Over the last years, installed capacity of wind power is increasing rapidly worldwide and wind power is applied in a wide range varying from few kilowatts to several megawatts [V], [VI]. The speed of the electrical generator used in wind turbines is fixed or might be variable. Among the different wind turbines characterized by variable speed, specifically DFIG has gained remarkable attention due to its smallcapacity converters, high energy efficiency, low cost and flexibility in power control [VIII], [XVI], [XI]. Doubly fed induction generator carries a DC link capacitor and an electronic converter with successive power supply attached with generator rotor whereas the stator device is affixed to grid without an intermediary. As the stator and grid are directly coupled, DFIG is generally sensitive to fluctuations in grid. Any kind of faults in the grid results in voltage dip in stator, which increases the stator current that in turn increases the rotor current due to magnetic coupling. Increase in rotor current during fault condition will not just destroy the power converter components but DC-link capacitor as well. A large wind power in flow though power system results in the revision of grid codes. Among the fundamental grid codes, Low Voltage Ride Through [XII] is noteworthy, according to which the systems for conversion of the wind energy must be grid-connected in the event of grid faults.
Several hardware-and software-based solutions were put forth [VII], [XIV] to augment LVRT potential of DFIG turbines. Crowbar circuit has been considered a successful hardware-based solution to protect the wind turbine [VII], [XIII], [XIV] but it cannot provide voltage support because it bypasses converter at rotor side (RSC) and DFIG acts as normal squirrel cage induction generator. A major disadvantage of this protection technique is that DFIG takes upper active power present in grid during fault, causing additional reduction of voltage in the grid. Considering this drawback, improved crowbar techniques which includes that united with a DC-link chopper [IV], limiting the rotor current with crowbar together with a series dynamic braking resistor, are proposed [II].All these techniques mainly focus on reducing the crowbar operation time. Voltage stability problem was resolved with Dynamic Voltage Regulators (DVR) [X] or Static Synchronous Compensator (STATCOM) installed at PCC [III]. To overcome the disadvantages of hardware-based fault ride through featuresa nd devices used for injecting reactive power, upgraded controllers such as hysteresis-based current regulator and model predictive control are applied [XV]. One drawback of the existing software-based solution is its computational complexity. In the proposed system, an advanced intelligent controller is designed for the purpose of predicting the fault correction parameter, which subsequently augments the LVRT by i) protecting the converter at the rotor side against rotor over current and (ii) safeguarding the DC-link capacitor from the negative effects of overvoltage to ensure a stable operation without disconnecting the wind turbine from grid with minimal hardware requirements.
The paper is divided in to several sections. A DFIG wind turbine model and a schematic presentation of the proposed controller are presented in the next section. Design of fault confrontation controller with fuzzy logic is presented in section3.Section 4 details the identification of fault correction parameter with neural network. In section 5,an ANFIS-based design of fault confrontation controller is given. The results obtained from three techniques are described in the concluding remarks.
II. DFIG wind Turbine Modeling for LVRT Study
Modeling of DFIG DFIG consists of dual voltage source converters. These components are coupled to the grid and rotor. DFIG control constitutes the RSC and GSC (grid-side converter) controllers. Stator active power, in addition to reactive power, is restrained with an RSC controller, whereas the DC-link voltage through GSC controller. DC link capacitor that consecutively connects the dual converters reduces voltage ripple. Both converters regulate rotor voltages using PWM strategy, which in turn helps control rotor currents.
The d-q reference frame exhibiting synchronous rotation was selected for mimicking the dynamic nature of DFIG. The detailed mathematical modeling was illustrated in the studies [IX, I]. Only the equations necessary for LVRT study are mentioned here. The wind turbine's output power can be written in the following form: In the equation, the variables ρ and ν denote air density and wind speed, respectively. R represents rotor radius, while Cp the power coefficient.
The following equations represent a d-q or synchronous reference frame related tostator voltage as well as rotor circuits: In the above, the parameter λ indicates flux linkage. ω r and ω s stand for rotor angular speed and synchronous speed, respectively. R denotes resistance. R and s given in subscript represent the quantities of rotor and stator, respectively.
Description of proposed controller
An illustration of the intended FCC is depicted The roposed controller attenuates the system disturbance caused by the fault with appropriate coordination of (FUZZY/BPN/ANFIS) is used for System should not be affected Conventional RSC controller is modified by adding the FCC block as shown in the figure. The block functions only when Vs (stator voltage) differs by>10% from reference value. However achieved by properly controlling the rotor overcurrent along with overvoltage during fault and restoration. Extra energy that was produced transient state should be transferred together with DC-link voltage for rotor current is damped quickly, there will be a sudden increase in DC Instead, if it is reduced slowly, there are chances unfavorable values. Therefore, respective DC voltage values.As depicted in the figure, Vrq output of conventional RSC controller is adjustedby Ufcr derived by intelligent controller(Fuzzy/BPN/ANFIS). Inputs of the controller Vdc* and ir* are given as follows:
Description of proposed controller
An illustration of the intended FCC is depicted in Fig. 1.
Schematic diagram of fault confrontation controller
The roposed controller attenuates the system disturbance caused by the fault with coordination of both the converters. Computational Intelligent controller (FUZZY/BPN/ANFIS) is used for achieving efficient control in a very short period.
ould not be affected by measurement noise or the absence of information. Conventional RSC controller is modified by adding the FCC block as shown in the figure. The block functions only when Vs (stator voltage) differs by>10% from value. However, GSC control remains unaltered. Successful LVRT can be achieved by properly controlling the rotor overcurrent along with overvoltage during fault and restoration. Extra energy that was produced be transferred via converters to grid, to restore the rotor current link voltage for achieving normal values. In cases during which rotor current is damped quickly, there will be a sudden increase in DC Instead, if it is reduced slowly, there are chances for the rotor current to reach alues. Therefore, rotor current's correction signals must account for the respective DC voltage values.As depicted in the figure, Vrq output of conventional RSC controller is adjustedby Ufcr derived by intelligent controller(Fuzzy/BPN/ANFIS). Inputs of the controller Vdc* and ir* are given as dcss dcss V V wheress indicator denotes the value in steady state, while mav indicator the maximum pacified by manufacturer. In proposed system Vestas 2MW wind turbine ratings are used. r i stands for the rms current of the rotor. In order to ensure 3, March (2020) pp 125-139
Schematic diagram of fault confrontation controller
The roposed controller attenuates the system disturbance caused by the fault with both the converters. Computational Intelligent controller achieving efficient control in a very short period. of information. Conventional RSC controller is modified by adding the FCC block as shown in the figure. The block functions only when Vs (stator voltage) differs by>10% from the control remains unaltered. Successful LVRT can be achieved by properly controlling the rotor overcurrent along with DC-link overvoltage during fault and restoration. Extra energy that was produced under the the rotor current achieving normal values. In cases during which link voltage. or the rotor current to reach rotor current's correction signals must account for the respective DC voltage values.As depicted in the figure, Vrq output of conventional RSC controller is adjustedby Ufcr derived by intelligent controller(Fuzzy/BPN/ANFIS). Inputs of the controller Vdc* and ir* are given as (6) (7) while mav indicator the maximum In proposed system Vestas 2MW wind the rms current of the rotor. In order to ensure equal participation to the modulation of Fault Confrontation controller output,the deviations from values of steady state for the two quantities are divided by the deviations that are maximum acceptable. Only positive deviations are considered and negative deviations are taken as zero. The concept of proposed controller can be applied in different ratings of the machines. Rules applicable for the controller are the same. However, the only difference would be the highest values fixed by manufacturer.
III. Design of Fault Confrontation Controller with Fuzzy Logic
Fuzzy logic is nothing but the Boolean logic's extension. It is characterized by an appreciable flexibility allowing reasoning, and thus to consider uncertainties and inaccuracies. It can handle non-linearity and does not need an accurate mathematical model. A Fuzzy inference system appropriate for the controller proposed is shown in Fig. 2.Fuzzy rules of Fault confrontation controller are given in table 1. The following parameters are used to determine the efficiency of fuzzy logic control: Standard Deviation, RMSE (root mean square error), and Minimum and maximum error.
IV. Fault Confrontation Controller Design with BPN
Back propagation network The BPN learning algorithm is popularly used forthe purpose of training an (multi-layered neural network),so it can identifya function which can best map inputs to their appropriate output. Architecture of proposed system network con neuron and 3 hidden layers with 10:5:2 neurons. Learning process is started with the random initialization of the model with two inputs Vdc* and Ir*. Equations for calculating these values are expressed in Eqs. (6) and ( inputs are sentin to network layer, forward propagation. Loss function is defined to contrast actual output and desired output and to generate outputs that are system, three layers are used in neural network achieve more differences in loss function the error is propagated back from the end to the start. Delta rule is used for the weight updation. to ensure that he weight gets updated in a smooth and slow manne samples, 4797 rows have been taken for training the neural network and remaining for testing. Figure 8 and Figure 9 show the relationship between actual and desired value for trained data set and for the test data set.
Learning process has ta updated by gradient descent force towards a much lesser global loss function. The system's performance is measured with maximum and minimum error and root mean square error, and the values have been t square error of trained Maximum and minimum error is minimum for the trained data set, data set the error is maximum. But when compared to Fuzzy propagation network gives minimum error.
Fault Confrontation Controller Design with BPN
Back propagation network allows preparing the artificial neural networks. The BPN learning algorithm is popularly used forthe purpose of training an layered neural network),so it can identifya function which can best map inputs to their appropriate output. Architecture of proposed system network consists of 2 input neuron, one output neuron and 3 hidden layers with 10:5:2 neurons. Learning process is started with the random initialization of the model with two inputs Vdc* and Ir*. Equations for calculating these values are expressed in Eqs. (6) and (7).After initialization, the network layer, while the model's actual output is estimated forward propagation. Loss function is defined to contrast the difference between actual output and desired output and to understand how well Neural Network could generate outputs that are almost identical to the preferred values. In the proposed system, three layers are used in neural network separating the output and inputs to differences in neural network's functionality. From the derivative of loss function the error is propagated back from the end to the start. Delta rule is used Learning rate 0.2 and the momentum parameter 0.3 are he weight gets updated in a smooth and slow manner. Among 9594 samples, 4797 rows have been taken for training the neural network and remaining for testing. Figure 8 and Figure 9 show the relationship between actual and desired value for trained data set and for the test data set.
Learning process has taken 65 iterations. Following an iteration, the weight is descent force towards a much lesser global loss function. The system's performance is measured with maximum and minimum error and root mean square error, and the values have been tabulated as shown in table 3. Root mean of trained and test data set remains at the same value of 0.0315. Maximum and minimum error is minimum for the trained data set, while data set the error is maximum. But when compared to Fuzzy logic system the back propagation network gives minimum error.
3, March (2020) pp 125-139
Performance measures of Fuzzy logic control with different membership neural networks. The BPN learning algorithm is popularly used forthe purpose of training an MNN layered neural network),so it can identifya function which can best map inputs sists of 2 input neuron, one output neuron and 3 hidden layers with 10:5:2 neurons. Learning process is started with the random initialization of the model with two inputs Vdc* and Ir*. Equations for 7).After initialization, the the model's actual output is estimated by the difference between well Neural Network could to the preferred values. In the proposed output and inputs to om the derivative of loss function the error is propagated back from the end to the start. Delta rule is used Learning rate 0.2 and the momentum parameter 0.3 are used r. Among 9594 samples, 4797 rows have been taken for training the neural network and remaining for testing. Figure 8 and Figure 9 show the relationship between actual and desired , the weight is descent force towards a much lesser global loss function. The system's performance is measured with maximum and minimum error and root mean abulated as shown in table 3. Root mean and test data set remains at the same value of 0.0315.
while for the test logic system the back
V. Design of Fault Confrontation Controller with ANFIS
ANFIS represents a popular soft computing process in control area which works on the basis of Takagi-Sugeno fuzzy inference system. This technique is characterized by the benefits of neural network in addition to fuzzy logic system. FIS (Fuzzy Inference System) is a main element of ANFIS, and thus FIS acasts the model fo rmapping the characteristics of an input to its membership functions. These functions are then mapped to rules which in turn are mapped to the output characteristics. Finally, these characteristics are mapped to their membership functions, which in turn are mapped to an output with single value or an outputassociated decision. Several studies have used ANFIS for designingor regulating onlinear systems.
The proposed ANFIS architecture has 2inputs consisting of 5 layers. The inputs of ANFIS, Vdc* and ir* are given in Eqs. (6) and (7). An ANFIS output represents control signal Ufcr for fault correction to enhance the LVRT capability. Three cases with Triangular, Gbell, and PI membership functions are used, while the performance is again measured with Standard Deviation, RMSE, and minimum and maximum error. Comparative result between the performance measures is given in table 4.
ANFIS with Triangular Membership Function
Fuzzy inference system is created with 2inputs and 1output. Three triangular membership functions are used for both input variables. ANFIS output is measured with root mean square value of 0.0407 and standard deviation is 0.0335. Maximum and minimum error is 0.2119 and 3.0609e-06. For same triangular membership function, number of variables are increased to 5,7 and 9 and the results are tabulated in Table 4. Triangular membership function with 7 variables gives the optimal root mean square error value of 0.0078. The performance of the discussed ANFIS system is evaluated in Fig. 10.
VI. Simulation Results
Fault Correction parameter with the proposed fault confrontation controller is predicted using three soft propagation and ANFIS. Performance parameters of the three techniques are tabulated in table 6, and the results are
Simulation Results
Fault Correction parameter with the proposed fault confrontation controller is using three soft computing techniques namely fuzzy logic, back propagation and ANFIS. Performance parameters of the three techniques are tabulated in table 6, and the results are presented as a graph in Fig. 11.
LVRT Protection with CROWBAR
Crowbar protection technique is implemented for protecting the converter against overcurrent during transient condition. The DFIG Matlab/Simulink with crowbar is presented in Fig. 12. During the times rotor current surpasses its threeshold, crowbar circuit becomes activated; this also applies to dc-link voltage. There will be a huge increase in rotor current at the event of fault. If it flows through the RSC, then the converter will fail. Hence, crowbar is activated and excess energy will be dissipated in the crowbar resistor (Fig. 13). In the considered study, symmetrical fault is created at t = 3s. Hence rotor inrush current of about4000A is made to flow through the crowbar resistor in addition torotor side converter bypass. During crowbar activation, the rotor circuit is disconnected from the grid, which leads to the absorption of reactive power present ingrid. As a result, there occurs additional alleviation of grid voltage, which is not encouraged by the present grid code. Hence soft computing technique based modified vector control is implemented to RSC to avoid the stoppage of wind turbine during low voltage or grid fault conditions.
ANFIS based modified vector control for LVRT enhancement of DFIG
Fault confrontation controller is designed with three soft computing techniques namely Fuzzy control, Back propagation network and ANFIS. Among these techniques, ANFIS has given the best result with a minimum root mean square error value of 0.0078. Hence, ANFIS technique has been adopted for predicting fault correction parameter as well as modifying the vector control of RSC. Stator voltage measurement with ANFIS controller during fault condition is shown in Fig. 14.Voltage dip of 85% is created at 3 s and cleared at 3.5 s. Voltage rebuilds to the post fault state at t=4.1s. Rotor current during voltage dip is presented in Fig. 15.Fault is created at 3s. Hence, the rotor current increases to the value of 2000A which is below the threshold limit which converter can withstand. But with conventional control it has reached the value of 4000A.Stator current variation during transient condition is also reduced to the lower value around 7000A at t= 3s. At t=3.1s, stator current reaches the low value and attains the prefault value around t=3.5s. With the modified vector control technique, crowbar circuit is disabled. Hence, no current flows through the crowbar circuit (Fig. 16).
VII. Conclusion and Future work
Computational Intelligence-based Fault confrontation controller is used to predict the Fault correction parameter to augment the LVRT of DFIG turbine attached to grid requiring minimal hardware and less computational complexity. The operational efficiency of three soft computing techniques is analyzed in the present paper. Simulation results show that the ANFIS with Triangular membership function gives the best result in 7 levels. ANFIS method is therefore the most promising solution for LVRT enhancement. ANFIS Fault confrontation controller is incorporated in power system network and stator voltage responses and rotor current responses at the transient state are given.During fault condition, increased rotor current of about 4000A will flow through the crowbar resistor that disconnects the RSC and gives protection. But in ANFIS-modified vector control, crowbar circuit is eliminated, and therefore, no current flows through it, while RSC is still connected with grid and ensures power system stability. In future, Storage system (Battery/Flywheel/Super Capacitor) can be implemented across the DC link for energy storage. | 2020-04-09T09:05:17.345Z | 2020-03-29T00:00:00.000 | {
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252983031 | pes2o/s2orc | v3-fos-license | HERV-K Envelope Protein Induces Long-Lasting Production of Autoantibodies in T1DM Patients at Onset in Comparison to ZNT8 Autoantibodies
Human endogenous retroviruses (HERVs) have been thought of as silent passengers within our genomes, but their reactivation has been linked with several autoimmune diseases, including type 1 diabetes (T1DM). In order to evaluate the potential role of HERVs, in addition to the recognized role of HERV-W, we focused on the debated role of the HERV-K family in T1DM. Therefore, we performed a serological evaluation of IgG antibodies against HERV-K Env epitope (HERV-K Env19–37) in comparison to an important β-cellular autoimmunity biomarker, ZnT8, from plasma samples of Sardinian children at the onset of T1DM, different T1DM groups (1–5 and 6–12 years since diagnosis), and healthy controls (HCs), by an indirect enzyme-linked immunosorbent assay (ELISA). A significant antibody response was observed against HERV-K Env19–37 (p < 0.0001) in T1DM patients compared to HCs, and significantly higher IgG responses were detected in the group at the onset compared to the other T1DM groups and HCs. Unlike the trend of the β-cellular autoimmunity autoantibodies, for HERV-K Env antibodies we observed positive values that persist over time up to 5 years since the onset of T1DM. Our results add new evidence about the presence of antibodies against HERV-K in T1DM, but further investigations are necessary to relate these results with the established role of HERVs, considering the contrasting results for HERV-K.
Introduction
Human endogenous retroviruses (HERVs) constitute approximately about 8% of the human genome. The integrations of the provirus genome into the DNA of germinal cells are the remnants of infections that occurred over several million years, which guaranteed their vertical transmission in a Mendelian fashion [1].
The role of retrovirus in different diseases has remained unknown, particularly for autoimmune diseases that involve a combination of genetic and several environmental factors; HERVs are not infectious but can contribute to the development of inflammatory conditions. A number of factors, such as deletions, mutagenesis, and DNA methylation, can modulate their transcription levels [2].
Some diseases are linked to the transcription of certain HERV genes, such as cancer and multiple sclerosis; in addition, the HERV proteins may act as superantigens, which in turn may lead to the development of antibodies against them.
Given their potential pathological effects, HERVs have been proposed as possible cofactors in the etiopathogenesis of various diseases, recently including type 1 diabetes (T1DM) [3,4]. T1DM is one of the most common autoimmune diseases in children and adolescents, caused by the autoimmune response against pancreatic β-cells that results in the local inflammation of pancreatic islets (insulitis) and the progressive loss of pancreatic β-cells, leading to insulin deficiency [5,6]. Both adaptive and innate immunity are involved in the disease process.
These represent the best biomarkers currently available to identify early immunity and to predict the clinical onset of the disease [9]. ZnT8 is the latest to be identified as a novel autoantigen in T1DM; it may precede the clinical symptoms of the disease and increases sensitivity when analyzed in relation to the other T1DM Abs [10]. Autoantibodies detection before the manifestation of clinical symptoms is a primary diagnostic target for defining the onset of T1DM and may also be useful for the evaluation of immunological therapies, with interventions targeting the autoreactivity of T and B lymphocytes. Different families of HERVs have been associated with T1DM onset as HERV-W, HERV-K. In particular, the most plausible evidence for a functional association with T1DM pathogenesis was found with the HERV-W family [4,11,12].
The HERV-W envelope protein (HERV-W Env) has been detected in patients with T1DM and, in particular, in pancreatic acinar cells near the pancreatic lesions of patients with T1DM. HERV-W Env was observed to promote macrophage recruitment within the pancreas and beta-cell dysfunction, as demonstrated by the inhibition of insulin secretion by HERV-W Env in primary cultures of human islets of Langerhans [12]. It was the first study highlighting the detection and the pathogenic properties of HERV-W Env in T1DM.
In previous studies, we portrayed the association of Mycobacterium avium subspecies paratuberculosis (MAP) and HERV-W in T1DM etiopathogenesis in children from Sardinia, supporting for the first time the hypothesis that it may be possible that both pathogens contribute to the onset of T1DM [13]. Moreover, we highlighted a correlation between anti-HERV-W and proinsulin (PI) autoantibodies in the sera of children with T1DM collected at different times after onset [14].
A different story is associated with HERV-K, where basic questions have been raised regarding the association of T1DM with HERV-K given the contradictory results reported [15][16][17].
In this study, we aimed to investigate the humoral response against HERV-K in association with the detection of classical autoantibodies such as those against ZnT8 in sera from children collected at T1DM onset and different times after diagnosis.
Sample Collection and Processing
In this retrospective study, samples were obtained from T1DM and healthy controls (HCs) participating in studies. Informed consent was obtained from subjects according to the Institutional Ethical Committee of Sassari University. A total of 70 Sardinian pediatric patients with T1DM were included, including twenty-six patients recruited at the time of the onset. Fifty-seven age-matched controls were recruited for the HCs group during routine check-up visits from the Department of Endocrinology of the University Hospital (AOU) of Sassari.
Clinical data about the patients and HCs are shown in Table 1.
Blood was taken from each individual using ethylenediaminetetraacetic acid (EDTA) tubes; the blood was processed with Ficoll-Paque ® (Sigma-Aldrich, St. Louis, MO, USA) to collect the plasma. Following centrifugation, the resulting supernatant is designated plasma. According to the protocol, the plasma was immediately transferred into a polypropylene tube, slipped into aliquots, stored, and transported at -20 • C.
Ninety-six-well Nunc immune-plates were incubated overnight at 4 • C with 0.05 M of carbonate-bicarbonate (pH 9.4, Sigma-Aldrich, St. Louis, MO, USA) and the respective peptides at 10 µg/mL. The plates were incubated for 1 h in a blocking solution with 5% non-fat dried milk (Sigma-Aldrich, St. Louis, MO, USA) and phosphate-buffered saline (PBS), then washed twice in a solution with PBS and 0.05% Tween-20 (PBS-T). Plasma samples were added at a 1:100 concentration and incubated for 2 h at room temperature. After a washing step, each plate was incubated for 1 h with 100 µL of PBS and an alkaline phosphate-conjugated goat anti-human IgG polyclonal antibody (1:1000, Sigma-Aldrich, St. Louis, MO, USA). After washing, each plate was washed in PBS-T and then incubated in Milli-Q water and p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO, USA) for 8-10 min in a dark environment. The optical density was read at 405 nm using a SpectraMax Plus reader (Molecular Devices, Sunnyvale, CA, USA).
Each sample was run in triplicate, and normalization was performed with a positive control setting with the absorbance reactivity at 1.0 arbitrary units (AU)/mL. Negative control sera were also included in all experiments. ELISA precision was determined by calculating both the inter-and intra-assay coefficients of variation.
Statistical Analysis
Statistical analysis was performed with commercial software GraphPad Prism 9.1 (GraphPad Software, San Diego, CA, USA). Statistical significance was defined as a p value < 0.05.
Data distribution was analyzed using the D'Agostino-Pearson omnibus normality test and the Shapiro-Wilk test. Non-parametric data were analyzed using the Mann-Whitney U test and the Kruskal-Wallis test with Dunn's multiple comparisons test to compare the antibody levels against Znt8 178-186 and HERV-K Env 19-37 -derived peptides between the different groups. Receiver-operating characteristic (ROC) was used to choose the cut-off value to assess the sample positivity, which was consequently tested through Fisher's exact test. A Spearman correlation test was performed among levels of antibodies on HERV-K Env and ZnT8-derived peptides.
To deepen the significance of the results regarding of the HERV-K Env19-37 humoral response, we decided to stratify the population to investigate a potential change in humoral response related to disease duration and in relation to the trend of the Concerning HERV-K peptide, we found for the first time a significant difference in the antibody response between T1DM and HCs and at T1DM onset ( Figure 1C,D). In T1DM patients, 27 out of 48 were positive (56.25%) in comparison to HCs (5.26%), where 3 out of 57 were positive (cut-off value 0.40; AUC = 0.84; p < 0.0001 Figure 1C). In the onset group, 16 out of 26 were found to be seropositive to HERV-K Env 19-37 (61.54%), whereas only 4 out of 50 were positive in HCs (8%,) (cut-off value 0.39; AUC = 0.84; p < 0.0001 Figure 1D).
To deepen the significance of the results regarding of the HERV-K Env 19-37 humoral response, we decided to stratify the population to investigate a potential change in humoral response related to disease duration and in relation to the trend of the autoantibody against ZnT8 178-186 . The results obtained from testing the four groups investigated (onset, 1-5 yr, 6-12 yr, HCs) were analyzed by Kruskal-Wallis test and using Dunn's post hoc analysis.
Discussion
T1DM is considered an autoimmune disease, caused by immune responses in genetically susceptible individuals, characterized by T cell infiltration against pancreatic b-cells, resulting in their destruction and marked by the production of Abs. The first objective of this study was to determine the humoral response against HERV-K Env19-37. A further aspect investigated was the change in the prevalence of these Abs at the diagnosis of the disease and at different years since diagnosis, and whether these changes
Discussion
T1DM is considered an autoimmune disease, caused by immune responses in genetically susceptible individuals, characterized by T cell infiltration against pancreatic b-cells, resulting in their destruction and marked by the production of Abs. The first objective of this study was to determine the humoral response against HERV-K Env [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] . A further aspect investigated was the change in the prevalence of these Abs at the diagnosis of the disease and at different years since diagnosis, and whether these changes follow a trend referable to an important β-cellular autoimmunity marker, such as ZnT8, which plays an important role in the progression of the disease and which has been identified as one of the main biomarkers for type 1 diabetes. These Abs are present months or years before the clinical manifestations [18,19]. ZnT8 is a protein encoded by the SLC30A8 gene on chromosome 8 that is expressed selectively on the insulin granular membrane of pancreatic b-cells; its function is to maintain the zinc balance in beta cells by importing and exporting zinc ions [20,21]. It plays a key role in the regulation of insulin signaling and glucose homeostasis, and it is also involved in insulin synthesis, storage, and secretion in humans [22].
In addition, ZnT8-derived peptides have been identified as important biomarkers of diabetes autoantigens that may trigger a self-reactive CD8+ T lymphocytes response, suggesting that this antigen plays an important role in the progression of the disease [23].
The secretion of insulin granules increases the exposure of the autoantigen ZnT8 to the cell surface, which can initiate ZnT8 epitope-specific T cell-mediated beta cell destruction, resulting in T1DM.
This mechanism is amplified when beta cells are destroyed, as this increases the release of autoantigens, which thus intensifies the T cell-mediated immune response.
Several peptides of Znt8 were previously identified by our group, which share more than 50% of their identity with Mycobacterium avium subspecies paratuberculosis (MAP). The potential involvement of MAP in diabetic pathology may result from molecular mimicry with the ZnT8 178-186 epitope, which leads to autoimmune responses. The cross-reactivity to common target sequences and the specificity of anti-MAP-ZnT8 Abs has been verified by competition assays in previous studies [24].
It is known that the innate immune cells can be specifically activated by the envelope surface proteins of HERVs through the pattern recognition receptors CD14 and TLR4 [25].
Among the different HERV families, the largest evidence of a functional association in the pathogenesis of T1DM has been presented for HERV-W Env [12].
This association was also confirmed by our group. HERVs-encoded proteins may be the principal trigger of the disease when they are expressed or overexpressed. It is possible that these proteins could induce the overproduction of anti-HERVs antibodies by B lymphocytes with a protective role, as observed in ALS recently [26]. Several studies observed the detection of HERV-K Env in the cerebrospinal fluid (CSF) and brain in amyotrophic lateral sclerosis (ALS) patients, noting its specific neurotoxic effect in vitro and in vivo [27][28][29]. Significantly increased levels of Abs targeting HERV-K Env in ALS patients were initially reported by our group [26,30].
Conflicting opinions regarding the role of HERV-K in T1DM have been published [15,16]. Past studies based on viral sequence expression IDDMK1 [31,32] initially in favor of such a link have subsequently excluded the association between HERV-K and T1DM [15,16].
The marked decline of ZnT8 already 1 year after onset probably reflects a reduction in these autoimmune processes over time due also to the lack of the antigen.
In conclusion, our results provide further evidence of the association of antibodies against HERVs and autoantibodies and lead us to question if these autoantibodies against HERV-W and HERV-K may be useful biomarkers in T1DM progression.
Further studies are needed to clarify the role of HERV-K, as well as the diagnostic and prognostic value of serum Abs, in order to have a more accurate picture of disease. Additional investigations on the expression levels of the HERV-K envelope protein in PBMCs might be useful. | 2022-10-19T15:05:31.810Z | 2022-10-01T00:00:00.000 | {
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212535934 | pes2o/s2orc | v3-fos-license | Hardening Effects of In-Situ Aging for a Laser Welded
Corresponding Author: Milton Sergio Fernandes de Lima Photonics Division, Institute for Advanced Studies, 12.228-001 São Jose dos Campos, Brazil Email: msflima@gmail.com Abstract: Maraging steels are ultra-high strength alloys which have been successfully laser welded to obtain structural components. For most of the applications these steels are in an aged state to attain tensile strength up to 1.5 GPa, although welding induces local softening due to dissolution of precipitates. This paper aims to investigate the effect of in-situ aging of the maraging steel plates after laser welding to reduce the local softening. For a preheating at 40C, just below the martensite finish line, the hardness of the Fusion Zone (FZ) attained between 440 and 490 HV and the Heat-Affected Zone (HAZ) attained a maximum of 570 HV compared to 300 HV of the Base Material (BM) for aging temperatures between 450C and 520C and periods between 10 and 30 min. The intercritical aging (570C/1 h) also promoted an increase in local hardness of FZ 320 HV and HAZ 400 HV. Using an intercritical aging, the hardness situated between room temperature and quenched and aged coupons and was more homogeneous considering FZ, HAZ and BM. The microstructure of the intercritical aged welds is marked by duplexferrite and martensite micro-constituents.
Introduction
Maraging alloys belong to the Ultra-High Mechanical Strength Steels (UHSS) as could attain tensile strengths up to 3.4 GPa depending on composition and the thermomechanical treatments (Rohrbach and Schmidt, 1993). Different from ordinary UHSS, Maraging steel martensite is ductile and the maximum strength is obtained by aging (Silva et al., 2019). The phase transformations for produce the steel ingots are the origin of the name MARtensite and AGING. Bieber (1969) was the first to prove that a Fe-25%Ni-4%Ti-4%Al steel attained a maximum tensile strength of 1 GPa after aging at 480C during 160 min.
Although Maraging steels proved their usefulness in high demanding applications such as rocket bodies, high pressure tanks and landing gears, welding is still a challenge (Rajkumar et al., 2014). Fusion welds solubilizes the precipitates into the austenite matrix and produce softened weld zones (Fanton et al., 2014). Even with solid state processes failed to prevent softening of the joint because annealing produces fully martensite in cooling (Kellogg, 2012). Silva et al. (2019) heat treated a class 300 Maraging steel from 1 to 100 h at 440C obtaining hardness ranging from 300 HV to 620 HV. These authors recorded the Moessbauer spectra for each aging time and noted that a high mobility of Mo and Ti atoms for times under 3 h.
The present investigation proposes an inductive heating together with the laser weld of Maraging 300 steels in order to produce aging directly in the final product. One difference of the current approach to the conventional heat treating is that the material is not normalized prior to aging. It is expected that Base Material (BM) and the Heat-Affected Zone (HAZ) austenitize rapidly during laser heating. However, the Fusion Zone (FZ) of the welds present microsegregation which will produce composition modulations retained during aging. These assumptions are corroborated by Casati et al. (2016).
Inductive heating in laser welding had been already used to increase the cooling period of EN 10025-6 S690QL fine-grained structural steel (Lahdo et al., 2014) and to produce bainite in hot-stamping steels (Lima et al., 2017). However, to the best knowledge of the authors, it is the first time aging is applied concomitant to laser welding of maraging steels. Krasnikova et al. (1986) reported that the interdendritic region of as-cast ingots of 250 maraging are rich in Ti and Mo. These segregations induce the formation of -ferrite precipitates and to a depletion of these elements in the dendrite trunks. Linnert (1967) stated that reducing these elements in the Fe-Ni tempered martensite reduces the mechanical strength due to a decrease in precipitation hardening potential. These findings were also pointed out by Pektas and Atala (1998) which indicated a high temperature solution treatment, e.g., 1000C/4h, aiming to reduce the elements segregation.
The temperatures for the isothermal treatment, i.e., 450C, 480C e 520C, were determined in a previous study (Lombardo et al., 2016). According to Pektas and Atala (1998), 18%Ni maraging steels present a good balance between tensile strength and toughness when aged at 480C per 4 h. The maximum temperature of austenite precipitation was chosen according to Kapoor et al. (2003) at 570C as the maximum density of precipitates measured by dilatometric tests in 18wt.% Ni maraging steel of grade 350.
One way to understand the martensitic transformation in maraging steels is to start with the metastable phase diagram, Fig. 1. From the solidification, there is no transformation of the austenite phase in the steel before it reaches the Martensite Start (Ms) temperature. Some studies show that the martensitic transformation always operates on 300 maraging steels, even when the cooling rate is quite slow, as well as in large parts. The resulting martensite has a center body cubic structure, as opposed to the hexagonal structure of the common martensites of the steels, due to the low carbon content. Martensite is relatively ductile,0 ~ 30 HRC and has a high dislocation density without significant twining. Alloy elements change Ms but do not alter the fact that the martensitic transformation is independent of the cooling rate (ASM Handbook Committee, 1990a). The great majority of the alloying elements and in particular the Ni, decrease the transformation temperature Ms. Cobalt is an exception because it increases Ms. The addition of Co is precisely to balance the decrease in Ms of the alloying elements, e.g., Ni, Ti, Mo, which are required for precipitation during aging.
According to common practice, aging hardening is carried out at temperatures between 455 and 510C for 3 to 9 h, followed by air cooling. From the phase diagram, blue lines in Fig. 1, 42% of ferrite is obtained for an 18.5% Ni composition and a temperature of 500C. A reversion to an austenitic-ferrite structure would hinder precipitation hardening. However, the enthalpy for precipitation of intermetallic phases is smaller than the reversal for a ferrite-austenite mixture. For example, for the current alloy and according to ThermoCalc computations, the equilibrium phases at 475C are: Iron ferrite (92%mole), Ni3Ti (3%mole), FeCr (3% mole) and Cu0.76Ti0.23 (1%mole) (Sha, 2000). Therefore, there is a precipitation of phases inside the grains with martensitic structure in plates aided greatly by the high density of dislocations. The distribution of particles consistent with the matrix is uniform and the main hardeners are Ti-based. The phase with the least distortion (misfit) in the CCC matrix of martensite is Ni3Ti and therefore privileged in the initial states. The precipitation has a limit given by the solute diffusivities and high aging times tend to form the so-called reversed austenite. Overaging causes an expressive reduction of the hardness of the steel and is fundamentally the reversion to the thermodynamic equilibrium in the intercritical zone (Fig. 1). The most probable mechanism of reversed austenite formation is the dissolution of nickel-rich precipitates, which enriches the matrix and renders the austenite more thermodynamically stable. The red lines in Fig. 1 presents the experimental limits (ASM Handbook Committee, 1990a) for Austenite reversion on Heating (Ah) and Martensite formation on Cooling (Mc) together with the current initial Ni composition.
Experimental
The material was received as an 18%Ni class 300 Maraging steel from Böhler Co. in the form of 3.3 mm thick plates. The composition is given in Table 1 and agrees with the SAE AMS 6514F standard (SAE Mobilus, 2019). The material was hot rolled and air-cooled resulting in a relatively soft martensite (330 HV).
The laser workstation is composed of an YLR-2000 IPG Photonics fiber Laser, a CNC table, a furnace and auxiliary systems. The laser minimum spot is 0.1 mm and the beam quality is M 2 = 9. The CNC table is driven by three step motors with maximum speed of 160 mm/s and processing area of 400500 mm 2 . The furnace was conceived for inductive heating during the welds as previously reported in the literature (Lima et al., 2017). The auxiliary systems comprise water cooled jackets and nitrogen cross jet to protect the optics.
The welds were realized bead-on-plate with the focus on the top of the upper surface. The surfaces were grounded and cleaned with ethanol prior to the welds. After the focus position was calibrated 5050 mm 2 samples of the steel were positioned in the center of the furnace. The samples were inductively heated up to a given temperature and maintained at these temperatures with the use of a manual switch. One thermocouple welded to the bottom surface of the plate recorded the temperature profile. The furnace and laser setup were reported elsewhere (Braga et al., 2018).
Since the steel plates were inserted in a furnace, a protective atmosphere was not possible. However, previous results have shown that the porosity due to the atmospheric contamination is high only for full penetration welds, i.e. when the fusion zone attains the lower surface of the plate. In view of this, the laser power and weld speed were conceived for partial penetration. For the current experimental conditions, the laser power was fixed at 1800 W and the weld speeds were 1.8, 2.4 and 3.0 m/min in accordance to a previous work (Sakai et al., 2015). The microstructure of the samples was analyzed by a Light Optical Microscopy (LOM) using an Imager.2M Zeiss microscope and Nital 2% etching. Scanning electron microscopy was carried out using a Hitachi TM-3000 microscope. An equipment model FM800 (Futuretech) was used for Vickers hardness tests using 100 gf load and a dwell time of 10 seconds.
For thermodynamic calculations using the ThermoCalc© software, the TCFE6-steel database was used (Andersson et al., 2002). The thermal simulation was carried out using Sysweld© Software (Esi-Group, 2019). Sysweld is a Finite Element Modeling (FEM) software specially designed for welding and heat treatment of metals and alloys. For the current purpose a mesh refined around the laser path was designed with a lateral resolution of about 0.001 mm. The exact maraging 300 properties are not available in the Sysweld database, therefore X5CrNi1810 (EN 1.4301) steel properties were used instead. The model dimensions and welding procedure followed those described in the experimental design, starting with the material at room temperature and heating it up to a given temperature T*. The software produces many valuable outputs, but those of interest here are the time-temperature evolution in different regions of the plate.
Results and Discussion
Ambient Temperature Welds Figure 2 presents cross-section images of representative welds obtained for speeds of 1.8, 2.4 and 3.0 m/min. In a general view, the welds were thinner and deeper as the speed varies from 1.8 to 3.0 m/min. The weld obtained at 1.8 m/min has a top width of 2.2 mm and penetration of 2.5 mm, compared to the weld obtained at 3.0 m/min with a top width of 1.2 mm and penetration of 2.7 mm. As the welds were performed without gaseous protection, the plasma generated on the surface of the plate is more intense with slower speed, since the interaction time increases and more metallic ions are present. Additionally, as speed increases, the plasma plume tends to move back from the center of the keyhole, allowing the incident beam to penetrate further into the plate. This effect is known and reported as a function of the gas flow, causing the plasma to move away from the center of the beam (Joseph et al., 2017).
The extent of HAZ is a function of the welding speed, although it varies depending on the region where it is measured. In the case of the macrographs shown in Fig. 2 the HAZ ranges between 0.7 and 0.4 mm, from 1.8 to 3.0 m/min. This region is equivalent to the limit of dissolution of intermetallic precipitates responsible for the hardening of the material. From the FEM simulations, the limit of the HAZ represents an isotherm of 900C.
It is expected that microsegregation appears among the dendritic branches of the fusion zone (Fig. 3). In the figure, the central region is composed of equiaxed dendrites spaced 3 µm and where 39% were interdendritic material. These values did not change significantly in the current speed interval. Figure 4 presents the time-temperature profile for each speed condition estimated in the middle of the fusion zone. Each curve is displaced in time for better visualization. According to the thermodynamic database Thermocalc (Andersson et al., 2002), the liquidus temperature is 1417C, also displayed in Fig. 4. For every condition, the liquid was superheated above 1900C and the cooling rates at liquidus were estimated 16'300, 22'500, 25'700C/s for 1.8, 2.4 and 3.0 m/min, respectively. Near to 760C, the cooling rates are 4'000, 5'700, 6'900C/s for 1.8, 2.4 and 3.0 m/min, respectively, which are large enough to produce martensite transformation with a Vickers hardness of 313 HV (ASM Handbook Committee, 1990b).
The actual hardness profiles of the samples are presented in Fig. 5. The Fusion Zone (FZ) hardness (open squares in Fig. 5) situated between 280 and 300 HV, consistent with an unaged martensite structure. The Heat-Affected Zone (HAZ) hardness (crossedsquares in Fig. 5) is typically above the FZ Vickers hardness because of the solid-state transformation implies in the absence of microsegregation. Finally, the Base Material (BM) is delimited by the temperature below the austenite reversion on heating (Fig. 1). The rise in HV for the BM near to the HAZ is due to local ageing of the original martensite.
Quenching and Aged Welds
Quenched and aged welds are those heat-treated subsequent to complete quenching, as presented in Fig. 6. The temperature profile came from Sysweld simulation (laser weld) and from the thermocouple records when the time is above 10 seconds. For the welding, only one condition (3 m/min) had been chosen, although the time and temperature for isothermal aging was changed. Figure 6 also presents the temperatures for austenite reversion in heating (Ah) and Martensitic transformation in Cooling (Mc). The isothermal aging was conceived to occur between the Ah and Mc lines, in accordance to previous works (Fanton et al., 2014). Figure 7 presents the macrographies of the welded regions and surroundings as a function of the aging time and temperature. The weld shapes resemble those obtained in ambient temperature conditions (Fig. 2), although the penetration attained the bottom surface in a majority of cases. Since the furnace configuration does not support a protective gas nozzle, the enhanced weld penetration could be associated to a better plasma coupling or to the absence of surface cooling induced by the flowing argon. Also, the HAZ is more distinctive in Fig. 7 The intermetallic precipitation noticed for the ambient temperature welds (Fig. 3) is still visible in the welds after aging (Fig. 8a). Although a partial solubilization of the microsegregation after an aging of 520C per 10 min promoted a skeleton-type distribution. The aged martensite is also visible in Fig. 8a, with the precipitates removed by the chemical etching. Figure 8b shows a region near the Fusion Line (FL) where the same features of the weld centerline could be seen at the right. At the left, aged martensite grains are free of massive precipitates as those observed in the FZ.
The hardness profiles for each aging condition are plotted in Fig. 9 as a function of the region: FZ, HAZ and BM. As reported before (Sakai et al., 2015), the general tendency is an increase in HV when increasing aging time or the temperature. However, the FZ values are distinctly below the other regions HV. As presented before, Fig. 8, the heat treatment was not sufficient to homogenize the as-solidified precipitates at the interdendritic region. Some important elements for nucleation during aging, particularly Ti and Mo, are depleted in the martensite matrix and therefore the FZ tempered martensite is softer than other regions. The hardness obtained in BM and HAZ are the same because of the initial homogeneous structure.
After 30 min the average hardness of BM and HAZ attained approximately 510, 520 and 570 HV for temperatures of 450, 480 and 520C, respectively. For the same temperatures the FZ attained considerably lower values of 440, 470 and 490 HV, respectively.
Intercritical Aging
Instead of quenching and aging the laser welded coupons, the intercritical aging aimed to heat the steel plated between Mc and Ah in order to create a duplex structure α+ (Fig. 1). Figure 10 is a time-temperature plot composed of Sysweld results (laser weld) and the thermocouple record data (heating, isothermal aging and cooling). The temperature path was chosen slightly above the previous temperatures (Fig. 7) to maximize diffusion of the elements, but keep it below the austenite reversion on heating. The average isotherm is 570C, with a little period above Ah due to the laser heat input. The weld macrostructure (Fig. 11a) and FZ microstructure (Fig. 11b) is very different from previous observations. The entire coupon was heat treated creating a flatter etching contrast (Fig. 11a). The FZ microstructure is composed by a light gray (ferrite) and dark gray (martensite) phases with calculated surface phase amounts of 43% ferrite and 57% martensite. Scanning electron images (Fig. 12a) indicated the ferrite phase occupies the former interdendritic spaces growing at expense of microsegregated phases. Figure 12b is an image obtained near to the fusion line of the weld. In the figure, the HAZ ferrite growth occurred with a Widmanstätten morphology from the grain boundaries.
The hardness of the coupons (Fig. 13) lay between the room temperature welds (Fig. 5) and welded and aged samples (Fig. 9). The hardness range of all welding speeds was narrower than the welds without heat treatment, which could eventually lead to a decrease in stress concentrators.
Conclusion
This work compared the microstructure and hardness of maraging 300 plates obtained from room temperature weld, quenching and aging and intercritical aging with the use of a furnace coupled to a laser workstation.
The microstructure of room temperature welds is marked by dendrite growth, hence they present some microsegreation. This solute partitioning is responsible for the depletion in hardness in the weld compared to surrounding regions. The Fusion Zone (FZ) hardness situated between 280 and 300 HV, consistent with an unaged martensite structure. The Heat-Affected Zone (HAZ) hardness are typically above the FZ Vickers hardness because of the solid-state transformation implies in the absence of microsegregation. Finally, the Base Material (BM) is delimited by the temperature below the austenite reversion on heating.
When the welded coupons were quenched and aged in the range of temperatures 450 to 520C, with times from 10 to 30 min, the aged martensite was observed. After 30 min the average hardness of BM and HAZ attained approximately 510, 520 and 570 HV for temperatures of 450, 480 and 520C, respectively. For the same temperatures the FZ attained considerably lower values of 440, 470 and 490 HV, respectively, due to the composition modulation.
Using an intercritical aging, the hardness situated between room temperature and quenched and aged coupons and are more homogeneous considering fusion zone, heat affected zone and base material. The microstructure of the intercritical aged welds is marked by duplex-ferrite and martensite microconstituents. The hardness range of all welding speeds was narrower than the welds without heat treatment, which could eventually lead to a decrease in stress concentrators. | 2019-12-19T09:20:03.802Z | 2019-01-01T00:00:00.000 | {
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62797395 | pes2o/s2orc | v3-fos-license | The Nilgiri Tahr ( Mammalia : Cetartiodactyla : Bovidae : Nilgiritragus hylocrius Ogilby , 1838 ) in the Agastyamalai range , Western Ghats , India : population status and threats
The Nilgiri Tahr (Nilgiritragus hylocrius Ogilby, 1838) has not been comprehensively surveyed in the southern Western Ghats, India. Here we present results of a survey conducted in 2012 and 2013 in 25 sites where Nilgiri Tahr was reported in Agastyamalai range south of the Shencottah gap. The objectives of the survey were to assess population status; evaluate threats and propose conservation measures. In each site the geographical coordinates were noted. If Nilgiri Tahr (=Tahr) were sighted, the number and herd structure were recorded. Indirect signs of Tahr presence such as faecal pellets and feedback from local informants were noted in sites with no direct sightings of Tahr. The total sightings were 247 Tahr in 10 sites, and indication of Tahr presence in seven sites. Only two populations viz. Kalamalai-Varraiattumudi and Muthukulivayal-Balamore were large (>30 individuals). Tahr were not present in eight sites: of which four had earlier records of Tahr presence, and the other four had no prior data. There was a significant positive association between percentage of young (kids and yearlings) and number of Tahr sighted. Illegal hunting was widespread in the past, and continues to be a serious threat. Loss of Tahr grazing habitat to successional processes resulting in increased tree cover, is a long term threat that could increase with climate change. A landscape level management plan to reconnect small populations, rehabilitate Tahr in sites where they have disappeared, use fire to restore short grass habitats, and stringent curb on illegal hunting is required for the long term viability of the Nilgiri Tahr in this region.
INTRODUCTION
An endangered species is vulnerable to extinction due to small population size, declining numbers, habitat changes and overexploitation (Mace & Lande 1991).A recovery plan for an endangered species should be based on population monitoring, improving habitat quality and controlling threats (Campbell et al. 2002).
The Nilgiri Tahr (Nilgiritragus hylocrius Ogilby, 1838), called hereafter 'Tahr', is a mountain ungulate endemic to the Western Ghats of India.The Tahr is classified as 'Endangered' due to its restricted geographical distribution and declining population (Alempath & Rice 2008).Its present population is estimated to be around 3,000 (Schaller 1970;Davidar 1978;Predit et al. 2015).The geographical range of the Tahr extended to the state of Karnataka over 50 years ago.It was last reported in Agumbe Ghat at around 13 0 30'N latitude in 1954 (Davidar 1978).At present, Its northernmost range limit is the Nilgiri Mountains, at around 11 0 30'N latitude.The largest population of around 800 animals, is found in the Eravikulam National Park, Kerala (Abraham et al. 2006).The Nilgiri population has been annihilated by hunting since the early 20 th century (Fletcher 1911) and only a few hundred animals remain in the southwestern region in the Mukurthi National Park (Davidar 1978;Madappa 2012).
The increasing fragmentation and isolation of Tahr population is a cause for concern (Davidar 1978;Rice 1984;Mishra & Johnsingh 1998;Vergis 2011) as small isolated populations have greater risk of extinction due to stochastic factors (Soulé 1987).Rice (1984) indicated that 15 of the 17 sites with Tahr populations held fewer than 100 animals.There were no signs of the Tahr in six of 20 sites (30%) (Mishra & Johnsingh 1998;Vergis et al. 2011) where they had been documented earlier (Davidar 1978;Abraham et al. 2006).Loss of these small populations will result in genetic homogenization and further endanger the long term survival of the species.
Small isolated populations can help maintain the metapopulation structure of the species, if gene flow is restored through habitat connectivity or genetic rescue.Therefore it is important to document the status of smaller, isolated population of Nilgiri Tahr for conservation planning.
The southern Western Ghats south of the Shencottah Gap, termed the Agastyamalai range, has been relatively less investigated for the presence of Tahr.The early documentation of the species was by Webb-Peploe (1946-47) in and around the Naraikadu estate in Kalakad, where he recorded around 40 individuals.Later surveys by Daniel (1970), Davidar (1978), Daniel (1987), Lal Mohan et al. (1998) and Vergis et al. (2011), were not extensive, but indicated that many of these populations were small, and hunted.
Threats to Tahr in this region are several.Illegal hunting (Daniel 1987;Davidar 1978) was the most direct and obvious cause for the decline of this species.More insidious but permanent threats emerged such as human disturbance, roads and other obstructions (Davidar 1978).Strict protection against fire promoted tall grass succession which reduces availability of short grasses favoured by herbivores (Sankaran 2005).Livestock grazing reduces forage availability for wild herbivores (Davidar 1978;Madhusudan 2004) and could be agents for diseases such as rinderpest (Schaller 1970).
In order to check the status of the Tahr in this region, we conducted surveys during the dry seasons of 2012 and 2013.The objective of this study was to (i) identify sites where Tahr is present south of the Shencottah Gap, (ii) see whether the recruitment rate as indicated by the proportion of young Tahr is related to its population size, and (iii) qualitatively evaluate threats in the context of its present status.We tested the null hypothesis that small populations of the Tahr have not changed over the years.
Study area
This study was conducted over several sites south of the Shencottah gap in the Ashambu Hills and Kadayam ranges in Tirunelveli District, and in Kodayar in Kanyakumari District of Tamil Nadu.This region is generally termed the Agastyamalai range.This is the southernmost range of the species, and has extensive cliffs, undulating grasslands, steep gradients and deep gorges favourable to the Tahr.The largest population is along the western flank of the Agastyamalai adjoining Kerala (Vergis 2011;Hopeland 2012).
This region receives rainfall from the south-west and north-east monsoons, and rainfall decreases from the western crest of the Ghats towards the east.The topographical diversity and varied climate supports rich and diverse vegetation ranging from tropical wet evergreen forests, dry deciduous forests, savannah woodlands and grasslands (Roy et al. 2015).
METHODS AND MATERIALS
The survey was carried out in the dry season (January-June) of 2012 and 2013 in Kalakad-Mundanthurai Tiger Reserve (KMTR) and Kanyakumari Wildlife Sanctuary (KWS), covering an area of 1,220km 2 .We included the results of an earlier survey in 2009-2010 in the Ponmudi range of Kerala which is part of the Agastyamalai range (Vergis et al. 2011).A total of 24 sites known to support populations of Tahr, including sites surveyed at different times by various investigators (Daniel 1970;Davidar 1978;Daniel 1987;Mohan et al. 1998) were surveyed on foot.
In each site, transects were walked along the ridge or cliffs, where several vantage points were chosen which gave a good perspective on the landscape.The habitats surrounding these vantage points were thoroughly surveyed between 0600 to 1800 hours with an Olympus 10 x 50DPS I binoculars and a Celestron C 70 Mini Mac spotting scope with 90X magnification.
The surveys were carried out over consecutive days within a short period of time.Time taken to cover a site was considered a session.Each effort day had a minimum of one session and a maximum of two depending on the size of the site.The number of effort days per site ranged from one to five.This was replicated at every site possible over two seasons.
A Nikon D200 SLR camera with 55-300 mm lens was used to photograph Tahr whenever sighted.Detailed perusal of photographs can aid in enumerating animals in larger herds, avoiding double counts, and classifying animals by sex and age.Notes on location, time of sighting and herd structure, together with photographic database helped avoid recounts of individuals.Data were omitted where there were possibilities of double count of individuals or herds.After these precautions, the highest number sighted in a site in any single session was taken as the final count.
The geographical coordinates and elevation (amsl) of each site were recorded using a global positioning system.Signs of Tahr such as fresh faecal pellets were noted and indicated on the map (Fig. 1).In some sites where only Tahr faecal pellets were available, the pellets were compared with those of sympatric ungulates such as the Sambar Rusa unicolor and Barking Deer Muntiacus vaginalis for similarity.Tahr faecal pellets can be distinguished as they are smooth, compact and slightly elongated or elliptical shape as opposed to coarse, dark and larger pellets of the Sambar and the latrine type defecation piles of Barking Deer (Jayson & Easa 1997).In addition, the habitat in which the pellets are found, i.e., rocky outcrops are indicative of Tahr presence.However, there are possibilities of error and therefore only definitive records are included.
In addition to Tahr sightings, past estimates for each site were obtained from literature if available, and by discussion with local informants familiar with the study area.The sightings and estimates were tabulated.The population was designated as "large" if the sightings were over 30 and "small" if lesser than 30 animals.
The animals were classified as pre-reproductive (kids less than one year old and yearlings between one and two years old) and reproductive, based on size.Kids were from the present breeding season and yearlings from the previous breeding season.Larger animals approximately 80cm at the shoulder were classified as adults (Rice 1984).We correlated the number of sightings in each site with the proportion of young animals using Spearman's rank correlation to see whether larger populations had proportionately more young than smaller populations.
In each site the major threats were noted from personal observations and discussions with local people.Present threats were illegal hunting, livestock grazing, tall grass cover and human disturbance through noise, harvesting of forest products, tourism, feral dogs, mining and other activities.Information on past threats was obtained from informal and open-ended interviews with local people particularly elders who had a good knowledge of the area.The threats were categorised qualitatively as being present or absent.
We mapped the sites with currently observed populations and all other sites formerly surveyed and resurveyed by us, with or without evidence of Tahr present using ASTER-GDEM satellite data, a product of NASA and METI.We compared our sightings to estimates provided in literature.We correlated the number of sightings with percentage of young (kids and yearlings) using a Spearman rank correlation.
RESULTS
A total of 247 Tahr was sighted in 10 of the 25 sites surveyed and faecal pellets were recorded in seven additional sites.There was no Tahr in four sites where they had been reported earlier.Fourteen sites had no clear published information on presence of Tahr, and of these four had no current evidence of Tahr (Table 1, Fig. 1).
Thirty and more individuals were sighted in only two sites: Kalamalai-Varraiattumudi and Muthukulivayal-Balamore in Kodayar.The rest of the sites held fewer than 30 animals (Table 1, Fig. 1).
Totally there were 49 young animals in six sites which constituted 20% of the population.The highest proportion of young was in the Tiruvanamalai mottai (36%) and Muthukulivayal-Balamore (26%).Young animals were not recorded in four sites with fewer than 30 Tahr.There was a significant positive association between the total number of sightings and percentage of young Tahr (Spearman rank correlation = 0.73, n=9, p <0.05).The major threats were tall grass cover in 20 sites (80%), illegal hunting of wildlife in 16 (64%) including all sites with Tahr presence, and human disturbance in 16 (64%) sites (Table 1).Livestock grazing was prevalent in 6 (24%) sites.No threats were reported from Kottanguthati and Kanuni (Table 1).
DISCUSSION
This study represents the first comprehensive survey of the Tahr in its southernmost range in the Western Ghats.Our surveys recorded Tahr in only ten out of 25 sites.Thirty and more individuals were sighted in only two sites: Kalamalai-Varraiattumudi and Muthukulivayal-Balamore in Kodayar.The former is contiguous with Neyyar Wildlife Sanctuary in Kerala, where Vergis et al. (2011) recorded 76 individuals in 2010 and 103 in the present survey.The remaining eight sites had small numbers consisting mostly of a single herd.
This historically widespread population in the Agastyamalai landscape is now isolated forming four clusters with the Ponmudi range in the north-west, the Kadayam range in the north-east, Kodayar range in the south-west and Tiruvanamalai Peaks in the south-east.
We could not find any evidence of Tahr in four sites where they had been reported earlier, which includes Kottanguthati and Kanuni (Webb-Peploe 1946-47), where pellets were observed but no definitive presence as local residents had not sighted any Tahr there for decades (P.Davidar pers.obs.).Hence it was included among sites with no Tahr presence.The loss of Tahr in many sites could be because of several reasons: Hunting was widely prevalent in the past (Davidar 1978;Daniel 1987) and was also reported in 16 (64%) out of 25 sites by local informants.There are eyewitness reports of illegal hunting of Tahr in this region in the past (Daniel 1970;Davidar 1978;Mohan et al. 1998) and could be among the reasons for its absence in certain sites.Illegal hunting is a major threat to Tahr over its range (Davidar 1978;Mishra & Johnsingh 1998;Vergis et al. 2011).Unless stringent steps are taken to curb illegal hunting, many small populations will be decimated.
The Tahr in the Agastyamalai landscape occurs from an elevation of 650-1,400 m.Larger populations seem to thrive at elevations above 1000m where there are extensive grasslands, cliffs and protection from low elevation habitats.This suggests that inaccessibility protects populations from threats such as poaching and other human caused disturbances.Loss of short grassland habitats due to successional processes has been previously reported (Vergis et al. 2011).In regions with high rainfall, forests can replace grasslands in about 20-30 years in the absence of fire (Bond & Parr 2010).This process can make the habitat unsuitable for Tahr due to lack of forage and increased susceptibility to ambush predators such as the tiger and leopard.Sankaran (2009) has found that tall grass communities were least used by herbivores at all elevations in KMTR.Cymbopogon flexuosus, the dominant tall grass species is highly resistant to burning and can recover quickly to its original state if herbivore grazing pressure is low (Sankaran 2005).The low densities of wild grazing herbivores in KMTR are probably another reason for the dominance of tall grasses (Sankaran 2005).However, tall grass cover can be controlled with fire management.
Livestock grazing was extensive in the past, although has considerably declined.
Livestock could have transmitted diseases like rinderpest and foot and mouth to wild ungulates (Sankar 2008).However, evidence is lacking.
In conclusion, Tahr was sighted in only 10 of 25 sites over the course of this study, and eight of these populations were very small.Pellets were noted in some sites, but here again it is not a definitive indicator of Tahr presence.Illegal hunting appears to be the major cause for decimation of the Tahr, and continues to be a threat to remnant populations.
Landscape level planning using scientific tools and models can help identify areas where corridors can be placed with minimum impacts, and sites where Tahr could be reintroduced to maintain metapopulation dynamics at the landscape scale.
F
fi g u r e 1 .T h e s t u d y r e g fi o n w fi t h l o c a fi o n o ff N fi l g fi r fi T a h r s fi t e s , fi n d fi c a fi n g p r e s e n c e , fi n d fi r e c t e v fi d e n c e a n d a b s e n c e .T h e s am e n um b e r fi n g o ff s fi t e s fi s ff o l l ow e d fi n T a b l e 1 a n d F fi g . 1 ff a c fi l fi t a fi n g c r o s s r e ff e r e n c e .S fi t e S fi g h fi n g s o r p e l l e t s T h r e a t s E a r l fi e r e s fim a t e s 1 K u d fi r a fi v efi C l fi ff s N A H D G L N A : l o c a l fi n ff o rm a n fi r am a l a fi -P e r fi n j a v fi a l -N o o lm u d fi -K a l am a l a N A H D G L 3 0 : M o h a n e t a l . 1 6 ) s u r v e y fi n 2 0 0 0 -2 0 0 1 . 1 1 : V e r g fi s ( 2 0 1 1 ) 2 5 C h emm u n j fi p e a k s N A H D N A T a b l e 1 .S umm a r y t a b l e w fi t h l fi s t o ff s t u d y s fi t e s .S e r fi a l n um b e r s a r e s am e ff o r F fi g u r e 1 .T a h r s fi g h fi n g s o r ff a e c a l p e l l e t s ( p ) a r e fi n d fi c a t e d .T h r e a t s a r e : t a l l g r a s s c o v e r ( G ) , fi l l e g a l h u n fi n g ( H ) , h um a n d fi s t u r b a n c e fi n c l u d fi n g n o fi s e , p l a n t h a r v e s fi n g a n d b a r r fi e r s ( D ) a n d l fi v e s t o c k g r a z fi n g ( L ) .N o t a v a fi l a b l e ( N A ) . | 2018-12-29T16:30:10.459Z | 2016-06-26T00:00:00.000 | {
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13859523 | pes2o/s2orc | v3-fos-license | Maximal displacement in a branching random walk through interfaces
In this article, we study a branching random walk in an environment which depends on the time. This time-inhomogeneous environment consists of a sequence of macroscopic time intervals, in each of which the law of reproduction remains constant. We prove that the asymptotic behaviour of the maximal displacement in this process consists of a first ballistic order, given by the solution of an optimization problem under constraints, a negative logarithmic correction, plus stochastically bounded fluctuations.
Introduction
We study a branching random walk on R.This process starts with one particle located at the origin at time 0.Then, at each time k, every particle currently in the process dies, leaving a certain number of children, which are positioned around the position of their parent, according to independent versions of a point process, whose law may depend on the generation of the parent.It is supposed that each particle makes at least one child, so the process survives almost surely.
When the law of the point process (which we also refer to as branching mechanism) does not depend on the generation of the individual, and satisfies some integrability conditions, the asymptotic of the maximal displacement is fully known.Kingman [11] and then Biggins [6] proved this maximal value grows at linear speed.Later, Hu and Shi [9] proved the existence of a logarithmic correction in probability, with almost sure fluctuations ; and Addario-Berry and Reed [2] showed the tightness of this quantity around this correction.Finally, Aidékon [3] proved the convergence in law of the recentered value.
Recently, Fang and Zeitouni [16] introduced a new model of branching random walk through an interface.For all n ∈ N, they studied branching random walks with binary Gaussian branching mechanism, first with variance σ 2 1 until the generation n 2 , then with branching mechanism σ 2 2 between the generation n 2 and the generation n.They observed that the behaviour of such branching random walks depends on the chosen order for the variances, and that the logarithmic correction exhibits a phase transition.It is possible to extend their results to more general time-inhomogeneous environments.Let t ∈ (0, 1), and L and L two branching mechanisms.For all n ∈ N, the model is defined as follows: during the first p = ⌊nt⌋ generations, individuals reproduce according to independent copies of L. During the next n − p generations, they follow L. We find three possible regimes for this model.In the first case, which we call "slow regime", the rightmost individual at time n is the descendent of one of the rightmost individuals at time p and the resulting log-correction is the sum of two log-corrections of homogeneous branching random walks, one for each environment.
In the second case, the "mean regime", the behaviour is similar to the one of a homogeneous branching random walk, and the interface has no effect.
Finally, in the third case, the "fast regime", an anomalous spreading is observed.The first order is then larger than in the other two cases, and the log-correction is much higher too.In this case, the maximum of the branching random walk behaves as the maximum of a large number of independent random walks: the branching structure is forgotten.
Description of the model and notation
Let T (n) be a tree of height n; for all x ∈ T (n) • V (n) (x) ∈ R is the position of the individual x; • |x| is the generation of x; • x k is the ancestor at generation k of x; the displacement of the children of x with respect to its position; • for all y ∈ T (n) , x ∧ y is the most recent common ancestor of x and y.
We also write M n = max |x|=n V (n) (x) the maximal displacement in a branching random walk.Let (L k , k ≤ n) be a set of branching mechanisms.A branching random walk (V (n) (x), x ∈ T (n) ) in time-inhomogeneous environment (which we refer to as BRWtie), is defined as follows: {L (x) , x ∈ T (n) } is a set of independent branching mechanisms, and L (x) has the same law as L |x| .The BRWtie is said to have branching mechanism L k at generation k.
In this article, the studied model is the branching random walk through an interface (BRWi), whose law we now define.Let t be a fixed number in (0, 1), for all n ∈ N, set p n = ⌊nt⌋, writing p if n is clearly defined in the context.Let L and L be two branching mechanisms ; the BRWi is a BRWtie with branching mechanism L during the first p generations, and L during the next n − p ones.The interface is said to be at position t.
In the sequel, it is assumed that for all branching mechanism L P(L = ∅) = 0 and In particular, under these assumptions, the population never gets extinct and grows exponentially fast.It is also assumed that the branching mechanisms are non-lattice, i.e.
We now introduce some functions linked to the branching mechanism L. For all θ > 0, set its Laplace transform and for all a ∈ R its Cramér transform.We later write κ the Laplace transform of L and κ * k the Cramér transform of L k without necessary recalling the notation.
In the context of branching random walk, we write which is the speed of a branching random walk with branching mechanism L (see Biggins [7]).Similarly, v is the speed of the branching random walk with branching mechanism L.
Let L and L be two given branching mechanisms.We make the assumption that there exist θ * > 0 and θ * > 0 such that Moreover, set which we assume to be finite.We finally add the following second order integrability condition, which is not necessary, but simplifies the proofs: Throughout the paper, the notation O P (1) stands for stochastic boundedness: let X n be a random sequence, then X n = O P (1) if for all ǫ > 0, there exists A > 0 such that P(|X n | ≥ A) ≤ ǫ for all n ∈ N.
Remark 1.1.To simplify notation, we denote in the sequel c, C two generic positive constants which may change from line to line, or even within line.Those constants are respectively small and large enough, and only depend on the branching mechanisms and the constant t > 0. .Under these assumptions, for a time-homogeneous branching random walk, the following theorem holds.Let (U (x), x ∈ T) be a branching random walk with branching mechanism L, and Theorem 1.2 (Addario-Berry and Reed, [2]).The position of the rightmost individual at time n in U is given by For BRWi, same kind of theorems holds.First, for the "slow regime", in which two logarithmic corrections adds.
For the "mean regime", in which the behaviour is no different for a timehomogeneous branching random walk, the log-correction is significantly smaller in absolute value.
In the case of the "fast regime", as the first order is modified, we need an extra notation.We suppose there exists θ > 0 such that and we write a = κ ′ (θ), b = κ ′ (θ).Observe that in this case Theorem 1.5 (Fast regime).If θ * > θ * , then the position of the rightmost individual is given by These three theorems give a full description of the possible behaviours of BRWi.This behaviour shows a phase transition for the log-correction.While the speed varies continuously with respect to the two branching mechanisms, there is a discontinuity in the second order, when θ * is getting close to θ * .
Heuristics
Biggins [7] proved that in the case of a time-homogeneous branching random walk with branching mechanism L, for all a ∈ R, e −nκ * (a) represents either an approximation of the number of individuals in a neighbourhood of na at time n, or the probability that there exists one such individual, depending on whether κ * (a) > 0 or not.
We now consider a BRWi with branching mechanisms L and L. For all a ≤ v, there are at time p about e −pκ * (a) individuals close to pa.Each of these individuals begins an independent time-homogeneous branching random walk with branching mechanism L. The probability to have an individual to the right of (n − p)b at generation n − p for one of those branching random walks is of order e −(n−p) κ * (b) .As a consequence there are individuals in a neighbourhood of pa + (n − p)b at time n with an ancestor at time p close to pa if tκ * (a) + (1 − t) κ * (b) < 0, and a < v.
We then expect that the first order for the maximal displacement in the BRWi is given by v = max with To describe the logarithmic correction in M n , it is enough to study the path which leads an individual to the rightmost position.Set (a, b) ∈ A attaining the maximum in (11).The decomposition into three regimes is made in the following way: if κ * (a) < 0 (or equivalently a < v), then v > tv + (1 − t) v and we are in the fast regime.In this case, the path followed by the individual which realise the maximal displacement at time n stay away from the boundary of the process most of the time, and the logarithmic correction is small.
If κ * (a) = 0, we are either in the mean or the slow regime.Set If v T = v, we are in the mean case.The restriction κ * (a) ≤ 0 is not important in the optimization problem (11).As a consequence, the path leading to the rightmost position at time n stay close to the boundary of the process at all time, as in the homogeneous case, so the logarithmic correction are the same.
In the slow regime, v T > v, therefore, the restriction κ * (a) ≤ 0 is crucial in (11).For the individuals, this means that being close to the boundary at time p gives an important advantage to the descendants.The rightmost individual at generation n is a descendant of one of the rightmost individuals at generation p.As a consequence, the logarithmic correction is the sum of the two timehomogeneous corrections obtained in the two phases of the process.
The next section presents some preliminary lemmas; Section 3 contains the proof of Theorem 1.3, Section 4 the proof of Theorem 1.4 and Section 5 the proof of Theorem 1.5.An extension of these results to multiple interfaces is discussed in the final section.
Some computations in the branching random walk
In this section, we make some computations that are valid for a general tieBRW with branching mechanism L k at generation k written (V (x), x ∈ T).We recall that κ k (θ) is the Laplace transform of L k .
The many-to-one lemma
Let θ ∈ R + ; we suppose that for all k ≤ n, κ k (θ) < +∞.We denote by (X k , k ≥ 0) a sequence of independent random variables such that for all measurable nonnegative function f For all n ≥ 0, set S n = n k=1 X k .The many-to-one lemma links moments of some functionals of the branching random walk and of (S n , n ≥ 0).This result has been stated in many forms, going back at least to the article of Kahane and Peyrière [10].
Lemma 2.1 (Many-to-one lemma).Let n ∈ N and f : R n → R be a measurable non-negative function, we have Proof.This result can be proved by induction.For n = 1, the statement is exactly the definition of the law of S 1 = X 1 .We assume that the statement is true for some n ∈ N. Let f : R n+1 → R be a measurable non-negative function.Using the branching structure, we get where Finally, using the induction hypothesis, we obtain which ends the proof.
Remark 2.2.Under nice integrability conditions, As a consequence, writing
Bounds for the probability of the existence of certain paths in the branching random walk
In this section, we apply the many-to-one lemma to compute the moments of the number of individuals which remain in some specific paths.These moments allow to bound the probability that M n is above a certain level.For all x ∈ T, we denote by H(x) = (V (x j ), j ≤ |x|) the path followed by x.For all n ∈ N and k ≤ n, let φ n (k) ∈ R be the deterministic path to follow.Let y ≥ 0 and θ > 0, we suppose that for all k ≤ n, κ k (θ) < 0. We write S the time-inhomogeneous random walk obtained using the many-to-one lemma with the parameter θ, and In a first step, we compute the probability there exists an individual which crosses at some time the upper frontier (φ n (k) + y, k ≤ n).To do so, denote the set of paths which can be followed by individuals crossing for the first time at time k the upper frontier, and the probability that S belongs to this set.Proof.By the Markov inequality and the many-to-one lemma This lemma can be used to prove the upper bound in Theorem 1.2 Corollary 2.4.Let (U (x), x ∈ T) be a branching random walk with branching mechanism L such that ( 6), (7) and (2) are satisfied.We denote There exists C > 0 such that for all y > 0 Proof.Observing that it is enough to only prove the second inequality.Applying Lemma 2.3 to the branching random walk U and the function φ n , there exists C > 0 such that where and ξ n,k (y) = P(S k > φ n (k) + y, S j ≤ φ n (j) + y, y < k), for S a classical random walk.By use of Corollary 2.9, proved in the next section, there exists (n + 1) < +∞, so there exists C > 0 such that In a second step, we bound from below the probability there exists an individual to the right of φ n (n) + y at time n.To do so, let the admissible interval sequence, in which we are looking for individuals which arrive in a neighbourhood of φ n (n) at time n.We write, for all n ∈ N and k ≤ n It is then assumed that there exists C = C(y) > 0 and c n,k such that for all We also suppose that there exists a constant Φ such that and we finally denote The following estimate holds: Lemma 2.5.For all y ≥ 0, there exists C > 0 such that for all n ∈ N we have This lemma can be used to obtain the lower bound for M hom n in Theorem 1.2, but we skip this proof, very similar to the one used for the study of the mean regime.
Proof.We write Y n (y) = |x|=n 1 {H(x)∈Ω φ n (y)} .Using the many-to-one lemme, we have We now bound from above the second moment of Y n (y) using the branching structure using the Markov property, where and U k,n is a branching random walk with the same law as the descendants of an individual at generation k in the branching random walk V , i.e. with branching mechanism L k+j at generation j.By use of the many-to-one lemma by assumption (12).Moreover, using assumption (13), there exists As a consequence, we obtain Using once again the many-to-one lemma, we obtain with ρ n,k the quantity defined in (14).Therefore, for any y ≥ 0, there exists C(y) > 0 such that Finally, by the Cauchy-Schwarz inequality, we have Lemmas 2.3 and 2.5 enable us to bound from above and from below the probability there are individuals in a neighbourhood of φ n (n), as soon as it is possible to obtain bounds for the probabilities ω, π and ξ.In the next part, we state some results which will allow us to obtain those, for a branching random walk through an interface.
Some random walks estimates
We first recall here some classical results for random walks, then state some extensions that applies to branching random walks through an interface.Let (X n , n ∈ N) be i.i.d.non-lattice random variables (i.e.there exists no (a, b) ∈ R 2 such that X 1 ∈ aZ + b a.s.) such that E(X 1 ) = 0 and 0 < Var(X 1 ) < +∞.
For all n ∈ N, denote T n = n j=1 X j .This first lemma, which is often called the ballot theorem, computes the probability for a random walk to stay above a constant.It is found in Kozlov [12], see also [1] for a review on such results.Lemma 2.6.There exist 0 < c < C such that for all y ∈ R + and n ∈ N, we have The next lemma is a consequence of the Stone local limit theorem [15], which bounds the probability for a random walk to end up in a window of finite size.Lemma 2.7.There exist 0 < c < C, and L > 0 such that for all h ≥ L and n ≥ 1 We now state an extension of Lemma 2.6, to estimate the probability for a random walk to stay above a barrier of the form −j α at any step j ≤ n, for some 0 < α < 1 2 .Lemma 2.8.Let α ∈ (0, 1 2 ), there exists C > 0 such that for all y > 0 and n ≥ 1, we have The proof of this result is postponed to Appendix A. We end this section with a bound for the probability for a random walk to make an excursion over some given line.A corollary, which can be found under a modified form in [5], Annex A, compute the probability the random walk makes a bridge above some barrier.
Corollary 2.9.Let X, Y, Z be three independent random variables such that We denote (X k ), (Y k ) and (Z k ) three sequences of i.i.d.random variables with respectively the same law as X, Y and Z.For all p, q, r ∈ N 3 , we denote Z l else .
There exist c, C > 0 such that for all y, h > 0, and p, q, r ∈ and moreover Proof.These bounds are obtained by dividing the event into three parts.We begin with the upper bound.First, using the Markov property at time p, we have where By use of Lemma 2.6, We write W j = W n−p − W n−p−j the time-reversal random walk, using the Markov property at time r, and then Lemmas 2.8 and 2.7.
To obtain the second upper bound, it is enough to condition with respect to the value of The lower bound is obtained using Lemma A.2 in [5], and similar arguments as above.
Slow regime in the branching random walk through an interface : proof of Theorem 1.3
Recall that (V (n) (x), x ∈ T (n) ) is a BRWi, with branching mechanism L during the p = ⌊nt⌋ first steps, and L in the n − p next ones.In this section, it is assumed that θ * < θ * , the characterization of the slow regime.We denote Theorem 1.3 can then be proved using the estimates obtained for a timehomogeneous branching random walk.
Proof of Theorem 1.3.Let ǫ > 0. We begin with a proof of the lower bound.The first p steps of the BRWi are the same steps as those of a time-homogeneous branching random walk, with branching mechanism L. Consequently, using the Theorem 1.2, there exists y 1 > 0 such that In the same way, if there is an individual x 0 to the right of vp − 3 2θ * log p − y 1 at generation p, its descendants reproduce using a branching mechanism L. Using again Theorem 1.2, there exists y 2 > 0 such that with probability greater than 1 − ǫ 2 a descendant of x 0 at generation n is to the right of m n − y 1 − y 2 .
To conclude, there exists y = y 1 + y 2 such that In a second step, we study the upper bound.We denote, for all k ≤ p Using Corollary 2.4, there exits C > 0 such that so for all y > 0 large enough, with high probability, individuals do not hit this barrier.We compute the number of individuals which remain below this boundary and arrive in a neighbourhood of φ n (p) + y at generation p. Set By the many-to-one lemma, we have for all thanks to the upper bound in Corollary 2.9.
Let ( U (x), x ∈ T) be a time-homogeneous branching random walk with branching mechanism L. Using once again Corollary 2.4, we observe there exists K > 0 such that for all y > 0 We denote by M n−p a random variable with the same law as max |x|=n−p U (x), and b n = v(n − p) − 3 2 θ * log(n − p).We finally bound from above the probability that there is an individual at time n to the right of m n + y by looking at the position of its ancestor at time p.We have Using these two bounds, we finally deduce
Behaviour in the mean regime : proof of Theorem 1.4
In this section, we consider a BRWi verifying θ * = θ * =: θ, so we are in the mean regime.As a consequence, in this case Set where we denote k ∧ p = min(k, p) and (k − p) + = max(k − p, 0).Using lemmas in Section 2, we prove that with high probability no individual crosses that boundary.
Lemma 4.1.
There exists C > 0 such that for all y ∈ R and n ∈ N Proof.First, observe that Then, using Lemma 2.3 and by use of Corollary 2.9, we obtain ≤ C(1 + y)e −θy .
WE will now prove that with positive probability, there exists an individual which remain below the curve φ n (k) + y at time k ≤ n and arrives at time n above m n .
Lemma 4.2. There exists
Proof.We use Lemma 2.5 with functions b n (k) = 0 and There exists C > 0 and y > 0 such that In this formula, which can be bounded using Corollary 2.9 .
Moreover, using this same corollary, we define and observe that for all u ≥ 0 Recall also that ρ n,k = E e θS k −(k∧p)κ(θ)−(k−p) + κ(θ) 1 {Sj≤φn(j)+y,j≤k} , by decomposition of the expectation in two parts, whether or not S k ≥ kv − 3 2θ log k.In the same way, for all k > p .
As a consequence which is bounded uniformly in n.
These two results give an upper and a lower bound for the position of the rightmost individual at time n.We now prove Theorem 1.4.
For the lower bound, we denote by (U (x), x ∈ T) and ( U (x), x ∈ T) two independent branching random walks with branching mechanism respectively L and L. Lemma 4.2 proves that there exists c > 0 such that for all n ≥ 0 and thanks to (1) there exists h ∈ N and y 1 ∈ R such that Let n ∈ N, we decompose the branching random walk V (n) as follows : • it begins with the h first steps of the branching random walk U , which gives, with probability at least (1 − ǫ 3 ), N individuals to the right of y 1 ; • at least N independent branching random walks with the same law as V (n−⌈ h t ⌉) all starting to the right of y, with probability at least 1 − ǫ 3 at least one of them makes a child to the right of m n−⌈ h t ⌉ + y 1 , • at most k steps of one branching random walk U starting from m n−⌈ h t ⌉ + y 1 , which gives, with probability at least 1− ǫ 3 a child at position As a consequence, there exists y > 0 such that which ends the proof.
As usual, m n is defined by and we begin by proving there is no individual to the right of m n at time n with high probability.
Lemma 5.1.There exists C > 0 such that for all y > 0 we have This result is the same as the one we could expect for large deviations in a simple random walk.As a consequence, there is no need to keep track of the branching structure here.
Proof.By use of the many-to-one lemma, we have and using Stone's local limit theorem we obtain that As a consequence, using the Markov inequality, which yields and this conclude the proof.
We now look for a lower bound for the position of the rightmost individual.To do so, we prove that there are individuals which stay, at any time k ≤ n in a neighbourhood of the path Lemma 5.2.There exists c > 0 such that Proof.Let A > 0, and Applying Lemma 2.5 with function φ n , b n (k) = −a n (k) = r n (k) yields that there exists y > 0 such that .
We recall that I n (j, y) = [φ n (j) − r n (j), φ n (j) + r n (j) + y], and By Lemma 2.7, we have Secondly, c k,n is defined in a way such that but using once again Lemma 2.7, we have In a third step, we compute and if k > p using Lemma 2.7 Using inequality (15), we obtain Adding up these inequalities, there exists c y > 0 such that , which ends the proof.
Those last two lemmas are enough to prove Theorem 1.5, using the same argument as in the proof of Theorem 1.4.
Proof of Theorem 1.5.Let ǫ > 0. Using Lemma 5.1, there exists y > 0 large enough such that for all n ∈ N P(M n ≥ m n + y) ≤ ǫ.
For the lower bound, we use exactly the same cutting argument as in the study of the mean regime.In a few steps, a large number of individuals are created, each of them having a descendent at generation n to the right of m n with positive probability.The few remaining steps do not change the order of the position.As a consequence, there exists y > 0 such that which ends the proof.
Concluding remarks
We have exhibited here a phase transition, already known to Zeitouni and Fang [16] in the case of binary Gaussian branching random walks.We have Using the strict convexity of κ * and κ * we notice that this decomposition is unique.We write θ = (κ * ) ′ (a) and θ = ( κ * ) ′ (b).Using Lagrange theory, we may notice that θ ≤ θ and θ < θ only if κ * (a) = 0.
With these notations, we resume the results in the following way This result is easily extended to a succession of interfaces.Let 0 We consider a branching random walk with K − 1 interfaces at positions (α i , i ≤ K).In other word, for all n ∈ N, V (n) is a branching random walk with branching mechanism L i at any generation k such that α i−1 ≤ k n < α i , for all i ≤ K. Writing Using once again Lagrange theory, this quantity is equal to and if the maxima are reached respectively at (a We denote by L the quantity 2φ p .
We finally have, under suitable integrability hypothesis Heuristically, the reason for this result is that the optimal path leading to the rightmost individual sticks to the frontier of the branching random walk whenever θ k increases (slow interface).As a consequence, the total logarithmic correction is the sum of the logarithmic corrections in each of the "eras" with constant optimal parameter.The logarithmic parameter in one of these eras depend on whether the optimal path stay close to the frontier at the beginning or at the end of the era (mean regime) or not (fast regime).
Finally, observe that the integrability conditions we asked in (13) are far from being optimal.These are used to compute the second moments, but we could have restricted our sets of individuals to those with few children.This was done in Aidékon [3], using the spinal decomposition.However, such truncation arguments require more technical details which we skipped.
A Proof of the Ballot extension
Proof of Lemma 2.8.Let y ≥ 0 and α < 1 2 .We compute the probability for a random walk to stay above a frontier −j α − y.To do so, we decompose this path into a sequence of hitting times on different levels.In the sequel, S k = min j≤k S j , and for all z ≥ 0, T z = inf{k ≥ 0 : S k ≤ −z}.
Let 0 < λ < 1 and k ∈ N.For all n ∈ N, by using the Markov property at time T (2k) α +y ∧ λn, we have As a consequence, choosing λ < 1 3 and k large enough such that K < 1, and then p ≥ − log n log K , this last inequality can be rewritten which ends the proof. | 2017-06-12T13:31:31.000Z | 2013-05-27T00:00:00.000 | {
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225973368 | pes2o/s2orc | v3-fos-license | In-Vitro Antioxidant Activity and Total Phenolic Content of Ruta montana L. Extracts
The aim of this study is to evaluate in vitro antioxidant activities of Ruta montana L. extracts. This activity was evaluated by three methods : DPPH (2, 2'-diphenyl1picrylhydrazy), bleaching of β-carotene and chelation of ferrous iron. Results showed that ethyl acetate extract (EAE) represents the highest amount of total polyphenols, tannins and flavonoids with 257,1 ± 0,703μg gallic acid equivalent/mg of extract, 251 ± 1.41 μg tannic acid equivalent /mg of extract,117,4 ± 3,451 μg quercetin equivalents/mg of extract, 139,5 ± 4,107 μg rutin equivalents/mg of extract, respectively. In the DPPH assay, ethyl acetate extract showed the higher scavenging capacity (IC50 = 0.044 ± 0.001 mg/ml) followed by methanol, aqueous and chloroform extract. Whereas, AqE showed the best chelating effect and the best inhibitory capacity of the coupled oxidation of linoleic acid/ β-carotene.
INTRODUCTION
Free radicals are continuously produced by the body's normal use of oxygen such as respiration and some cell mediated immune function [1]. Oxygen is an element indispensable for life. When cells use oxygen to generate energy free radicals are produced by the mitochondria. These by-products are generally reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) that result from the cellular redox process. The free radicals have a special affinity for lipids, proteins, carbohydrates and nucleic acids [2]. Some of these free radicals play a positive role in vivo such as energy production, phagocytosis, regulation of cell growth and intercellular signaling and synthesis of biologically important compounds [3]. At high concentrations, free radicals leads to damaging the lipids in the cell membranes, proteins in tissues as well as enzymes, carbohydrates, and DNA to induce oxidation. This oxidative damage may play a causative role in aging and several diseases which are cancer, cardiovascular disease, cataracts, and cognitive dysfunction [4]. propagation of free radical reaction [5]. Algeria. The plant has been used in Algeria as a cure for emmenagogue, antispasmodic and rubefiant [6].
The aim of the present study was the evaluation of the antioxidant activity of different extracts from the aerial part of Ruta montana L. using different in vitro tests and the total phenolic, flavonoids and tannins contents.
Plant material
Ruta montana L. was collected in October, from Beniaziz region, Wilaya of Sétif in Northeast of Algeria.
Aqueous extract
The aerial parts of plant material were cleaned with tap water, dried in the shade at room temperature for 2 weeks and ground into powder using an electric grinder. Briefly, 100g of Ruta montana L. powder was mixed with 1L of boiled distilled water (100 °C) and was placed at room temperature during 72h, The resulting mixture was filtered using Wattman filter paper n°3 and then evaporated in rotary vacuum evaporator at 45°C.
Methanolic extract (Crude extract)
The methanolic extract was obtained by maceration in
Fractionation of methanolic extract
Fractionation of the crude extract is performed using a series of solvents of increasing polarity. The crude extract was initially mixed with the hexane (V / V) to eliminate lipids, and after separation, the upper organic phase was recovered.
This step is repeated several times with renewal of the solvent until it becomes transparent. The hexane is then evaporated and the resulting extract is considered as the fraction of hexane. The lower aqueous phase was subjected to another fractionation with chloroform to give the chloroform extract (ChE), and finally by ethyl acetate to give the fraction of ethyl acetate (EAE). All fractions were preserved at -20 o C until use.
Determination of total polyphenol content
Total phenolic content was determined using Folin-Ciocalteu method, according to Li and al, [8] with slight modifications.
A volume of 100 µl of the extract was mixed with 500 µl of Folin-Ciocalteau (diluted 10% in distilled water). After 4 min, 400 µl of sodium carbonate solution Na2CO3 (75 g/l) was added to the mixture, the reaction mixture was incubated at room temperature for 1h 30 min and the absorbance of the mixture was measured at 760 nm, Gallic acid (20-140 mg/l) was used as standard for the calibration curve. The total polyphenols content was expressed as micrograms of gallic acid equivalents (GAE) per milligram of extract. All samples were analyzed in three replications.
Determination of total flavonoids contents
The total flavonoids in plant extracts were determined using the aluminum trichloride (AlCl3) method [9]. Briefly, 1 ml of 2% AlCl3 in methanol was mixed with 1 ml of the extract.
After incubation in dark at room temperature for 10 min, the absorbance of the reaction mixture was measured at 430 nm. and RE, respectively) per milligram of extract.
Determination of tannins contents
The test of haemoglobin precipitation by tannins compounds was used [10]. Briefly, a volume of each plant extract was mixed with an equal volume of heamolysed blood (absorbance (576 nm ) = 1.6). After 20 minutes incubation, this mixture was centrifuged for 10 minutes at 4C and the absorbance of the supernatant was measured at 576 nm.
Different concentrations of tannic acid were also mixed with an equal volume of heamolysed blood and the absorbance was measured in the same manner. The tannins content was expressed as micrograms tannic acid equivalent per milligram of extract.
DPPH free radical-scavenging assay
The free radical scavenging activity of the extracts was measured by 2,2-diphenyl-1-picrylhydrazyl(DPPH) assay [11]. After dissolving the aqueous extract in distilled water, the methanol, chloroform and ethyl acetate extracts in methanol, the solution of DPPH in methanol (0.04mg/ mL) was prepared and 1250 µL of this solution was added to 50µL of extracts solution at different concentration. The mixture was shaken vigorously and then kept in the dark for 30 minutes at room temperature. Then, the absorbance was measured at 517nm. BHT, rutin, quercetin and gallic acid were used as standards. All tests were performed in triplicate.
Radical-scavenging activity was calculated using the following equation: radical scavenging activity (%) = (A blank -Asample / A blank) × 100 A blank: Absorbance of the control.
A sample: Absorbance of the reagent with extract.
β-carotene/linoleic acid assay
In this test, the antioxidant capacity of the extracts was determined by measuring the inhibition of the oxidative degradation of β-carotene (discoloration or bleaching) by the oxidation products of the acid linoleic [12]. Absorbance in the presence of positive control BHT.
Chelation of ferrous iron
The chelating ability of the extracts is determined according to the method of Le and al, [13]. Moreover, the negative control containing all reagents except that test sample is replaced by the same volume of methanol.
EDTA was used as reference chelator.
The chelating effect was calculated as a percentage, using the same equation as that in the DPPH assay.
Statistical Analyses
The results are expressed as the mean ± standard deviation.
One-way analysis of variance (ANOVA) followed by the Tukey test was performed to assess differences between groups.
Differences were considered significant at p < 0.05.
Total polyphenols, flavonoids and tannins contents
The contents of total polyphenols, flavonoids and tannins in extracts are shown in
β-carotene/linoleic acid bleaching assay
As can be seen in figure 1 and figure 2, all the extracts were capable of inhibiting the bleaching of β-carotene by scavenging linoleate derived free radicals. cancer and diabetes which occur due to the deregulation of free radicals generation in the cells [15].
Metal chelating activity
There have been two opinions on the correlation between phenolics and total antioxidant activity. Some reports demonstrated positive correlation between them [16,17] and the others showed no correlation [18]. Our results agree with the latter, it seems that no correlation exists either between the percentage inhibition (% scavenging effect) assayed by DPPH and total phenolics, between the antioxidant activity assayed by β-carotene blenching method and total phenolics, or between the antioxidant activity assayed by ferrous ion chelating ability and total phenolics. [19] thought this phenomenon can be explained on the basis of high antioxidant activity of some individual phenolic units, which may act as efficient antioxidants rather than contributing to high total phenolics, while [20] thought that total phenolics content did not include all the antioxidants, such as ascorbic acid, carotenoid and tocopherol. [21] held the idea that the synergism among the antioxidants in the mixture made the antioxidant activity not only dependent on the concentration of antioxidant, but also on the structure and interaction among the antioxidants. This probably is the reason why samples with similar concentrations of total phenolics may vary remarkably in their antioxidant activity.
In general the antioxidant activity of phenolic compounds reportedly varies with the structure and degree of hydroxylation of the aromatic ring [22,23]. It is associated with the number of hydroxyl groups and the most active possess from 3 to 6 hydroxyl groups. Hydroxylation in the C3 position seems to be detrimental for their antioxidant potency [24]. [25] reported that for benzoic and cinnamic acid Catechol ring and C2 -C3 double bond conjugated with 4-keto moiety are essential for electron delocalization from the ring B, and it increases the antiradical activity [26]. Many studies showed that when these structural features were removed from flavonoids structure their antioxidant activity was decreased [27,28].
CONCLUSION
The present study aimed to evaluate the Antioxidant activity of extracts prepared from the aerial parts of Ruta montana L. , this activity was tested in vitro using three methods : DPPH (2, 2'-diphenyl-1-picrylhydrazy), bleaching of β-carotene and chelation of ferrous iron. The results showed that Ruta montana L. has an important antioxidant activity. The ethyl acetate extract exhibited the highest scavenging activity, whereas aqueous extract showed the best chelating capacity and the best inhibitory capacity of the coupled oxidation system of linoleic acid/ β-carotene. (ANDRS). We express our gratitude to these organizations. | 2020-06-11T09:10:37.964Z | 2020-03-15T00:00:00.000 | {
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54743492 | pes2o/s2orc | v3-fos-license | Effect of Fruit Addition on the Quality Characteristics of Tahini Halva
The purpose of this study was to make Tahini Halva which incorporated with 10, 15 and 20% fruits (raisins and apricots) and to investigate effect on sensory and chemical properties of Tahini halva of addition of fruit during storage. Moreover, tahini halva produced plain compared with halva that added fruits. Dry matter, ash, crude fiber, protein, sugar, fat, acidity in oil which was extracted, peroxide and refractive index determinations were analyzed in Halva. According to statistical evaluation of the results, tahini halva containing 10% raisin had the best flavor for panelists. Change in flavor was found less than expected according to added fruit content, in this, it was thought due to the fact that flavor of tahini has intense enough to eliminate the fruit flavor. The amount of fat can be reduced proportionally with addition of fruit in halva, therefore, reduction in worth of the calories can be achieved.
Introduction
There are lots of traditional foods consumed through regional scale in Turkey (Tan, 2004). Tahini halva is one of the Turkish traditional confectionary foods and it is produced at industrial scale in Turkey. Tahini helva is also the most popular food in the Mediterranean and Middle East Countries such as Greece, Iran, Iraq, Jordan, and Saudi Arabia (Özdemir ve et al., 2006).
Non-fat fraction of sesame oil includes sesamol, sesame and sesamin compounds which do not find in other oils. These compounds maintain their properties after hydrogenation (Nas, 1998). Sesame oil shows a significant antioxidant effect level according to the amount of tocopherol in the composition. Sesamol, sesame and sesamin which is a natural antioxidant compounds also influence the stability of oil (Altug, 2001).
Cleaned and roasted sesame seeds are pressed to obtain sesame oil. Oil quality is influenced by the duration and temperature of the roasting. Sesame oil may be stored without deterioration for longer time than other vegetable oils (Özcan, 1993).
In our country, tahini consumed lovingly is the product obtained by crushing at the mill after coat of sesame seeds which is leaved is dried and parched (Anonymous, 1977).
To prepare sugar syrup is the first stage of making tahini halva. Crystal sugar joining 10-15% water is melted by mixing in a steam boiler. The desired structure wax is obtained by removing water from solution. To provide a bleaching solution of sugar, color return fully to white color by adding 0.1% soapwort extract. The resulting network in 1: 1 ratio mixed with tahini and flavoring agents are added. When the desired structure is, tahini halva is molded and packaged (Birer, 1985).
Problem of oil leakage is an undesirable condition for the manufacturers of halva in tahini havla. Storage temperature is a major factor on the amount of oil leaking in the tahini halva which has a physical mixture. In order to prevent this situation, soapwort extract which is within the group of substances in emulsifying property is used. Soapwort extract is used both to bleach the color of the sugar syrup and as an emulsifier (Anonymous, 1999).
Licorice extracts as well as soapwort extract can be also used as a bleaching agent and emulsifying (Yurdagel and Baysal, 1996) They are equivalent in terms of nutrients with the egg because of the fact that tahini and tahini halva have high levels of methionine and lysine amino acids and essential amino acids. It has also rich fat and carbohydrates (Güngör, 1993).
Tahini Halva which is produced as sesame paste sweetened in the Middle East is a good source of nutrients in terms of fat and protein. Sesame paste sweetened has a more streamlined structure than the tahini halva. However, it has been reported that it caused the problems in the filling stage because of high viscosity (Abu-Jdayil, 2004).
The aim of this study was to investigate the effects of addition dried fruit (raisin and apricot) and storage period on physicochemical and sensorial properties of tahini Halva.
Materials and Methods
Tahini halva of 200 kilograms was produced. Its composition was given in table 1. Tahini halva which prepared with the addition of raisins and dried apricots in ratio of 10-15-20% in the study were stored for 6 months at room temperature.
Preparation of tahini halva added dried fruit
Tahini halva which produces from tahini and sugar as the main ingredient is a thin fibrous product view. It is also obtained by adding drinking water, citric acid, soapwort extract and when need addition of flavoring agents (Anonymous, 1993).
Statistical analysis
The data were analyzed statistically using SPPS software (SPSS PASW 18.0) and the means were separated using the Duncan's multiple range test (p<0.01). Chi-Square test was used for sensory analysis (SAS, 2005).
Results and Discussion
In the amount of dry matter in the halva was observed a declining trend with the increase of fruit during storage due to amount of moisture in the fruit used and their hygroscopic as shown in Table 2.
When examining the effect of the rate of fruit on dry matter, it was shown that the effect of the fruit content on the dry matter in Table 3. As a result of analysis during storage, the amount of ash in the halva was determined to vary between 1.8170% and 1.9522%. It was thought that amount of ash in halva increased with addition of fruit. The amount of ash of halva with apricot and raisin, plain halva were shown in Table 3 according to Duncan multiple comparison results. TS 2590 foresees the presence of protein at least 11%. As shown in Table 4, % value of protein determined for our examples was determined to be appropriate to standard. As shown in Table 5, amount of sugar in Halva varied by depending on the added fruit and ratios of fruit.
The amount of sugar in tahini halva was shown in Table 5. As seen in Table 5, it was evaluated that amount of sugar was increased when amount of fruits were increased.
It has been reported to be maximum 47% by restricting the use of sugar in standard of halva. No significant change was observed in the amount of sugar during storage period. Amount of sugar in halva varied by depending on the added fruit and ratios of fruit.
It was identified that the amount of raisins was significant in p <0.01 level to effect on the amount of sugar in tahini halva with raisins, storage period was not significance in p <0.01 level (Table 6). Change of oil and tahini of halvas were shown respectively in Table 7, 8.
It was found that amount of oil in halva with raisins and plain halva were different each other and % oil value changed according to the fruit content. It has been reported in TS 2590 that at least 27.5% oil and 52% tahini should be found in halva.
Tahini halva added fruit was found suitable standard for the amount of oil and tahini ( As a result of Duncan multiple comparison test, It was shown in Table 9 that oil ratios of samples were shown close to each other during storage for halva added raisins. It was not observed to be a significant change in the amount of % oil in samples of halva by depending on the storage period. The differences between the products of halva was believed to be due to changes in the dry matter. *:Means within a column followed by the same letter were not significantly different at the 0.01 probability level, according to Duncan's multiple comparison test *Duncan çoklu karşılaştırma testine gore aynı sütunda aynı harfle gösterilen ortalama değerler 0.01 ihtimal düzeyinde birbirinden farklı değildir. % acidity values ranged between 0.7833 and 0.3067 during storage period and fatty acid values of the halva increased by depending on the storage time (Table 10). It was determined that developing acidity of Halva with raisin was different from plain halva during storage time. It was thought that addition of fruit were influenced the development of acidity because acidity of plain halva was lower than halva with apricot and raisins. It has been reported in TS 2590 that oil extracted from standard of tahini halva contains acid up to 2%. Changes of acidity in tahini halva did not show any change in the deterioration (Table 10).
When plain halva and Halva with apricot compared, it was determined that % acidity value was different for each sample. In this case, it was believed that development of acidity in halva with apricot was affected from the rate of the storage period and fruit. It has been reported in TS 2590 that peroxide number which is a measure of active oxygen from the oil is at most 10 meq kg -1 in tahini halva. Peroxide values ranged from 8.6413-0.7995 during storage time. It was observed to increase the number of peroxide during storage period. It was determined that halva contained more fruit content had higher peroxide number (Table 11). Peroxide number of halva with apricot and plain halva were different from each other (Table 11). It was thought to be effective of addition and the rate of fruit on different from each other of number of the peroxide determined for samples of halva.
As seen in Table 12, Plain halva had the lowest values for all parameters.
Duncan's multiple comparison test results of the peroxide value in the simple halva and halva with raisin was shown in Table 12. In results of evaluation, It was determined that number of peroxide of halva contained 15-20% raisin were the same each other. Because number of peroxide of halva contained 10% raisin was lower than other halva with raisins and plain halva had the lowest values of peroxide, it was thought that addition and the rate of fruit affected on increase of values of peroxide.
Taste-Aroma and Structure-Texture of halva were given in Table 13 during storage period.
Refractive index did not show changes during storage. It was determined that the refractive index of the fat obtained from halva added fruit was 1.472 at 25ºC. The refractive index for the sesame oil was reported to be between 1470 and 1474.
Color, odor, flavor-aroma and structuretexture were used for sensory analysis As a result of ANOVA made for the color were significant at the p< 0.01 level.
As a result of evaluation as statistical was determined that the difference between the smell of halva in p <0.01 level was not significant.
As a result of analysis of variance, the datas obtained for structure-texture were significant at the 0.01 level.
As a result of evaluation as statistical was determined that the difference between the smell of halva in p <0.01 level was not significant.
As a result of analysis of variance, the datas obtained for structure-texture were significant at the 0.01 level.
As a result of Duncan test performed on the datas obtained for the structure-textural, it was determined that structure of halva with | 2018-12-14T09:58:04.651Z | 2016-12-29T00:00:00.000 | {
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25926005 | pes2o/s2orc | v3-fos-license | Liver atrophy after percutaneous transhepatic portal embolization occurs in two histological phases: Hepatocellular atrophy followed by apoptosis
AIM To clarify the histological changes associated with liver atrophy after percutaneous transhepatic portal embolization (PTPE) in pigs and humans. METHODS As a preliminary study, we performed pathological examinations of liver specimens from five pigs that had undergone PTPE in a time-dependent model of liver atrophy. In specimens from embolized lobes (EMB) and nonembolized lobes (controls), we measured the portal vein to central vein distance (PV-CV), the area and number of hepatocytes per lobule, and apoptotic activity using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Immunohistochemical reactivities were evaluated for light chain 3 (LC3) and lysosomal-associated membrane protein 2 (LAMP2) as autophagy markers and for glutamine synthetase and cytochrome P450 2E1 (CYP2E1) as metabolic zonation markers. Samples from ten human livers taken 20-36 d after PTPE were similarly examined. RESULTS PV-CVs and lobule areas did not differ between EMB and controls at day 0, but were lower in EMB than in controls at weeks 2, 4, and 6 (P ≤ 0.001). Hepatocyte numbers were not significantly reduced in EMB at day 0 and week 2 but were reduced at weeks 4 and 6 (P ≤ 0.05). Apoptotic activity was higher in EMB than in controls at day 0 and week 4. LC3 and LAMP2 staining peaked in EMB at week 2, with no significant difference between EMB and controls at weeks 4 and 6. Glutamine synthetase and CYP2E1 zonation in EMB at weeks 2, 4, and 6 were narrower than those in controls. Human results were consistent with those of porcine specimens. CONCLUSION The mechanism of liver atrophy after PTPE has two histological phases: Hepatocellular atrophy is likely caused by autophagy in the first 2 wk and apoptosis thereafter.
INTRODUCTION
The interruption of portal blood flow by portal vein embolization or tumor thrombosis, for example, causes liver atrophy [1] . However, the mechanisms responsible for this effect have not been fully elucidated. Using pig models of percutaneous transhepatic portal vein embolization (PTPE) with absolute ethanol, we previously observed the temporary elevation of serum levels of liver enzymes immediately after ethanol injection. Moreover, in our previous report, macroscopic liver atrophy accompanied by an increased future liver remnant (FLR)/total estimated liver volume ratio was evident 2 wk after PTPE [2] . These observations suggest that the mechanisms responsible for liver atrophy likely commence soon after the disruption of portal blood flow. Consequently, histopathological changes would likely also be observed soon after PTPE.
In pigs that had undergone PTPE using a combination of coils and polyvinyl alcohol particles, the lobule size in the embolized lobe relative to normal liver reportedly decreased gradually to 23% at 12 d; after 12 d, the size of the embolized lobe remained constant [3] . Therefore, to clarify the mechanisms responsible for liver atrophy, pathological analysis should be carried out within this time period. However, to the best of our knowledge, such time-course studies have not yet been carried out.
To assess microscopic changes in liver tissues, it is important to study liver lobules, the smallest functional units of the liver. The observation of clear histological changes would be expected when hepatic blood inflow is disturbed and would be dependent on lobule metabolism, which varies in different zones of the lobule. In particular, we focused on the zonation associated with different levels of metabolism, as illuminated by immunohistochemical (IHC) staining for glutamine synthetase (GS) [4] and cytochrome P450 2E1 (CYP2E1) [5] . Both markers were observed in the pericentral zone of lobules.
Recently, the relationship between apoptosis and autophagy has been extensively reported [6] . The molecular mechanism of autophagy was illuminated by the discoveries of the membrane protein autophagy-related gene 5 in yeast and the microtubule-associated protein 1 light chain 3 (LC3) in mammals [7] . Consequently, IHC staining for these proteins can be used to evaluate levels of autophagy [8,9] . Recent studies have used lysosomalassociated membrane protein 2 (LAMP2) to evaluate autophagy because it is related to autolysosomes for some kinds of autophagy [10] . Autophagy in the liver is reportedly caused by starvation and is related to hepatocellular atrophy [11] . The interruption of portal blood flow, which contains a wealth of nutrients [12] , is considered a form of starvation. Therefore, autophagy may be related to both cellular shrinking and apoptosis. However, the relationship between portal venous obstruction and autophagy has not been reported.
The aim of this study was to investigate, using specimens from a previously reported porcine PTPE model [2] , the microscopic changes associated with apoptosis and autophagy in the days and weeks following portal venous obstruction and to clarify the mechanism by which interrupted portal blood flow causes liver atrophy. Furthermore, we sought to verify the integrity of our pig results by performing the same histopathological investigations in specimens resected from human patients who had undergone PTPE [13][14][15][16] .
Animal specimens
Liver specimens obtained from seven female domestic pigs (Saitama Experimental Animal Supply, Saitama, Japan) weighing 30.0-35.0 kg were used in this study. All pigs underwent segmental PTPE under fluoroscopic guidance with injection of 10 mL absolute ethanol, as we described previously [2] . Specimens from two pigs were excluded from this study because their quality was unsuitable for pathological analysis. Finally, specimens from five pigs were selected for analysis; one pig was sacrificed on day 0, two pigs at week 2, one pig at week 4, and one pig at week 6 ( Figure 1).
The removed pig livers were observed macroscopically (Figure 2A and B). No pig livers exhibited bleeding, degeneration, or necrosis. To evaluate the pure histological changes of the embolized area compared with those of the nonembolized area without histological regenerative reactions, formalin-fixed paraffin-embedded specimens were produced from samples resected from the embolized segment and a nonembolized lobe (control) far from the lobe containing the embolized segment.
Patients
Formalin-fixed paraffin-embedded specimens obtained from 111 patients who underwent major hepatectomy with preoperative PTPE between 2004 and 2010 were collected at the Hepatobiliary Pancreatic Surgery Division of the National Cancer Center Hospital, Tokyo, Japan. Of these 111 patients, 21 had colorectal liver metastases without preoperative jaundice or viral hepatitis. To facilitate the histological evaluation of liver lobules, 11 patients were excluded because of steatosis. In total, 10 patients with comparable embolized lobe and nonembolized liver parenchyma samples (e.g., the caudate lobe or partial hepatectomy from contralateral lobe of PTPE) were selected ( Figure 1). All patients (male-to-female ratio: 4:6, median age: 59 years, range: 43-76 years) underwent hepatectomy with PTPE based on their individual clinical status (Table 1), and samples were collected between 20 and 36 d later (median: 22 d). All patients underwent PTPE via the ipsilateral approach using a 21-G needle (Top, Tokyo, Japan) under ultrasonographic guidance. A 5-Fr sheath (introducer set, Medikit, Tokyo, Japan) was introduced into a branch of the portal vein under fluoroscopic guidance and a 5-Fr balloon catheter (Selection Balloon Catheter, Terumo Clinical Supply, Gifu, Japan) was used for the injection of absolute ethanol (99.5% ethanol, Fuso Pharmaceutical Industries, Osaka, Japan). The study was approved by the Ethics Committee of our Pig specimens (n = 7) Human cases (n = 111)
PTPE
Colorectal liver metastasis cases without preoperative jaundice or viral hepatitis (n = 21) Excluded because of histological conditions (n = 2)
Histological examination Verification
Patients with comparable embolized lobe and nonembolized liver parenchyma samples (n = 10) Histological examination institution. All patients gave written informed consent for inclusion in this study (ID: 2007-022).
Histological examination
All histological examinations were carried out using digital images scanned by Nanozoomer Digital Pathology (NDP, Hamamatsu Photonics, Hamamatsu, Japan) evaluated by two experienced pathologists (Yasuhito Iwao and Hidenori Ojima) who were blinded to all experimental and clinical data. The pathologists conferred if the original evaluations differed.
Morphological study of the lobule
Sections were stained with hematoxylin and eosin (HE), and the morphological changes in embolized and nonembolized lobules were evaluated at 50 random locations on NDP images. The distance between the endothelium of the portal vein in the portal triad and the associated central vein in the same lobule (PV-CV) and the cross sectional area of the lobule (which has a convex shape around a single central vein) were recorded. After the median lobule size (median, ± 0.100 mm 2 ) of each group was determined, the number of hepatocytes in each lobule was counted for 20 randomly selected lobules ( Figure 2C and D). Hepatocyte density was calculated by dividing the number of hepatocytes by the area of the counted lobule for pig specimens. For human specimens, the hepatocyte density was counted within 20 randomly selected 1-mm-diameter circles.
Evaluation of apoptotic activity
Apoptosis of hepatocytes was quantified by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay (In situ cell
Immunohistochemical staining
Sections (4-mm thick) were deparaffinized and incubated in an autoclave for 10 min at 121 ℃ and 1.5 bar. IHC staining was performed using a polymer system (Dako, Glostrup, Denmark) with 3,3′-diaminobenzidine (DAB/ Tris tablets, Muto Pure Chemicals, Tokyo, Japan) as the chromogen. A mouse monoclonal antibody (1:50, sc-271625, clone G-2, Santa Cruz Biotechnology, Santa Cruz, CA, United States) was used for LC3, a rabbit polyclonal antibody (1:100, bs-2379R, Bioss, Beijing, China) was used for LAMP2, a mouse monoclonal antibody (1:2000, MAB302, clone GS-6, Millipore, Billerica, United States) was used for GS, and a rabbit polyclonal antibody (1:100, bs-4562R, Bioss, Beijing, China) was used for CYP2E1. The sections were incubated for 2 h at room temperature. After sections stained for LC3 were scanned and captured by NDP, the digital images were analyzed using ImageJ version 1.48 (National Institutes of Health, Bethesda, Maryland, United States). To facilitate comparisons between pig specimens, the IHC intensity of LC3 was evaluated for each lobule and then divided by the IHC intensity of nerve in the same portal area as the positive control.
Electron microscopy
Formalin-fixed pig liver specimens were analyzed using a Hitachi H-7650 (Hitachi, Tokyo, Japan) transmission electron microscope. Magnification at 80 kV achieved a clear depiction of the hepatocyte organelles.
Statistical analysis
Statistical analysis was performed with the Statistical Package for Social Sciences version 22 (SPSS Inc., Chicago, IL, United States). The Mann-Whitney U test was used to assess differences between embolized and nonembolized samples at each time point. For nonparametric multiple comparisons, the Kruskal-Wallis test was applied. Differences were considered significant at P < 0.05. Data are expressed as medians unless otherwise indicated.
Animal care and use statement
The animal experiment protocols were described in our previous report [2] . All protocols were approved by the Committee for Ethics in Animal Experimentation and were conducted in accordance with the Guidelines for Animal Experiments of our institution (ID: K03-004).
The number of hepatocytes in lobules of median size did not differ significantly between embolized and control specimens until 4 wk after PTPE (week 4: 1459 and 2055, respectively, P = 0.025; week 6: 1494 and 2642, P < 0.001). At weeks 4 and 6, the number of hepatocytes per median-sized lobule in embolized specimens was significantly smaller than those in embolized and in control specimens at day 0 and week 2 (P < 0.001 for all) ( Figure 3C). Therefore, the hepatocyte density in embolized specimens peaked at week 2 (5878/mm 2 ) ( Figure 3D).
Evaluation of apoptotic activity
The fraction of TUNEL-positive hepatocytes was higher in embolized than in control specimens at day 0 and week 4 (11.1% vs 2.37% on day 0 and 5.51% vs 0.493% at week 4, P = 0.018, P = 0.009, respectively; Figure 3E).
Transition of LC3/LAMP2 IHC intensity in the lobule and GS/CYP2E1 zonation
The IHC intensity, as measured by Image J, for LC3 and LAMP2 in embolized specimens at week 2 (0.994 and 45.4, respectively) was significantly higher than that in control specimens (0.486, P = 0.046 and 27.0, P = 0.014, respectively). Moreover, the LC3 and LAMP2 intensities of embolized specimens at week 2 were significantly higher than those in all other specimens (P ≤ 0.025 and P ≤ 0.014 for all; Figure 3F, G and Figure 4). GS and CYP2E1 staining intensities in embolized specimens were not closely associated with those of control specimens at day 0. The extent of the stained zones decreased after 2 wk (Figure 4).
Electron microscopy
Clear findings were hard to establish because of the poor condition of pig liver specimens that had been fixed in formalin some time previously. There was the suggestion of a peak of autophagic vacuoles in embolized samples at week 2 ( Figure 5), which was consistent with the IHC staining intensity of LC3.
PV-CV distance and hepatocyte density in human specimens
We sought to validate our findings in porcine samples by repeating the analytical procedures in human liver specimens from patients following PTPE. Because human lobule structures are not as well defined as those in porcine specimens, the PV-CV distance and hepatocyte density were assessed in a morphological study ( Figure 2E and F). PV-CV was significantly shorter in embolized specimens than in nonembolized specimens (0.455 mm vs 0.563 mm, P < 0.001) ( Figure 6A), as was also observed in porcine specimens 4 wk after PTPE. The hepatocyte density in embolized specimens was significantly higher than that in nonembolized specimens (2111/mm 2 vs 1772/mm 2 , P = 0.038) ( Figure 6B).
Evaluation of apoptotic activity, LC3 intensity, and GS and CYP2E1 zonation in human specimens
A significantly greater fraction of hepatocytes was TUNEL-positive in embolized specimens than in nonembolized specimens (2.804% vs 0.559%, P < 0.001) ( Figure 6C). However, the LC3 intensity did not differ significantly between embolized and nonembolized specimens ( Figure 6D, E and H). The extents of GS and CYP2E1 zonation were reduced in embolized specimens compared with nonembolized specimens ( Figure 6F, G, I and J); similar results were observed in porcine specimens collected 4 wk after PTPE.
DISCUSSION
Interruption of the portal blood flow causes shrinkage of the embolized lobe and compensatory enlargement of the nonembolized lobes. The effects of portal venous obstruction on hepatocyte volume and apoptosis have been previously reported [13][14][15][16] . However, these studies used only human specimens in which the atrophy process was complete. As a result, the process of liver atrophy could not be studied in detail. Our morphological study focused on changes in the lobules over time in porcine samples. We observed two distinct phases of liver atrophy following portal blood flow disruption. The first phase was characterized by lobule shrinkage without a fall in the number of hepatocytes and was accompanied by strong expressions of LC3 and LAMP2 in the first 2 wk after portal venous obstruction. The second phase, which occurred between 2 and 4 wk after portal venous obstruction, was characterized by a reduction in the number of hepatocytes without changes in lobular size. This reduction was accompanied by decreased LC3 and LAMP2 intensity and an increased fraction of TUNELpositive cells (Figure 7).
Soon after the injection of ethanol, the zonation of GS and CYP2E1 in embolized specimens expanded markedly. Increased GS zonation could represent accelerated ammonia metabolism resulting from the degradation of denatured proteins [17] . Moreover, it has been reported that CYP2E1 is directly associated with ethanol metabolism [18] . Furthermore, in this study, the fraction of TUNEL-positive hepatocytes was observed to increase in embolized specimens at day 0; this finding may reflect damage caused by ethanol. Hepatocytes in the embolized lobule may degenerate soon after ethanol injection. These changes are consistent with the clinical observation that circulating levels of transaminases are transiently elevated after ethanol injection to patients undergoing portal vein embolization [19] . In addition, we found that the proportion of TUNEL-positive hepatocytes decreased in the first 2 wk and did not differ between embolized and control specimens at week 2. However, hepatocyte numbers were reported to be restored in 3-4 d after partial hepatectomy [20] , and hepatocyte replication in the embolized lobe was reported to be slightly increased approximately 7 d after PTPE with coils and particles [3] . Perhaps the cellular damage observed at day 0 in our study was repaired via regeneration within the first few days.
The PV-CV distance and lobule size were reduced without the loss of hepatocytes in embolized specimens at week 2. This first phase could be considered a hepatocellular atrophic phase. Interruption of the portal blood flow (which is rich in nutrients from the gastrointestinal tract) may starve hepatocytes after embolization. Starvation reportedly causes autophagy and hepatocyte atrophy [11] . Interestingly, in our study, LC3 and LAMP2 expression was significantly increased in embolized specimens at week 2. Simultaneously, GS and CYP2E1 zonation were reduced at week 2 as starvation caused a reduction in metabolism. Moreover, we found an increase in the number of autophagic vacuoles in embolized specimens at week 2. Thus, we speculated that disruption of the portal blood flow caused hepatocyte shrinkage by activating autophagy.
Between weeks 2 and 4, the number of hepatocytes in embolized specimens decreased without significant changes in the lobule size. During the same period, LC3 and LAMP2 expressions fell and a larger proportion of hepatocytes became TUNELpositive. Consequently, this phase may be regarded as encompassing the deactivation of autophagy and the activation of apoptosis. Recently, autophagy was reported to induce cell death [21] . Therefore, the TUNELpositive cell death we observed might represent caspase-independent apoptosis, rather than caspasedependent apoptosis [22] . Because hepatocyte numbers decreased while TUNEL-positive staining increased after the activation of autophagy, we characterized the phase occurring 2-4 wk after PTPE as the "apoptotic phase". During this phase, the zonation of GS and CYP2E1 did not differ significantly from that observed in embolized specimens at week 2. Between weeks 4 and 6, no morphological or IHC changes were observed at the lobular level and no significant difference was observed in the proportion of TUNEL-positive cells between embolized and control specimens at week 6. Therefore, the liver atrophy process likely terminates between weeks 4 and 6. Our results corroborate that FLR hypertrophy usually takes 4 wk to complete after PTPE because the liver atrophy process is not complete until week 4, although it appears at the macro level to have resolved by week 2.
Hepatectomy is usually performed around 4 wk after PTPE. Consequently, we sought to validate our porcine model observations in human specimens. The observations we made concerning the PV-CV distance, hepatocyte density, TUNEL staining, LC3 and LAMP2 expression, and GS and CYP2E1 zonation in embolized and nonembolized specimens at 4 wk after PTPE in porcine samples also applied to human specimens taken between 20 and 36 d after PTPE. Moreover, the TUNEL results supported those already reported for clinical samples [14][15][16][17] . Furthermore, the pigs we used underwent the same PTPE protocol that humans undergo clinically. Because the histological observations made using this porcine model did not contradict the human results (Table 2), the mechanism by which the interruption of portal blood flow causes liver atrophy may be similar in pigs and in humans.
The limitations of this preliminary study were that the number of pigs was insufficient for a detailed histopathological study to provide unequivocal evidence of the relationship between hepatocellular atrophy and autophagy. Further we attempted Western blotting for LC3-II, but it was not successful. However, we believe that our results and speculations provide a basis for understanding the mechanism of liver atrophy after interruption of the portal blood flow and will facilitate further study. Future research will hopefully provide a sound theoretical basis for planning treatment strategies for acute portal obstruction-related liver dysfunction or disease and chronic ischemic-related liver diseases with liver atrophy.
In conclusion, to investigate the mechanism by which portal vein obstruction causes liver atrophy, we investigated the histological changes in pig livers following PTPE and observed two distinct phases. The first phase, termed the hepatocellular atrophic phase, is characterized by lobular shrinkage without hepatocyte loss and with high levels of LC3 and LAMP2 expression. This phase lasted for the first 2 wk following PTPE. The second phase, which occurs between weeks 2 and 4, is termed the apoptotic phase and is characterized by a reduction in hepatocyte numbers without a reduction in lobular size. This is accompanied by reduced LC3 and LAMP2 expression and increased TUNEL staining. Human liver specimens resected after PTPE had many similar characteristics Day 0 Week 2 Week 4 Week 6 Lobule shrinking with normal cellularity
Apoptotic phase
Hepatocyte reduction without lobular size change Hepatocellular atrophic phase (likely associated with autophagy) Figure 7 Schema of the histological changes occurring following interruption of portal blood flow. At 2 wk after obstruction of the portal vein, lobular shrinkage was observed without reduction in hepatocyte number, but with strong LC3 and LAMP2 expression. These changes may be associated with autophagy, and this process is termed the hepatocellular atrophic phase. At week 4, hepatocyte numbers fell, without a reduction in lobule size, but with an elevation of TUNEL staining. These secondary changes may be attributed to apoptosis occurring after autophagy, characterizing the hepatocellular atrophic phase. No significant histological changes were observed at week 6 compared with week 4.
to specimens collected from pigs at week 4. Therefore, our findings suggest that the mechanism by which the interruption of portal blood flow causes liver atrophy may be similar in pigs and in humans.
Research background
The interruption of portal blood flow by portal vein embolization or tumor thrombosis, for example, causes liver atrophy. However, the mechanisms responsible for this effect have not been fully elucidated.
Research motivation
The previous study suggested that the mechanisms responsible for liver atrophy likely commence soon after the disruption of portal blood flow. Consequently, histopathological changes would likely also be observed soon after percutaneous transhepatic portal embolization (PTPE). Recently, the relationship between apoptosis and autophagy has been extensively reported. Autophagy in the liver is reportedly caused by starvation and is related to hepatocellular atrophy, and, moreover, interruption of the portal blood flow, which contains a wealth of nutrients, is considered a form of starvation. Therefore, autophagy may be related to both cellular shrinking and apoptosis. However, the relationship between portal venous obstruction and autophagy has not been reported. To clarify the mechanisms responsible for liver atrophy, histopathological analysis should be carried out repeatedly within the first few weeks after PTPE. However, to the best of our knowledge, such time-course studies have not yet been carried out. The results and hypotheses will provide a basis for understanding the mechanism of liver atrophy after interruption of the portal blood flow and will facilitate further study.
Research objectives
The aim of this study was to investigate, using specimens from a previously reported porcine PTPE model, the microscopic changes associated with apoptosis and autophagy in the days and weeks following portal venous obstruction and to clarify the mechanism by which interrupted portal blood flow causes liver atrophy. Furthermore, to understand the mechanism of liver atrophy in humans after PTPE, the authors sought to verify the integrity of the pig results by performing the same histopathological investigations in specimens resected from human patients who had undergone PTPE.
Research methods
The authors performed histopathological examinations of liver specimens from five pigs that had undergone PTPE in a time-dependent model of liver atrophy. In specimens from embolized lobes (EMB) and nonembolized lobes (controls), the authors measured the portal vein to central vein distance (PV-CV), the area and number of hepatocytes per lobule, and apoptotic activity using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Immunohistochemical reactivities were evaluated for light chain 3 (LC3) and lysosomal-associated membrane protein 2 (LAMP2) as autophagy markers and for glutamine synthetase and cytochrome P450 2E1 (CYP2E1) as metabolic zonation markers. Samples from ten human livers taken 20-36 d after PTPE were similarly examined.
Research results
PV-CVs and lobule areas did not differ between EMB and controls at day 0, but were lower in EMB than in controls at weeks 2, 4, and 6. Hepatocyte numbers were not significantly reduced in EMB at day 0 and week 2 but were reduced at weeks 4 and 6. Apoptotic activity was higher in EMB than in controls at day 0 and week 4. LC3 and LAMP2 staining peaked in EMB at week 2, with no significant difference between EMB and controls at weeks 4 and 6.
Glutamine synthetase and CYP2E1 zonation in EMB at weeks 2, 4, and 6 were narrower than those in controls. Human results were consistent with those of porcine specimens. However the number of pigs was insufficient for a detailed histopathological study to provide unequivocal evidence of the relationship between hepatocellular atrophy and autophagy.
Research conclusions
To investigate the mechanism by which portal vein obstruction causes liver atrophy, the authors examined the histological changes in pig livers following PTPE and observed two distinct phases. The first phase, termed the hepatocellular atrophic phase, is characterized by lobular shrinkage without hepatocyte loss and with high levels of LC3 and LAMP2 expression. This phase lasted for the first 2 wk following PTPE. The second phase, which occurs between weeks 2 and 4, is termed the apoptotic phase and is characterized by a reduction in hepatocyte numbers without a reduction in lobular size. This is accompanied by reduced LC3 and LAMP2 expression and increased TUNEL staining. Human liver specimens resected after PTPE had many similar characteristics to specimens collected from pigs at week 4. Despite liver atrophy appearing to be mostly resolved 2 wk after embolization, the period after PTPE could beneficially be extended to 4 wk to ensure contralateral hypertrophy and to allow the completion of liver atrophy.
Research perspectives
Histopathological analysis is the best way to clarify the mechanisms responsible for liver atrophy. To assess microscopic changes in liver tissues, it is important to study liver lobules, the smallest functional units of the liver. The observation of clear histological changes would be expected. To clarify the more detailed mechanism of liver atrophy after interruption of the portal blood flow, the authors have to study the histopathological changes using not only the pig model but also small animal models, e.g., mouse models, because such animals are easy to handle. After such detailed studies, future research will hopefully provide a basis for understanding the mechanism of liver atrophy after interruption of the portal blood flow and also give a sound theoretical basis for planning treatment strategies for acute portal obstruction-related liver dysfunction or disease and chronic ischemic-related liver diseases with liver atrophy. | 2018-04-03T04:02:10.907Z | 2017-11-18T00:00:00.000 | {
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8560804 | pes2o/s2orc | v3-fos-license | Statistical Approach of Functional Profiling for a Microbial Community
Background Metagenomics is a relatively new but fast growing field within environmental biology and medical sciences. It enables researchers to understand the diversity of microbes, their functions, cooperation, and evolution in a particular ecosystem. Traditional methods in genomics and microbiology are not efficient in capturing the structure of the microbial community in an environment. Nowadays, high-throughput next-generation sequencing technologies are powerfully driving the metagenomic studies. However, there is an urgent need to develop efficient statistical methods and computational algorithms to rapidly analyze the massive metagenomic short sequencing data and to accurately detect the features/functions present in the microbial community. Although several issues about functions of metagenomes at pathways or subsystems level have been investigated, there is a lack of studies focusing on functional analysis at a low level of a hierarchical functional tree, such as SEED subsystem tree. Results A two-step statistical procedure (metaFunction) is proposed to detect all possible functional roles at the low level from a metagenomic sample/community. In the first step a statistical mixture model is proposed at the base of gene codons to estimate the abundances for the candidate functional roles, with sequencing error being considered. As a gene could be involved in multiple biological processes the functional assignment is therefore adjusted by utilizing an error distribution in the second step. The performance of the proposed procedure is evaluated through comprehensive simulation studies. Compared with other existing methods in metagenomic functional analysis the new approach is more accurate in assigning reads to functional roles, and therefore at more general levels. The method is also employed to analyze two real data sets. Conclusions metaFunction is a powerful tool in accurate profiling functions in a metagenomic sample.
Introduction
Metagenomics is the study of genetic material recovered directly from natural (e.g., soil or seawater) or host-associated (e.g., human gut) environmental samples that contain microorganisms organized into communities. The advancement of high-throughput next generation sequencing technologies provides a powerful way in metagenomic studies since they can be directly applied to an environmental sample without the need of isolating and culturing individual microbial species in a laboratory. More than 99% of millions microbial species on Earth cannot be cultured in a laboratory [1,2]. The massively parallel sequencing technologies, such as 454FLX, Illumina Genome Analyzer (GA), and ABI SOLiD, have enabled us to generate millions of reads (35-500 base pairs (bp), depending on the platform) at a time [3] The initial computational analysis of metagenomics focuses on two main questions: who is out there and what they can do [1,2]. To answer the first question, scientists determine taxonomic compositions in a particular metagenomic sample and determine the abundance/proportions of the species. Many methods have been proposed [4][5][6][7], particularly, TAMER8], GASSiC [9], and TAEC [10] focus on the taxonamic analysis at a very low phylogentic level -species.
To answer the question ''what they can do'' scientists need to determine the gene contents, functional categories, and estimate the relative functional abundances contributed in the metagenomic sample. According to Overbeek et al. [11], a functional role corresponds roughly to a single logical role that a gene or gene product may play in the operation of a cell, such as 'Aspartokinase (EC 2.7.2.4)', and pathway or subsystem which is a collection of related functional roles (Figure 1). To characterize the functional capacity of a metagenomic community, therefore, researchers can perform analysis either at the functional role level or pathways/ subsystems level. Most recently published studies focused on pathways or subsystems level [12][13][14][15]. However, a number of questions about functional roles of microbial communities are still ambiguous, e.g., do microbial communities consist of extensive genetic diversity, how are they diverse in functional roles, how does the diversity in functional roles of microbial communities affect their interaction with environment? Performing function analysis of metagenomes at functional roles level, therefore, is an appropriate approach to addressing these issues. Through such type of analysis, functional roles can be detected and further metabolic pathways or subsystems that the functional roles are involved can be established [14].
Several tools have been developed to detect/annotate functional roles from a metagenomic sample [16]. Among the commonly used publicly available pipelines, most of them are homologybased tools, such as MEGAN [17], MG-RAST [18], IMG/M [19], and CAMERA [20]. In MEGAN the functional analysis of metagenomes is based on the SEED hierarchy [18]. The SEED has consistent and accurate microbial genome annotations of any publicly available source [11]. To perform a functional analysis, MEGAN assigns each read to the functional role of the highest scoring gene in a BLAST comparison against a protein database (e.g., NCBI-NR), and then different functional roles are grouped into SEED subsystems. The SEED classification can be represented by a hierarchical tree, where the internal nodes represent subsystems and the leaves denote the functional roles ( Figure 1).
However the MEGAN program has several disadvantages. First of all, the best score assignment might miss putative functions. Because of the existence of sequencing error [21], a sequence read could come from a gene/function with aligned matches of 32 out of 33 codons and could also from a gene/function with aligned match of 31 out of 33 codons. The MEGAN method misses the second or even the third best scoring functions that the read may have. Furthermore, a gene could play multiple functions at the same time. However MEGAN just assigns one function (with the best match value) to the short read even when multiple functions show the same best match values (e.g., the e-value, bitscore, or the number of matched codons). For example, blastx output for a short read shows two functions ''Argininosuccinate lyase (EC 4.3.2.1)'' and ''N-acetylglutamate synthase (EC 2.3.1.1)'' with the same best match values, but MEGAN only assigns the first function (alphabetically) to the read. Thus, MEGAN misses some functions existing in the community and therefore underestimates their abundance.
MG-RAST [18] can assign multiple functions to a read, but some flat cutoffs, e.g., e-value , 1.0e-5 and identity cutoff . 60% are used. Thus assignment of reads to different ranks of taxonomy tree greatly depends on the threshold of bit-score or Expect value used. As a consequence, the results lack specificity. IMG/M uses the best BLAST hits for function assignment [19]. In CAMER [20] open reading frames (ORFs) are clustered at a certain cutoff of identity (e.g., 60%) over a certain threshold (e.g., 80%) of ORF length. ORF clusters are then used for functional studies. Both the best-hit approach (in MEGAN and IMG/M) and objective cutoff approach (in MG-RAST and CAMER) lack of statistical support.
Motivated by both the advantages and limitations of these methods and inspired by the statistical model in Jiang et al. [8] we propose a two-step procedure to accurately assign functions to reads. In the first step sequencing error is estimated through a mixture model, which is proposed to model the translated sequence reads at the base of codons and detect the possible functions in a metagenomic sample. As a gene could be involved in multiple biological processes, the functional assignment is adjusted by utilizing an error distribution at the second step. The proposed two-step method is comprehensively tested on simulated metagenomic data with diverse complexity of microbial community structure, and also applied on two real metagenomic datasets. Compared with MEGAN and MG-RAST for functional metagenomic analysis, the proposed approach demonstrates greater accuracy in function identification and abundance quantification. The R package ''metaFunction'' is available for download at http://cals.arizona.edu/,anling/software.htm.
Methods
For each sequence dataset we use BLASTX to search for matched reference sequences (i.e., genes) in the NCBI-NR protein database. Then genes are classified into functional role categories as defined by the SEED classification. Based on the sequence reads we need to estimate: (1) the sequencing error rate and (2) functional roles contained in the metagenomic sample and their relative abundance (i.e., proportions). To answer these questions, we set up a mixture model based on the information from BLASTX results. And then a binomial model for the sequencing error (estimated by the mixture model) is proposed to adjust the function assignment, and therefore the proportion estimation for each function is adjusted accordingly. The adjustment on assigning functions is to incorporate the fact that a gene/short read could play multiple function roles. The flowchart for the proposed procedure can be found in Figure 2.
Estimate sequencing error
Suppose we have n sequence reads that are mapped to sequence homologs in the reference database (i.e., NR protein database) and return K functions (i.e., gene families) in the result of homolog research, e.g., BLASTX output. Let C ij denote the number of identical matched codons for read i under functional role j and L ij represent the corresponding aligned codon length. LetL i denote the maximum aligned codon length for read i across all candidate functions, i.e., L i~m ax j (L ij ) then we have C ij ƒL i . If the read i does not have matched sequences for function j, thenC ij~0 . We assume that the larger the C ij value, the more likely that the read i performs function j. Let R j denote the proportion of reads having function j, thus P K j~1 R j~1 . Even if the read i is from function j, it is also possible that C ij is not exactly as same as L i , the maximum aligned length. It may be due to the sequencing error and/or single nucleotide polymorphism (SNP) effect or various sources (i.e., organisms) for the same gene in the database. Let p denote the probability of observing a mismatched codon, then 1{p is the probability of observing an identity or conserved codon. Therefore the probability that the read i performs function j with C ij matched codons and L i {C ij mismatched codons is R j p Li{Cij (1{p) Cij . Then the probability to observe the read i in the dataset is Hence the likelihood function of the data is: In this likelihood function, the maximum aligned length L i and the matches C ij can be extracted from the BLASTX output. The parameters p and R j (j~1,2, Á Á Á ,K) are then estimated by Expectation Maximization (EM) algorithm [22]. As p is the probability for observing a mismatched codon, for simplicity, we just call p as sequencing error (rate) and a mismatched codon as a mismatch.
Multiple-function assignment
One read could get involved in multiple functional roles. For read i, assume its best mismatch (i.e., minimum number of mismatched codons) across all functions is M i0 , we can determine the maximum allowable mismatch M i1 for a given small probability e such that: where we assume that the mismatch m i follows a binomial distribution with parameters (L i ,p). Then read i can be assigned to all the functions with mismatch #M i 1 . The relative abundance R j will be updated by this new multiple function role assignment, i.e., the updated one becomes:R j 0~h j n , where h j is the number of short reads assigned to the function j after the adjustment, and n is the total number of short reads in the dataset. Thus we have P K j~1 R j 0 §1. The algorithm procedure for the multiple-function adjustment can be summarized as below: Based on the estimated sequencing error obtained in step 1 and a pre-specified small probability e: 1) for read i calculate its maximum allowable mismatch M i1 using eq. (3) 2) assign all functions with corresponding mismatch #M i1 to read i 3) repeat steps 1) and 2) for all reads 4) calculate the new roportion R j 0~h j n for each function based on these new assignments. therefore the read is finally assigned to these functions. The small probability e in equation (3) is suggested as one third of the sequencing error at the codon level (estimated in the first step) or just the sequencing error at the nucleotide level, if known or given. More details about the selection of e can be found in the simulation studies below.
Construct statistical inferences
None of the existing methods on functional metagenomic analysis could further assess the uncertainty of the proportion of assigned reads to functions. We propose to use bootstrap method [23] for constructing the confidence intervals for the estimates. We first draw a bootstrap sample by resampling the reads from the original sequence reads with replacement; the relative abundances are estimated using the described two-step procedure for the bootstrap sample. We repeat this resampling/bootstrap for a large number of times, e.g., 1000 times. Then the confidence intervals can be constructed based on these bootstrap estimates. Since we construct the confidence intervals for the abundances of the K functions, R j (j = 1,…, K) simultaneously, a multiple correction method, e.g., Bonferroni method [24], is applied to guarantee a pre-specified family-wise confidence level.
Simulation studies
Experimental data. Due to the complexity of metagenomic data, simulation studies with verifiable structure are crucial to benchmark the proposed approach and to conduct comparisons with other existing methods. So far there is no literature about how to set up a simulation study for functional metagenomics. We propose to use the SEED database (http://pseed.theseed.org) and conduct six different simulation studies. Basic information of these six simulation settings is listed in Table 1. Similar to the studies in MetaSim [25] which contain a small number of genomes in each setting we simulate a small number of functions in each study. Study 1 contains 10 function roles that are far away from each other in the SEED tree. For each function role, 20% of the sequences (i.e., FIGfams, very long sequences) from the SEED database are chosen and the sampling rate for this situation is 20%. Then a short segment of 100 bp is randomly chopped from the selected long sequence, and 2% sequencing error is added to it. The sequencing error could be due to the substitution, deletion and insertion. For the purpose of method illustration we only consider the substitution error. It is well know that some genes are involved in multiple functions in a microbial community. This is also reflected from the gene sequences in the SEED database, i.e., some long sequences are labeled with multiple functions. As expected, a few additional function names are obtained for the short reads in the 10 pre-selected groups. We name them secondary functions, and the 10 pre-selected functions as primary functions (see the Table S1 in File S1). The number of short reads for each function is also listed in the table S1 in File S1. Both types of functions are treated as true functions since the functions in either type are the true ones for the generated short reads.
Study 2 contains the same 10 primary functions as study 1 but with various sampling rate (see the Table S1 in File S1). The number of short sequence reads generated for each function is based on the total number of long sequences in the function group in the SEED database. Generally, the sampling rate varies between 20%,40%. In studies 3 and 4 we use another set of 10 functions (see the Table S2 in File S1). Different from the studies 1 and 2, the 10 function groups here are very closely related (i.e., some functional roles are belong to the same subsystems). Study 5 contains the same 10 primary function groups as studies 1 & 2 but the sampling rate is much larger, about 4,5 times of the first two studies; similarly, study 6 contains the same 10 primary function groups as studies 3 & 4 but the sampling rate is about 4,5 times of these two studies (see the Table S1 and S2 in File S1). The coverage, i.e., ratio of the number of simulated base pairs to the total number of base pairs for the selected functions in the SEED database, varies 2%,9% in these six studies.
Simulation Results. Three methods, MEGAN (best hit), MG-RAST (flat cutoff) and the proposed method metaFunction, are compared through these six simulation studies. The result for the first simulation study is shown in Figure 4 where it plots the relationship between the estimated (i.e., predicted) abundance for each function and its true (i.e., expected) abundance. If all the functions are detected and their abundances are correctly estimated then the Pearson correlation between the expected and predicted abundances is one. From the plot it is obvious that the proposed approach has the largest correlation. Table 2 displays the summary of the correlations in all six studies for these three methods. The new method outperforms the other two methods in all studies in terms of correlation between the true and estimated abundances. While MEGAN and metaFunction meth- We also evaluate the performance of three methods via the same simulations using another metric. A common measure for error is root mean square of relative error [7,26]. In this definition each feature group is assumed the same weight in the error calculation, regardless the abundance of features in each group. In function analysis of metagenomics a function group estimated with a tiny number of counts actually should much less likely exist in the sample than a group with large number of read counts. We modify the error measure to weighted root mean square of relative error (WRRMSE), i.e., , e j is the estimated number of reads for function j, a j is the estimated relative abundance (i.e., estimated proportion) and t j is the true relative abundance, and m is the number of true function groups. The WRRMSE results for six studies are shown in Figure 5. In each of subplots the x-axis is the SEED system level. Compared to the MEGAN and MG-RAST, the proposed method has the lowest error at any level of the subsystems and for all simulation studies. Decrease in Error on Sub 2 level in Figure 5 is due to the unnamed subsystems in the SEED tree. For example, a read is assigned to a level-3 subsystem but its parent node has no name (i.e., NULL) then the assignment to this unknown level-2 subsystem will be excluded in calculating the error. The decrease in error for sub 2 level is due to the removal of the NULL group that may contain some wrong assignments. The accuracy on estimation of relative abundance plays an important role in metagenomic analysis, the accuracy of assignment of short reads is also very interesting to biologists in functional metagenomics as they need the information of what reads do what kind of functions. As the MG-RAST does not give the information of the assignment we compare the performance of MEGAN and the proposed method metaFunction regarding the assignment details. In each of six simulation studies we calculate the proportion of correctly assigned (CA), wrongly assigned (WA), and not assigned (NA, i.e., not aligned to the reference database) across all functions. The assignment details are also examined at other levels of the subsystem. The results of the simulation study 1 are displayed in Table 3. At any level of the subsystems (including the function level) the proportions of NA using metaFunction are lower than those from the MEGAN result. The WAs for metaFunction are little higher than the ones for the MEGAN but they are comparable (all ,1%). The new approach results in much higher CA rate than MEGAN (about 90% vs 70%). Consistent conclusions are obtained for other simulation studies (data not shown).
Selection of e. The above results are based on e~0:01 in eq. (3). We conduct another study to investigate the effect of selecting different small probability e on the final result in terms of the error metric defined above. Let e take various values of 0.01, 0.05, or 0.1 for the multiple role assignment. Within each of the above six simulated experiments the WRRMSE values are very close for these three different e values. That is, the absolute difference on WRRMSE is less than 0.0001 between the two situations of e = 0.01 and e = 0.05; and less than 0.008 between e = 0.01 and e = 0.10. In terms of relative difference on WRRMSE, the values are (0,2%) for different e. Therefore, the final result is not sensitive to the selection of e. In the above six simulated experiments 2% error is added to each short read, any value between 0.01 and 0.05 is plausible for e.
Real Data Analysis
Real metagenomic data from an environmental study and a human health study are analyzed using the proposed method -metaFunction.
Environmental study
Metagenomic functions were compared between Lake Erie (North America) and Lake Taihu (China) [27]. Toxic cyanobac-teria blooms appear to be a global problem as toxins produced by bloom-associated cyanobacteria can have drastic impacts on the ecosystem and surrounding communities; in addition, the produced bloom biomass can disrupt aquatic food webs and act as a driver for hypoxia. Freshwater samples were collected from different lakes to examine the bloom associated microbial communities. We select two lakes -Lake Erie and Lake Taihu as they represent different continents -to examine the gene contents. After quality checking totally 750 thousands reads with an average length of 425 bp are aligned to the NCBI nonredundant database. Then the proposed method is applied to the alignment output. The original study used both MEGAN and MG-RAST for functional annotation and they addressed that the two results are highly consistent. We compare our result to the MG-RAST result in the original paper, which are downloaded from the MG-RAST online server (http://metagenomics.anl.gov/ ) under the identification numbers 4467029.3 (Erie), 4467058.3 (Taihu).
The functionality profiles of microbial communities in these two lakes by metaFunction and MG-RAST are summarized at the level 1 of subsystem ( Figure 6). Generally, the results from these two approaches are consistent: the subsystems found by one method with big proportions are also detected by the other with large amount. However there also exists some discrepancy between the two results. The subsystem ''Miscellaneous'' is found dominant by MG-RAST in both lakes but not ample by the new method; ''Virulence, Disease and Defense'', ''Virulence'', ''Membrane Transport'', and ''Cell Wall and Capsule'' are observed more abundant by the new approach than by MG-RAST.
When compare the results between two lakes we found that subsystems abundant in one lake by the MG-RAST often show plenty in the same lake by the new method. For instance, ''Amino Acid and Derivatives'', ''Carbohydrates'', ''Nucleosides and Nucleotides'', and ''Membrane Transport'' are found more abundant in Lake Taihu than in Lake Erie. Meanwhile ''Cell Division and Cell Cycle'', ''Regulation and Cell signaling'', ''Cofactors, Vitamins, Prosthetic Groups, Pigments'' are lower in Lake Taihu. A big difference tween the results from two approaches is that the new method can provide confidence interval information for the proportion estimation, which is displayed as the small bars in Figure 6. Thus the new method can provide more information about the group comparisons. Comparison between two lakes at a lower level of subsystems -level 3 -is shown in the Figure S1. Not surprising, the results from two approaches in Figure S1 are more disparate than at the higher level of subsystems in Figure 6.
Human Health study
Human oral microbial samples were studied for oral cavity problem using 454 pyrosequencing [28]. Two healthy samples and two cavity samples are selected for our analysis, with one at an intermediate stage and the other one at an advanced stage of caries development. After quality checking, 0.5 Gbp of sequence with the average read length 425 bp are BLASTXed to NCBI-NR protein database for searching matched reference sequences (i.e., genes). Then reads are classified into functional role categories as defined by the SEED structure using the proposed method. The results of functionality profiling for all four samples at the subsystem level 3 are shown in Figure 7.
In this plot the abundance of ''Conjugative transposon Bacteroidales'' is much higher in the cavity samples than in the healthy orals, which is also confirmed in other literature [29]; ''Fatty Acid Biosynthesis FASI'' also shows a higher value in the diseased samples than in the healthy samples, which is consistent with the finding in [30]; That the ''Flagellum'' is abundant in the cavity samples is also reported in Seshadri et al. [31]; high values of ''Glutamine Glutamate, Aspartate and Asparagine Biosynthesis'' and of ''Methionine degradation'' in the oral cavity samples are also mentioned in other publications [32,33]; the abundance of ''Universal GTPases'' is higher in the cavity samples than in the healthy orals, which is also found in other literature [34]. In conclusion, the results from the new method provide us the findings consistent with the previous literatures.
Discussion
One of the main challenges in metagenomic studies is how to accurately identify all possible functional roles present in an environmental sample and precisely estimate their abundance. Due to the complexity of metagenomics and the huge volume of sequencing reads of short lengths obtained from the next generation sequencing technologies, the need of efficient statistical tools to accomplish this challenge is increasing. We proposed a two-step procedure to perform functional analysis on a metagenome: mixture model coupled with the adjustment of multiple role assignment, to accurately assign reads to related functional roles by utilizing the SEED classification. Though this research is initiated for the SEED classification, actually the proposed method can be generalized to any type of function annotation system.
Compared to MEGAN and MG-RAST through comprehensive simulation studies, our procedure metaFunction demonstrates more effective in assigning reads to functional roles, thereafter, to subsystems. In the simulation study 1 and 2, the results show that MEGAN cannot assign any read to one of the true functional roles ( Figure 4) while in the simulation study 3 and 4, MG-RAST cannot assign any read to one of the true functional roles (plot not shown). This type of phenomenon has never happened to our approach. In addition, the proposed method can correctly assign higher percentage of reads to functional roles than MEGAN does. MEGAN utilizes the best bit-score for assignment. If a read returns with best scores for multiple functions in the BLAST output, then only the first function (alphabetically) is chosen for the assignment. In our method all of them with the same best score are assigned to the read. Different from other existing methods, the proposed method provides confidence intervals for the estimations of the proportions by using bootstrap.
We also applied the proposed method to two real metagenomic datasets and our results generally are consistent with the findings in the previous reports but provide more detailed information. A future work is to integrate the taxonomic analysis and functional analysis, in other words, to consider these two types of issues simultaneously, so that the power can be improved for both taxonomic and functional profiling a metagenomic sample. Figure S1 Proportions of the detected subsystems (level 3) by MG-RAST and metaFunction for the lake data. The top 66 subsystems with proportion .0.005 in at least one of samples are listed. The ''error'' bars represent the 95% confidence interval obtained by bootstrap method. Note: only the proposed approach can provide confidence intervals for the estimations of the proportions. (TIF)
Supporting Information
File S1 Table S1. Number of short reads generated from 10 primary function roles for the studies of 1, 2, and 5. The function names in italic are secondary functions. Table S2. Number of short reads generated from 10 primary function roles for the studies of 3, 4, and 6. The function names in italic are secondary functions. (DOCX) | 2017-07-28T05:23:07.856Z | 2014-09-08T00:00:00.000 | {
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232246547 | pes2o/s2orc | v3-fos-license | Detection of Representative Variables in Complex Systems with Interpretable Rules Using Core-Clusters
: In this paper, we present a new framework dedicated to the robust detection of representative variables in high dimensional spaces with a potentially limited number of observations. Representative variables are selected by using an original regularization strategy: they are the center of specific variable clusters, denoted CORE-clusters, which respect fully interpretable constraints. Each CORE-cluster indeed contains more than a predefined amount of variables and each pair of its variables has a coherent behavior in the observed data. The key advantage of our regularization strategy is therefore that it only requires to tune two intuitive parameters: the minimal dimension of the CORE-clusters and the minimum level of similarity which gathers their variables. Interpreting the role played by a selected representative variable is additionally obvious as it has a similar observed behaviour as a controlled number of other variables. After introducing and justifying this variable selection formalism, we propose two algorithmic strategies to detect the CORE-clusters, one of them scaling particularly well to high-dimensional data. Results obtained on synthetic as well as real data are finally presented.
Introduction
Discovering representative variables in high dimensional and complex systems with a limited number of observations is a recurrent problem of machine learning. Heterogeneity between the variables behavior and multiple similarities between variable subsets often make this process ambiguous. A convenient strategy to solve this task is to associate each representative variable of the complex system to a cluster of variables, and to model the relations between variables in a graph [1]. The complex systems are indeed typically modeled as undirected weighted graphs [2,3], where the nodes (vertices) represent the variables and the edge weights are a measure of the observed similarity between the variables of the dataset. The choice of a specific clustering algorithm over the wide variety of traditional methods often depends on the nature of the data (e.g., their structure or size). Determining the granularity level of the clusters is also a common issue in high dimensional data clustering. If the clustering granularity is high, some clusters have a high similarity rate between the nodes/variables they contain, but potentially, many other clusters only contain noisy relations. A large amount of selected representative variables can then be meaningless. Conversely, a low granularity leads to few large clusters with high internal heterogeneous behaviors, which makes it hard to identify the representative variables of the system in each cluster. Importantly, this issue is particularly critical when the number of observations n is lower than the observations dimension p, because of the instability related to high dimensionality and high complexity.
As in k-means clustering algorithms [4], we will use, in this paper, the notion of cores to address with an interpretable strategy the choice of the granularity level. Based on a distance function, the k-means algorithms indeed form a controlled number of clusters. This notion of core is also used in [5,6], where the graph is partitioned into a maximal group of entities, which are connected to at least k other entities in the group. The method of [7] is also related to the notion of coreness, as it hierarchically calculates the core number for each node with a complexity in the order of O(p). Strong connections also exist between the issues we address and the notion of coresets [8]. This notion has recently gained significant interest in the machine learning community, as it deals with finding representative observations, and not variables, in large datasets. As described in [9], it can be used in supervized learning to reduce the size of large training sets. In a similar vein, it can also be used to robustify the generalisation of the trained decision rules [10], for neural network compression [11] or for unsupervized learning [12]. Note that [12] is also particularly close to core methods, as it specifically deals with k−clustering, i.e., finding at most k cluster centers. The proposed method however does not address explicit interpretability issues.
The challenge of high dimensionality is clearly raised in [13][14][15], where the authors proposed different feature selection techniques with an explicit regularization in order to speed up a data mining algorithm and to improve mining performance. In this spirit, [16] recently developed an approach based on an iterative spectral optimization technique that improves the quality, computation time and scalability to high dimension of an existing alternative clustering method (kernel dimension alternative clustering). In [17], the authors also used a multinomial regression model to learn automatically the number of clusters, and then to limit strong assumptions required by the model in high dimension. Note that [18] also defined a strategy for the detection of representative variables in high-dimensional data, but did not explored a regularization strategy when the number of observations is much lower than the problem dimension. Those strategies require as well to make a decision about the number of clusters to determine.
Motivated by the above issues, we propose a new formalism to robustly and intuitively estimate the representative variables of complex systems. This is first made through a graph clustering strategy for which the clusters do not necessarily cover all nodes/variables. This clustering strategy specifically estimates CORE-clusters, which are connected subsets of variables having (1) a minimum number of nodes/variables, and (2) a minimal similarity level between all their variables. The representative variables are then those having the lowest average distance to all other variables in each CORE-cluster. The detection of representative variables is therefore regularized using a control on the minimal COREcluster size and not the number of representative variables to be detected, or a LASSOderived penalty term. This point of view has been originally considered in [19,20], We present here a totally reformulated version of this initial idea, which makes fully interpretable the selection of the representative variables by introducing the notion of CORE-clusters. Fine algorithmic improvements, described in this paper, also make the original clustering algorithm more efficient. A greedy version of this original algorithm, which turns out to scale particularly well to high dimensional data, is additionally proposed. New results on synthetic data as well as comparisons with other methods now shed light on how the proposed strategy is robust and explainable. Finally, we now demonstrate the validity of our formalism on two high dimensional datasets representing the expression of genes and a road network.
Our methodology is described in Sections 2 and 3 and is then tested both on simulated and real data in Section 4.
Graph-Based Representation of the Observations
Let us consider a complex system of p quantitative variables X = (X 1 , · · · , X p ) and n observations (X j 1 , · · · , X j n ), j ∈ {1, · · · , p} of these p variables. The driving motivation of our work is to detect representative variables out of X when n p. As mentioned in Section 1, the detection of these representative variables is regularized using a graph-based approach. We then model the relations between the variables using an undirected weighted graph G(N, E), where N = (N 1 , · · · , N p ) is the nodes set corresponding to the p variables, and E is the edges set. We also denote e i,j ∈ E the edge joining the nodes N i and N j with weight w i,j .
In order to handle the properties of application-specific similarity measures that can be encoded in the edge weights w i,j , we will consider in the remainder of the paper that all w i,j ≥ 0 and that the higher w i,j the closer the observed behavior of the variables X i and X j . The weights therefore represent a notion of similarity between the variables X i and X j . For instance, if the empirical correlations cor(X i , X j ) are measured between the pairs of variables X i and X j , with (i, j) ∈ {1, . . . , p} 2 , then w i,j = |cor(X i , X j )| can reasonably be used.
Coherence of a Variable Set
To define a notion of distance between two variables X i and X j , which are not directly connected in the graph, we use the notion of capacity (see [21,22]) of a path P between the corresponding nodes N i and N j in G(N, E). A path P of a graph G from X i to X j of length Λ is a list of indices {d 1 , . . . , d Λ } ⊂ {1, . . . , p} such that X i = X d 1 , X j = X d Λ , and w d l ,d l+1 is known and is not equal to 0, for all l = 1, . . . , Λ − 1. The capacity cap(P) of path P is then the minimal weight of its edges, i.e., We also denote by P i,j the set of all possible paths connecting X i to X j . The coherence c(X i , X j ) between X i and X j is then defined by considering the path P having the maximum capacity among the paths of P i,j [22], i.e., If the weight w i,j is known, it is interesting to remark that the coherence c(X i , X j ) is not necessarily equal to its value. For instance, both X i and X j may be very similar to a third variable X k , but not similar to each other. From a computational point of view, the similarity in w i,j may also be unknown if the edge e i,j is not stored in a sparsified version of the complete graph. Since n p, pertinent relations may finally be lost in w i,j but recovered in c(X i , X j ) thanks to other relations that would be captured. We believe that these points are particularly important to define coherent variable sets in the complex data case.
We now denote by S a connected subset of the variable set X. The coherence c(S) of this variable subset is the minimal coherence between the variables it contains: If all the variables of S have a coherent observed behavior, then c(S) is high. The use of this notion on synthetic data is illustrated in Appendix A. The coherence of a subset measures the strength of the variables it contains. The more coherent S, the more sense it makes to consider that its variables share common features measured by the chosen similarity. Decomposing the graph into maximal groups sharing a strong similarity, i.e., finding all the groups of variables with a large enough coherence is the core of the following subsections on CORE-clusters selection.
CORE-Clusters
We recall that the goal of our formalism is to detect the representative variables of complex systems. In our formalism, each representative variable is extracted out of a CORE-cluster, defined as: Definition 1. A CORE-cluster S ξ,τ ⊂ X is a connected variable subset with a size higher than τ and a coherence c(S ξ,τ ) higher than a threshold ξ.
The parameters τ and ξ ensure that each representative variable has a non-negligible coherence with at least τ − 1 other variables, which directly regularizes its selection: Large values of τ indeed lead to the detection of large sets of coherent variables. In that case, the representative variables are likely to be meaningful even if n < p. If τ is too large, each CORE-cluster may however contain several variables that would have been ideally representative. On the contrary, too small values of τ are likely to detect all meaningful representative variables, but also a non-negligible number of false positive representative variables, especially if the observations are noisy or if n < p. A good trade-off, which depends on n, p and the level of noise in the observations has then to be found when tuning τ.
CORE-Clustering
CORE-clustering consists of estimating an optimal set of CORE-clusters, so that the representative variables they contain explain as much information as possible in the observed complex system. We use the following definition: Definition 2. CORE-clustering with parameters ξ and τ consists of finding U variable subsets S = { S u } u∈{1,..., U} , where U is not fixed, by optimizing: under the two constraints: 1. All S u are connected variable sets having a size higher than τ and a coherence c(S u ξ,τ ) > ξ. They therefore correspond to CORE-clusters and can be denoted S u ξ,τ .
It may first seem that U should be as high as possible, so that the union of all S u ξ,τ contains all the variables of X. Each CORE-cluster S u ξ,τ must however have a coherence higher than the threshold ξ. As illustrated in Appendix A, the variables of X which are not coherent with at least τ other variables should ideally not be contained in any COREcluster, as they would make their coherence drop. The CORE-clusters in S should then only contain pertinent variables so that the optimal value for U is implicitly defined during the CORE-clustering procedure.
It is also important to remark that the potential number of subsets of X to find good CORE-cluster candidates is huge, even for moderate values of p. Moreover, computing Equation (4) is particularly demanding. The two optimization algorithms of Section 3 are then aggregative and divisive algorithms designed to optimize Equation (4) without explicitly computing it.
Representative Variables Selection
We now present how each representative variable is extracted out of a CORE-cluster S ξ,τ . As mentioned in Section 2.2, the pertinent relations between two variables X i and X j may be lost in w i,j , since n p and recovered by their coherence c(X i , X j ) using other relations. In this example, the similarity s captures true positive and false negative relations and the notion of coherence makes robust the detection of relations. For the same reasons, it may however also capture false positive relations, so the CORE-clusters may contain undesirable variables. The variables captured by CORE-clusters using false positive relations should however be located at the cluster boundaries if the data are not too noisy. False positive connections are indeed less common and on average weaker than true positive connections in this case.
In order to limit the impact of the false positive relations, we then define the representative variables as the CORE-cluster centers. More specifically, each representative variable minimizes an average distance with the other variables of a CORE-cluster S ξ,τ . Instead of using distances based on the maximum capacity Equation (2), we use a more standard notion of distance calculated as sums of weighted edges traversed by the optimal paths. This limits the phenomenon of having multiple variables with the same optimal distance due to the min-max strategy. The graph weights w i,j , which represent a similarity level, must however be converted into distances, which can be simply done by using The representative variable of a CORE-cluster S ξ,τ is then the one that has the lowest average distance to the other variables of S ξ,τ . The impact of selecting the representative variables as CORE-cluster centers is discussed Appendix A.
General Guidelines for the Choice of ξ and τ
The selection of the representative variables directly depends on the parameters ξ and τ. As explained in Section 2.3, a CORE-cluster S ξ,τ is indeed a connected variable subset with a size higher than τ and a coherence c(S ξ,τ ) higher than a threshold ξ. In order to estimate pertinent representative variables, we recommend to use the following guidelines: (1) First compute how the edge weights w i,j of the graph G(N, E) are distributed. The coherence c(S ξ,τ ) represents the weakest connection between the variables of S ξ,τ , so the threshold ξ should be relatively high regarding the different values of w i,j . A value of ξ equal to the 80th percentile of the edge weights w i,j appears to be reasonable. (2) Choosing a suitable minimal amount of variables τ in S ξ,τ is more subtle, as this choice both depends on the complexity of the relations expressed in G(N, E), and how the number of observations n is low compared with the observations dimension p. In all generality, tuning τ as equal to p/10 is reasonable as a first guess. (3) If no CORE-clusters are found with the initial parameters, the user may try to run again the CORE-clustering procedure with lower parameters ξ and τ.
From our experience, we recommend in all cases not using values of ξ lower than the 40th percentile of the edge weights or values of τ lower than 10. The CORE-clusters would be likely to contain variables with strongly heterogeneous behaviors or false positive connections in these cases. Note finally that when several connected CORE-clusters are identified with a given parametrization, it is interesting to test whether stronger COREclusters would be locally found by using higher values of ξ or τ. This is illustrated in Section 4.3.
Main Interactions Estimation
The very first step of our strategy is to quantify the similarity between the different observed variables. The similarity is first computed using the absolute value of Pearson's correlation and represented as a dense graph G(N, E), where N contains p nodes, each of them representing one of the observed variables, and E contains K E = p(p − 1)/2 undirected weighted edges that model a similarity level between all pairs of variables. In this approach, the variables with no connection are associated with a correlation coefficient of zero. The algorithmic cost of this estimation can be O(n 2 p), but it can also be easily parallelized using divide and conquer algorithms for reasonably large datasets, as in [23]. For large to very large datasets, correlations should be computed on sparse matrices, using e.g., [24] to make this task computationally tractable.
CORE-Clustering Algorithms
Inspired by [22], who solved the maximum capacity problem of [21] using optimal spanning tree, we estimate the CORE-clusters on optimal spanning trees. A spanning tree G(N, T) is a subgraph of G(N, E) with no cycle and T ⊂ E. The maximum spanning tree of G is then the spanning tree of G, having the maximal sum of edge weights. Using the maximum spanning tree to detect the CORE-clusters strongly limits the potential amount of node combinations to test, while preserving the graph edges that are likely to be good candidates for the optimal paths of Equation (2). Conversely, it is straightforward to show that the coherence of a variable subset in G(N, T) is lower or equal to the coherence of the same variable subset in G(N, E). The edges of T are indeed a subset of E, so Equation (2) between two variables X i and X j is lower or equal on T than on E. The CORE-clusters computed in G(N, T) are therefore eligible CORE-clusters on G(N, E). By discussing the algorithmic cost of our algorithms, we will make it clear that this reasonable domain reduction makes our problem scalable to large datasets. The impact of using maximal spanning trees on the measure of a cluster coherence is also discussed in Appendix A on synthetic examples.
Maximum Spanning Tree
The maximum spanning tree of G is the simple and reliable modeling of the relationship between the graph nodes (only p − 1 links). One of the most famous algorithms developed to find such trees is called Kruskal's algorithm [25]. The maximum spanning tree is built by adding step by step partial associations so that there will be no cycle in the partial graph.
We denote by G(N, T) the resulting graph, where T has a tree-like structure. Details of the algorithm are given in Algorithm 1. The algorithmic cost of the sort procedure (row 1) is O(K E log (K E )). Then, the for loop (rows 4 to 10) only scans the edges once. In this for loop, the most demanding procedure is the propagation of label L(N i ), row 8. Fortunately, the nodes on which the labels are propagated are related to N j in G(N, E). We can then use a depth-first search algorithm [26] for that task, making the average performance of the for loop O(K E log (p)).
Algorithm 1 Maximum spanning tree algorithm
1: Sort the edges by decreasing weights, so Initiate an edge list T as void. 4: for k = 1 : K E do 5: We denote N i and N j the nodes linked by edge E k . 6: if L(N i )! = L(N j ) then 7: Add edge E k to the list T 8: Propagate the label L(N i ) to the nodes that have label L(N j ).
9:
end if 10: end for 11: return Graph with a tree structure G(N, T).
CORE-Clustering Algorithm
In contrast with other clustering techniques, this CORE-clustering approach detects clusters having an explicitly controlled granularity level, and only gathers nodes/variables with a maximal path capacity. CORE-clusters are detected from the maximal spanning tree G(N, T) by gathering iteratively its nodes N in an order that depends on the edge weights in T (increasing weight order). A detected CORE-cluster has a size higher than τ, where τ is the parameter that controls the granularity level. Thus, Algorithm 2 first generates many small and meaningless clusters and parameter τ should not be too small to avoid considering these clusters as CORE-clusters. Then, the first pertinent clusters will be established using the edges of T with larger weights, leading to pertinent node groups. Finally, the largest weights of T are treated at the end of Algorithm 2 in order to split into several CORE-clusters the nodes related to the most influential nodes/variables.
Algorithm 2 CORE-clustering algorithm
Require: Graph G(N, T) with nodes N i , i ∈ 1, · · · , p; and edges T k , k ∈ 1, · · · , K T . Require: Weight of edge T k is W(T k ). Require: Granularity coefficient τ and threshold ξ. We denote N i and N j the nodes linked by edge E k .
7:
if L(N i )! = L(N j ) then 8: Propagate the label L(N i ) to the nodes that have label L(N j ).
It is worth mentioning that the Rows 20 to 25 of Algorithm 2 are the only ones that require to compute the coherence c of the estimated CORE-clusters. Computing a coherence Equation (3) is indeed demanding, so it is considered here as a post-treatment limited to pre-computed CORE-clusters. In practice, it is also performed on the maximal spanning tree G(N, T) and not on the whole graph G (N, E).
Remark too that the algorithmic structures of Algorithms 1 and 2 are similar. However, Algorithm 2 runs on G (N, T) and not on G (N, E). The number of edges K T in G(N, T) is much lower than K E , since T has a tree structure and not a complete graph structure. It should indeed be slightly higher than p [27], which is much lower than K E = p(p − 1)/2. Moreover, the propagation algorithm (rows 9 and 11) will then never propagate labels on more than 2τ − 1 nodes. The algorithmic cost of the sort procedure (row 1) is then O(K T log (K T )) and the average performance of the for loop O(K T log (τ)).
A Greedy Alternative for CORE-Clusters Detection
We propose an alternative strategy to the CORE-clusters detection algorithm: The edge treatment queue may be ordered by following decreasing edge weights instead of increasing edge weights. The nearest edges are then first gathered, making coherent CORE-clusters as in Algorithm 2, although one CORE-cluster may contain several representative variables. To avoid gathering noisy information, the for loop on the edges (row 6 of Algorithm 2) should also stop before meaningless edges are treated. This strategy has a key interest: It can strongly reduce the computational time dedicated to Algorithms 1 and 2. By doing so, Algorithm 1 and modified Algorithm 2 are purely equivalent to Algorithm 3.
1: Sort the edges by decreasing weights. 2: Define the number of edges γ having a weight higher than ξ. 3: Assign label L(N i ) = i to each node N(i). 4: Set CORElabel = −1. 5: for k = 1 : γ do 6: We denote N i and N j the nodes linked by edge E k .
7:
if L(N i )! = L(N j ) then 8: Propagate the label L(N i ) to the nodes that have label L(N j ).
9:
if number of nodes with label L(N i ) ∈ {τ, · · · , 2τ − 1} then 10: Label CORElabel is given to the nodes with label L(N i )
Again, the structure of this algorithm is very similar to the structure of the maximum spanning tree strategy in Section 3.1. The algorithmic cost of the sort procedure (row 1) is O(K E log (K E )). Then, the loop rows 5 to 14 scans γ edges, where γ is the number of edges having a weight higher than ξ. In most cases, γ should be much lower than K E , which strongly limits the computational impact of this loop. Labels propagation in this loop (rows 8 and 10) are also limited to 2τ − 1 nodes. The average performance of the for loop is therefore O(γ log (τ)).
Central Variables Selection in CORE-Clusters
Once a CORE-cluster is identified, we use a straightforward strategy to select its central variable: the distance between all pairs of variables in each CORE-cluster is computed using a Dijkstra's algorithm [28,29] in G(N, E). The central variable is then the one that has the highest average distance to all other connected variables in the CORE-cluster. As the computed CORE-clusters have less than 2τ nodes, the algorithmic cost of this procedure is O(τ 2 ) times the number of detected CORE-clusters, which should remain low, even for large datasets.
Core Clustering of Simulated Networks
In this section, we compare our CORE-clustering algorithms with other standard methods on simulated scale-free networks. Such complex networks are indeed common in the data science literature. They contain a little amount of highly connected nodes (hubs), that we will assimilate to representative variables, and many poorly connected nodes.
Experimental Protocol
To generate the synthetic networks, we first simulated the profile (observations) of representative variables and then the profile of remaining variables around these hubs. We then considered K different clusters of size p C 1 , p C 2 , . . ., p C K . A simulated expression data set X ∈ R n×p is then composed of p = p C1 + . . . + p C K variables. In the cluster k, the observations are then simulated as follows: (1) Generate the observations x (1,k) ) of a representative variable using a normal distribution N (0, 1). (2) Choose a minimum correlation r min and a maximum correlation r max between the representative variable and the other variables of the predefined cluster. In this paper, we always used r max = 1 and r min = 0.5. (3) Generate the profiles x (j,k) , with j ∈ {2, . . . , p C k }, such that the correlation of the j-th profile with the profile of . Three different types of networks were simulated using this protocol with different parameterizations. (a) The first type of network consisted in simulating n = 100 observations of 40 variables with K = 2 clusters of 20 variables. The additional noise was simulated with α, ranging from 0.25 to 1.5. (b) The same protocol was used for the second type of networks, but K = 5 clusters of 7 variables were simulated. (c) The third kind of networks consisted in varying the number of the observations from n = 5 to n = 30, with K = 5 clusters having 50 to 100 variables.
Note finally that an amount of 30 networks of each type was generated to assess the stability of our methodology. This will indeed make it possible to draw in Figure 1 the box-plots of the clustering quality for each type of network.
Measure of Clustering Quality
There exist various criteria to measure a clustering quality. External indices such as impurity and Gini indices measure the extent to which the clusters match externally supplied class labels. Internal indices like the modularity and the intra-cluster to intercluster distance ratio are also used to measure the quality of a clustering structure without any external information. Such criteria are however not suitable here, as we do not clusterize all variables, but rather extract core structures that emphasize representative variables. Thus, we propose the following criterion for simulated data for which the block structure of the similarity matrix is known: Let X i , i ∈ {1, · · · , p} be the variables and C j (j ∈ [1, K]) be the ground-truth CORE-clusters of variables, typically on synthetic data. As in Section 2, we also denote S u ξ,τ (u ∈ [1, U]) the CORE-clusters predicted by our algorithm. In order to evaluate the quality of the prediction, we compute a score R defined as: where 1 ≤ i ≤ p and R = 1 p U ∑ u=1 R u . To compute this score, each R u is equal to 0 if there is no overlap between C j and any S u ξ,τ , and is equal to the number of variables in C j if a S u ξ,τ contains all the variables of C j . A score R equal to 1 then means that a perfectly accurate estimation of the C j was reached, and the closer to 0 its values, the less accurate the CORE-clusters detection.
Results
We compared the standard and greedy CORE-clustering algorithms (Algorithms 2 and 3) on these simulated datasets with two other graph-based clustering algorithms: the spectral clustering [30,31], available in the R-package anocva, and Louvain method for community detection [32], available in the R-package igraph. Note that the Louvain method requires as input parameter a graph modeling the dataset (the correlation matrix is transformed upstream into a graph) but not the final number of clusters. Boxplots of the computed scores R (see Equation (5)) are shown in Figure 1. Note that in each boxplot, the dots represent the outlier scores, which are either lower than q 0.25 − 1.5(q 0.75 − q 0.25 ) or higher than q 0.75 + 1.5(q 0.75 − q 0.25 ), where q 0.25 and q 0.75 are the first and third quartiles of the scores, respectively. The subplots of Figure 1a,b show that Algorithm 2 is more robust than Algorithm 3, and gives slightly better results than spectral clustering and Louvain method, when the level of noise is high. The same applies when the sample size decreases in the subplots of Figure 1c.
Application to Real Biological Data
We now present the results obtained on the classic Yeast dataset (https://archive.ics. uci.edu/ml/datasets/Yeast (accessed on 19 February 2021)) [33]. With a total of about 1.3 × 10 6 weighted edges considered when representing the variables correlations in the graph G (N, E), the CORE-clustering procedure required about 160 and 3 seconds with Algorithms 2 and 3, respectively.
Yeast Dataset
The well-known synchronized yeast cell cycle data set of [33] includes 77 samples under various time during the cell cycle and a total of 6179 genes, of which 1660 genes are retained for this analysis after pre-processing and filtering. The goal of this analysis is then to detect CORE-clusters among the correlation patterns in the time series of yeast gene expressions measured along the cell cycle. Using this dataset, a measure of similarity between all gene pairs was measured with the absolute value of Pearson's correlation. A total of about 1.3 × 10 6 weighted edges are then considered when representing the variables correlations in the graph G(N, E).
Comparison of the Two CORE-Clustering Algorithms
In order to compare the two proposed CORE-clustering algorithms described (Algorithms 2 and 3) we tested them on the yeast dataset with τ = 30 and ξ = 0.75. We indeed empirically considered that CORE-clusters containing 30 to 59 variables are reasonable to regularize the problem, and that 0.75 is a threshold above which the absolute value of the correlation between two variables reasonably shows that their behavior is similar.
The clustering obtained using the standard algorithm, rows 1 to 19 of Algorithm 2, is shown in Figure 2. In this figure, the represented clusters 1 to 6 have a coherence c (see Equation (3) The computations were much faster using the greedy algorithm Algorithm 3. It indeed required about 3 seconds. An amount of 11 CORE-clusters was found. To interpret this result, we computed the coherence c (see Equation (2)) between the 4 representative variables obtained using Algorithm 2 and the 11 obtained using Algorithm 3. Interestingly, Algorithm 3 selected YLL026W = RV3 and YDL003W = RV5. Other variables very close to RV1, RV5 and RV6 (with c > 0.83 i.e., higher that the highest c within the CORE-clusters) were also selected: YHR219W, YLR103C, YLR276C and YLR196W. Results equal or close to those obtained with Algorithm 2 were then obtained. The representative variables YDR418W, YML119W, YJL038C, YNL283C and YGR167W were additionally found. In this experiment, Algorithm 3 therefore selected more representative variables and has then naturally a larger score (see Equation (4)) than by using Algorithm 2. However, it also obviously captured different representative variables that would be gathered in the same CORE-cluster using Algorithm 2. The two algorithms have therefore slightly different properties but lead to coherent results.
Impact of the Number of Observations
In order to evaluate the stability of the results with respect to the number of observations, we tested again Algorithm 2 with the same parameters, but by using only the 30 first observations of the yeast dataset out of the 77 observations. Interestingly, YDL003W = RV5 was selected and YML093W, which is very close to RV6 (c > 0.86), was also selected. The two other representative variables found, YER190W and YLL026W, were however not similar to RV1 or RV3. The information lost in the 47 observation that we removed therefore did not allowed to recover the influence of RV1 and RV3 on the complex system but the 30 remaining observations contained a sufficient amount of information to detect RV5 and RV6 as influent variables. This suggests that the strategy detects stable representative variables, even when the number of observations is very low compared with the dimension of the observations.
Comparison with Spectral-Clustering
In order to compare our CORE-clustering strategy with a standard clustering approach, we finally estimated representative variables in the Yeast dataset as the center of clusters estimated using spectral clustering. The standard version of the spectral clustering available in R was used. Its main parameter is the number of seeds η used in the k-means part of the spectral clustering. When using η seeds, an amount of 1 to η clusters (with more than one variable) are estimated using k-means and all variables are contained in a cluster. In order to fairly compare the spectral clustering and the CORE-clustering approaches, we then clusterized the variables of the Yeast dataset using η = {5, 30, 50, 70, 110}. Note that we only tested an η higher than 77, due to the fact that n = 77 observations are known. We tested η = 110 to evaluate the spectral clustering behaviour with η slightly higher than n.
An amount of {3, 3, 6} large clusters were obtained for η = {50, 70, 110}, respectively. For η = {5, 30}, only a single cluster gathering almost all variables was also obtained. The average coherence of the estimated clusters was 0.44 for a standard deviation of 0.06. The highest coherence was 0.63, which is clearly lower than the considered threshold of ξ = 0.75 that we used with the CORE-clustering. This makes ambiguous the interpretation of the role played by the representative variables.
Finally, it is worth mentioning that all representative variables (genes) obtained using Algorithm 2 have a known physiological function and only two variables out of eleven have an unknown function by using Algorithm 3. For the spectral clustering with η = 110, four selected variables out of six have known functions. For η = 70, three variables with unknown functions were selected. For η = 50, two variables out of three with known functions were selected. For η = 5 and η = 30, the single selected variable was the center of all variables with respect to the center definition in Section 3.3, and turns out to have a known function. It therefore appears in this experiment that the representative variables obtained using CORE-clustering are more interpretable than those estimated using spectral clustering.
Application to the U.S. Road Network
We now assess the CORE-clustering algorithms on the U.S. road network out of the 9th DIMACS Implementation challenge (http://users.diag.uniroma1.it/challenge9/ (accessed on 19 February 2021)). Our goal here is to discuss the pertinence of the detected CORE-clusters, and not specifically their representative variables, on a large scale and straightforwardly interpretable dataset. The graph contains here 2.4 × 10 7 nodes, each of them representing a crossing of the U.S. road/streets network, and 5.8 × 10 7 arcs, representing the part of the roads/streets between two crossings. Interestingly, the distance between the crossings is also associated with each edge of the graph.
To assess the CORE-clustering algorithms on the U.S. road network, we first transformed the distances between the crossings into weights that are higher and higher for increasingly closer crossings. A weight max (4 × 10 3 − l)/(4 × 10 3 ), 10 −4 was specifically given to each edge, where l is its original length in feet.
This means that each CORE-cluster contains a road network of 5 × 10 4 to 10 5 crossings, for which one can travel from any crossing to another one by only using streets of less than 3996 feet (about 1.2 km) between two crossings. As expected, the clusters represented in Figure 3 correspond to the 18 largest urban areas in the U.S. Note that two of them are made of three CORE-clusters (New York City and Los Angeles), and two other ones (Miami and Chicago) are made of two CORE-clusters. This is due to an algorithmic choice, discussed in Sections 3.2.2 and 3.2.3, which leads to the detection of CORE-clusters containing τ to 2τ − 1 nodes by using the proposed CORE-clustering algorithms. As discussed in Section 2.6, an interesting strategy if connected CORE-clusters are found consists in running again the CORE-clustering algorithms with higher values of τ or ξ, to potentially detect more robust CORE-clusters: By reproducing this test with τ = 10 5 instead of τ = 5 × 10 4 , we now try to find CORE-clusters containing 10 5 to 2 × 10 5 crossings, only 5 urban areas are found: New York City (2 CORE-clusters), Los Angeles, Chicago, Miami and San Fransisco. Now, by using τ = 5 × 10 4 and now ξ = 0.5, i.e., with distances between the crossings lower than about 2000 feet (about 0.6 km) instead of 3996 feet, only 3 urban areas are found: New York City (2 CORE-clusters), Los Angeles and Chicago. What is interesting here in terms of interpretability is that we have been able to select specific subparts of the U.S. road network by explicitly controling the size of the clusters or a specific level of density in the network. This notion of interpretability can be further discussed by observing the CORE-clusters obtained in the New York city urban area, as shown in Figure 3b-d. By using τ = 5 × 10 4 and ξ = 10 −3 , the three CORE-clusters split the densest parts of the New York City urban area into the New Jersey state, Long Island and the rest of New York state. When having larger CORE-clusters, i.e., with τ = 10 5 , the New Jersey cluster, expands and the densest parts of the two New York state clusters are merged. Now, when enforcing instead a stronger coherence inside of the clusters, i.e., with τ = 5 × 10 4 and ξ = 0.5, the New Jersey and Long Island CORE-clusters remain stable, but the Manhattan/mainland New York state cluster is not large and dense enough, so it is not captured as a CORE-cluster. Note that each CORE-cluster estimation required about 50 s and 2.4 GB here without any parallelization.
Conclusions
Although complex systems in high dimensional spaces with a limited number of observations are quite common across many fields, having efficient methods to treat the associated problem of graph clustering is an ambiguous task. Some of these techniques, based on assumptions in view of controlling the variables contribution to the global clustering, often do not allow to select the best graph partition. In reply to this issue, we developed a formalism based on an original graph clustering strategy with specific properties. This formalism makes it possible to robustly identify groups of representative variables of the studied system by tuning two intuitive parameters: (1) the minimum number of variables in each CORE-cluster, and (2) a minimum level of similarity between all the variables of a CORE-cluster. Its effectiveness was further satisfactorily assessed on simulated data and on real datasets.
From a methodological perspective, an interesting research direction would be to mix Algorithms 2 and 3 into a single hybrid top-down and bottom-up optimization scheme. Our goal would be to scale well to very high dimensional datasets, as when using Algorithm 3, while being as robust as Algorithm 2 when potential CORE-clusters are coarsely identified. When the CORE-clusters found by either Algorithms 2 or 3 are very large, a stochastic strategy could also make faster the detection of their representative variables. Although this secondary part of our methodology has been addressed by using a standard Dijkstra's algorithm (see Section 3.3), it could be addressed by using an extension of [34] where the optimal paths between all variables would not be pre-computed prior to the central variable detection. Application of our formalism in various fields such as gene regulatory networks, social networks or recommender systems would also be of interest. Our formalism is indeed sufficiently flexible to incorporate different types of similarity measures between the observed variables.
Note finally that our formalism was implemented in C++ and wrapped in a R package, which is freely available on sourceforge (https://es.sourceforge.net/projects/coreclustering/ (accessed on 19 February 2021)), so that these results can be easily reproduced.
Conflicts of Interest:
The authors declare no conflict of interest. Let us first interpret the results in Table A1. The coherences' first row on G 1 in tests T1 and T2 first show that similar coherences were obtained with CORE-clusters of size 7 and 3, although the coherences are slightly higher for smaller CORE-clusters. These coherences are also clearly higher for those obtained in tests T3 and T4 where all variables are not contained in the same simulated block. Interestingly, the results obtained on graph G 2 are similar to those obtained on G 1 . The only difference here is that the coherences of T4 are slightly higher than in G 1 , but still relatively low. Note that the tested CORE-clusters may lead to disconnected subgraphs when tested on the maximum spanning trees. Equation (2) does not make sense in this case. When the simulated subgraphs were connected (12 cases out of 16), we obtained the same coherences on the maximum spanning trees and the whole graph. As discussed in Section 2.4, the coherence of a given CORE-cluster on a maximum spanning tree is indeed lower or equal to the coherence of the same CORE-cluster on the whole graph. We however computed the representative variables in all tested cases, as the centrality measure makes sense even on disconnected subgraphs (see Section 3.3). The results in Table A2 show that the estimated representative variables are always in the reference sets S 1 Re f and S 2 Re f in the tested configurations. They also correspond to the simulated representative variables, X 3 and X 11 , in most cases, or are very close to these variables. Note that slightly inaccurate reference variables were detected in S 1 Re f , and we can clearly see ( Figure A1(top)) that the influence of its simulated representative variable is less obvious than in set S 2 Re f . We further study the influence of the undesirable relations between {X 1 , · · · , X 5 } and {X 9 , · · · , X 13 } in graph G 2 by simulating these relations with different strengths. In the previous subsection, undesirable relations were sampled following N (µ, 0.1), where µ = 0.37. Here, we sampled 100 graphs G 2 for each strength µ ∈ {0.1, 0.40, 0.60, 0.80, 0.90}. For each graph, we then measured the portion of representative variable estimates in the true reference block of variables and the portion of representative variable estimates that are the ground truth representative variables.
Results are given in Table A3 and show that the representative variables detection is particularly stable in these tests, even for large values of µ. All estimated representative variables are indeed in the true block of variables, except in Test 4 (where two variables of the reference sets are swapped) with µ = 0.9, i.e., with the same level of similarity as between the blocks representative variables and the other variables they contain. False estimations are however uncommon even in this case. Exact estimates of the representative variables are naturally less frequent, as this test is more strict. They are however always clearly higher than random estimations which would have portions equal to 0.14. The estimates of pertinent representative variables therefore appear as robust, even with strong undesirable relations in the variable similarities and tested CORE-clusters which contain undesirable variables. Table A3. Portion of representative variable estimates contained in the proper reference block of variables (main value) and corresponding to the ground truth representative variables (between brackets). Each portion was computed on 100 simulated graphs (G 2 ) and their corresponding maximum spanning trees (ST(G 2 )). For each group of 100 graphs, a different strength µ of the undesirable relations is tested. Test 2 Test 3 Test 4 | 2021-03-17T13:18:31.375Z | 2021-02-22T00:00:00.000 | {
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261847204 | pes2o/s2orc | v3-fos-license | Trust and Safety on Social Media: Understanding the Impact of Anti-Social Behavior and Misinformation on Content Moderation and Platform Governance
The Special Issue on Trust and Safety on Social Media delves into two pressing and interlinked concerns: the growing prevalence of anti-social behavior and the widespread presence of misinformation within and across various social media platforms. The collection of articles featured in the issue collectively examines factors that contribute to these concerns and proposes potential strategies to mitigate their negative impact on social media users and society. The articles included in the issue are extended versions of research first presented at the 2022 International Conference on Social Media & Society (#SMSociety), organized by the Social Media Lab at Toronto Metropolitan University.
Introduction
The Special Issue on Trust and Safety on Social Media is a collection of peer-reviewed articles that examine the rise of anti-social behavior, misinformation, and other forms of problematic content within and across various social media settings, contexts, and user groups.The special issue emerged from the presentations and deliberations by interdisciplinary scholars at the 2022 International Conference on Social Media & Society, organized by the Social Media Lab at Toronto Metropolitan University.The issue explores two dangerous and interconnecting trends: the rise of anti-social behavior and the spread of misinformation online.Its aim is to help the public, policymakers, and platform operators better understand the factors contributing to the rise of these minacious trends on social media.
The diverse targets of and motives for engaging in antisocial behavior make content moderation a challenge.
Platforms have been testing and building both automated and manual approaches to mitigate problems on the social web (e.g., Gibson, 2023;Horta Ribeiro et al., 2023;Papaevangelou & Smyrnaios, 2022).For example, Twitter tested (with some success) a feature that prompted users to reconsider their messages if they were potentially harmful (Katsaros et al., 2022).Other platforms, such as Reddit, rely more on community-led moderation and use jargon-free community rules to promote "healthier" conversations by enhancing civility and other pro-social behaviors (e.g., Del Valle et al., 2020;Trujillo & Cresci, 2022).
The situation surrounding the spread of misinformation online is not much different.Even though measuring the extent of misinformation on social media can be challenging due to its various forms and often ephemeral nature, researchers have extensively documented its impact, spread, and prevalence across social media platforms (e.g., Chen, Xiao & Kumar, 2023;van der Linden, 2022).This phenomenon is particularly evident in discussions of politically polarized topics, such as gun control (Williams, 2022), climate change (Falkenberg et al., 2022), abortion (Pagoto et al., 2023), vaccination (Gruzd et al., 2023), refugees (Zhen et al., 2023), and more recently, the COVID-19 pandemic (Gruzd et al., 2021).In its most dangerous form, misinformation turns into disinformation when it is deliberately used to deceive, polarize, or radicalize the population.Disinformation has been weaponized by malicious actors during events, such as the 2020 US election (e.g., Chang et al., 2021;Lee & Jones-Jang, 2022) and, more recently, Russia's full-scale invasion of Ukraine (Gruzd et al., 2022;Milmo, 2022).Previous efforts to understand the reasons for the spread of misinformation and propose mitigation strategies include identifying and flagging false claims, fake accounts, coordinated sharing of misleading links, and manipulated media (e.g., Calleja et al., 2021;Carnahan & Bergan, 2022;Gruzd et al., 2022).In addition, there have been some attempts to implement intervention strategies, such as accuracy prompts, debunking, friction, inoculation, media literacy, and self-reflection tools (e.g., Koch et al., 2023;Scharrer et al., 2022;Singh & Banga, 2022;Traberg et al., 2022;Vivion et al., 2022).Further research is necessary to fully grasp the phenomenon of misinformation and develop effective countering strategies, given the constantly evolving techniques of misinformation spreaders and changing nature of social media platforms and social norms.
Building on the previous research in these areas, the special issue proposes an integrated and multidisciplinary approach toward addressing the challenges posed by the growth of anti-social behavior and the proliferation of misinformation on social media.The articles in the issue demonstrate the complex and intertwined nature of these matters; for example, in cases where online harassment is employed as part of strategic disinformation campaigns, and vice versa.The issue incorporates a diverse range of works, including both conceptual and empirical case studies, and examines both human and algorithmic aspects of content moderation and platform governance.It also takes a critical view of user practices, community engagement, and the network effects of anti-social behavior.Below is a brief overview of the works included in the issue.
Platform Governance
The issue starts with three papers on platform governance (Haythornthwaite, 2023;Nagappa, 2023;Zuckerman & Rajendra-Nicolucci, 2023).By examining the role of platform and community governance, these studies offer potential strategies for improving online moderation practices and fostering healthier online spaces.
In "Moderation, Networks, and Anti-Social Behavior Online," Haythornthwaite discusses the challenges of moderating extreme content on major social media platforms.The author examines the questions of how to define and identify anti-social behavior online and how to effectively use automation and human review to manage offending content.The paper suggests a framework of three layers (environment, community, and crowd) and emphasizes the importance of understanding the network impact of antisocial behavior.
In "From Community Governance to Moderation and Back Again: Re-examining Pre-Web Models of Online Governance to Address Trust and Safety's Crisis of Legitimacy," Zuckerman and Rajendra-Nicolucci argue that the issues of content moderation on social media are central to democratic participation.They review early models of social media content moderation, considering whether the "free speech" and "public health" approaches to moderation might have obscured an earlier model of community-led content moderation.The authors advocate for a community moderation approach to social media, which they argue could address persistent challenges of social media moderation and provide valuable training in democratic participation.
In "Narratives of Change to Platform Governance on DTube, an Emerging Blockchain-based Video-sharing Platform," Nagappa discusses the emergence of blockchainbased social media platforms as alternatives to mainstream social media platforms.The paper focuses on the changes to one such platform called DTube and its governance structure.It highlights how user practices, rather than technology, steer platform functions.
Computational Techniques for Investigating Anti-Social Behavior and Disinformation and Misinformation
The next set of three articles are focused on methodology and present advanced computational techniques for investigating anti-social behavior and the dissemination of disinformation and misinformation on social media (Angus et al., 2023;Giglietto et al., 2023;Steinfeld, 2023).
In their paper, "Computational Communication Methods for Examining Problematic News-Sharing Practices on Facebook at Scale," Angus et al. present a novel approach for analyzing the spread of "fake news" and other problematic information on Facebook.By analyzing networks of content sharing between public pages, groups, and external sources, the proposed method can pinpoint the most prominent sources of problematic content.The result is a comprehensive understanding of the impact that misinformation and disinformation can have on societies that are interconnected through social media.
In "A Workflow to Detect, Monitor and Update Lists of Coordinated Social Media Accounts Across Time: The Case of 2022 Italian Election," Giglietto et al. propose a methodology for detecting coordinated social media accounts in the context of political elections.The authors applied their technique to uncover various instances of potentially coordinated accounts engaged in discussions about the 2022 Italian election, including politically motivated, click-driven, and religiously motivated operations.
In "How Do Users Examine Online Messages to Determine If They Are Credible?An Eye-Tracking Study of Digital Literacy, Visual Attention to Metadata and Success in Misinformation Identification," Steinfeld investigates how users assess the credibility of online information and the association between user attention to metadata, digital literacy, and the identification of misinformation.The author found that users with advanced digital literacy tend to focus more on information metadata and are better equipped at spotting online misinformation.
Complexities of Managing Online Content and Behavior across Various Platforms
The issue concludes with five case studies that offer insights into the complexities of managing online content and behavior across various platforms, contextual backgrounds, and user demographics.These case studies have been authored by B. Chen, Lukito, and Koo (2023), Salles et al. (2023), Morales (2023), Hodson and O'Meara (2023), and Musiyiwa and Jacobson (2023).
In their research article, "Comparing the #StopTheSteal Movement across Multi-platform: Differentiating Discourse on Facebook, Twitter, and Parler," Chen, Lukito, and Koo analyze the discourse around the #StopTheSteal movement on Facebook, Twitter, and Parler in the aftermath of the 2020 US Presidential election and leading up to the Capitol Riot.The authors employ Snow and Benford's Social Movement Frames typology and specifically explore the presence of violence cues.Their findings indicate that Parler, an alternate platform, was more inclined toward inciting violence through the use of aggressive language, thereby exacerbating the call to action, as opposed to Facebook or Twitter.
In "The Far-Right Smokescreen: Environmental Conspiracy and Culture Wars on Brazilian YouTube" by Salles et al., the authors examine how the conservative YouTube channel "Brasil Paralelo" used platform affordances and political alignment to gain social relevance on environmental conspiracies.The study, which used topic modeling and network analysis, discovered that far-right rhetoric opposing environmentalism is employed as a contemporary culture war weapon.This narrative often contains unfounded allegations concerning politics, gender, religion, and other ideological themes.
In the article on "Ecologies of Violence on Social Media: An Exploration of Practices, Contexts, and Grammars of Online Harm," Morales argues for the need to understand the ways that violence is performed and communicated on social media.Using a case study of young adults in Colombia, the author demonstrates the complexity of violence on social media platforms, which is often multifaceted, overlapping, and interconnected.Morales proposes a framework for understanding and addressing harmful practices as ecologies of violence, which includes examining the practices, contexts, and grammars that contribute to online harm.The author stresses the importance of recognizing and addressing this complexity to build online and offline "cultures of peace." Hodson and O'Meara's study, "Curating Hope: The Aspirational Self and Social Engagement in Early-Onset Cancer Communities on Social Media," examines online communities and discourse among young cancer patients and caregivers on social media.The authors note that these communities offer more than just information and emotional support, they also inspire hope and confront the traditional division between online authenticity and the aspirational self often present on social media.
Finally, in their work titled "Sponsorship Disclosure in Social Media Influencer Marketing: The Algorithmic and Non-Algorithmic Barriers," Musiyiwa and Jacobson explore the obstacles that hinder compliance with sponsorship disclosure on social media.The study reveals that both algorithmic and non-algorithmic challenges exist, making it difficult to ensure proper disclosure.Such challenges include the deprioritization of disclosure by algorithms and time-consuming disclosure procedures.The researchers suggest various strategies that influencers can employ to effectively disclose their sponsored content upfront and in a prominent manner.
Closing Thoughts
Overall, the diverse perspectives and innovative approaches presented in all 11 papers contribute to a deeper understanding of the challenges and opportunities in managing social media platforms, fostering healthier online spaces, and guiding future research in this rapidly evolving field.While the papers tackle various issues, their collective message emphasizes the importance of a comprehensive strategy that incorporates automated and manual review, community governance, and user literacy to effectively combat emerging challenges in digital spaces.
As the editors of the special issue, our hope is that it will reach a broad audience of social media stakeholders, including policymakers, developers, and platform owners and users.Researchers from a range of fields, such as communication, information technology, media studies, sociology, psychology, computer science, and other fields, will benefit from the theoretical frameworks, methodologies, and findings provided in these papers.Social media platform developers and managers can use the insights gained from the issue to inform design decisions related to content moderation, community governance, and user engagement.In addition, policymakers and regulators, educators, digital literacy advocates, and the general public may use this research to develop a deeper understanding of issues related to online behavior, moderate content, misinformation, and user safety.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. | 2023-09-15T15:03:08.611Z | 2023-07-01T00:00:00.000 | {
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219920332 | pes2o/s2orc | v3-fos-license | Repurposing the Native Type I-F CRISPR-Cas System in Pseudomonas aeruginosa for Genome Editing
SUMMARY Repurposing the broadly distributed native CRISPR-Cas systems in prokaryotes for genome editing is emerging as a new strategy for genetic manipulations. We recently reported the establishment of a single plasmid-mediated, one-step genome-editing technique in a multidrug-resistant genotype of the opportunistic pathogen Pseudomonas aeruginosa by harnessing its endogenous type I-F CRISPR-Cas system. The platform is readily applicable in additional type I-F CRISPR-containing clinical and environmental P. aeruginosa isolates. Herein, we provide the detailed protocol for the methodology. For complete details on the establishment and exploitation of this protocol, please refer to Xu et al. (2019).
array PA154197_2 located 1,376,188-bp downstream of PA154197_5. The 28-bp consensus repeat in the three arrays differs by only one nucleotide and their spacers are identical in size (32 bp).
PAM sequence characterization
Identification of protospacer adjacent motif (PAM) sequence is essential for assembling a guide mini-CRISPR array and repurposing the identified CRISPR-Cas systems for genome editing. To this end, all 26 spacers present in the three CRISPR arrays are manually extracted and aligned against the bacteriophage and plasmid databases using CRISPRTarget to acquire protospacer sequences (Biswas et al., 2013). Since PAM is located at the 5 0 -end of the protospacer in type I Figure 1. Bioinformatic Analysis for the Presence of Native CRISPR-Cas Locus and Its Spacers, Repeat, and PAM Sequences (A) Output results showing the presence of a type I-F CRISPR-Cas system by CRISPRCasFinder search following input of PA154197 genome sequence as the query. Blast of the identified CRISPR arrays and cas genes against the genome of PA154197 led to assembly of the complete native CRISPR-Cas loci in the strain. Diamonds and rectangles indicate the repeats and spacers in the CRISPR arrays, respectively. Number of spacers is indicated above each of the CRISPR arrays. Bended arrows (black) above the leader sequence (purple) indicate the orientation of CRISPR transcription. (B) Alignment of 8-bp upstream sequences of protospacers identified from the bacteriophage and plasmid databases led to prediction of the PAM sequence 5 0 -CC-3 0 (Blue). A frequency chart is plotted to visualize the PAM sequence by inputting all the available PAM-Protospacer sequences using WebLogo.
CRISPR-Cas systems, sequences 8-bp upstream of the identified protospacers are extracted and aligned to predict the PAM sequence ( Figure 1). The PAM sequence of type I-F CRISPR-Cas system was identified as 5 0 -CC-3 0 (Richter et al., 2014). A frequency chart is plotted to visualize the PAM sequence using WebLogo (Crooks et al., 2004). Authenticity of the PAM sequence is further validated by ''Self-targeting activity assay'' described in the Step-by-Step Method Details section. 3. Design mini-CRISPR and primers to assemble pTargeting and pEditing a) mini-CRISPR and pTargeting: A mini-CRISPR guide is required to construct the targeting and editing plasmids for self-targeting activity assay and genome editing, respectively. A mini-CRISPR encompasses a 32-bp spacer sequence derived from a selected target gene in the chromosome of PA154197 immediately downstream of a selected 5 0 -CC-3 0 PAM and two 28-bp repeat sequences flanking at both ends ( Figure 2). In our study, the 88-bp mini-CRISPR array (28+32+28) is synthesized commercially and is provided in a plasmid termed as pUC57-mini-CRISPR in which the synthesized mini-CRISPR is cloned between the KpnI and BamHI sites of the pUC57 vector (BGI, China) ( Figure 2). pTargeting is assembled by cloning the mini-CRISPR between the KpnI and BamHI sites in the platform plasmid pAY5211.
b) Editing Donor: In CRISPR-mediated genetic editing, various types of editing are achieved by the provision of desired donor sequences ( Figure 3). For gene deletion, an editing donor typically consists of 500-bp upstream (Up donor) and 500-bp downstream (Down donor) of the desired deletion region. For gene insertion, an editing donor typically consists of 500-bp upstream (Up donor) and 500-bp downstream (Down donor) of the insertion site and the intended insertion fragment (Mid donor). For single nucleotide substitution, an editing donor is consisted of 1-kb DNA sequence spanning the editing site with desirable single nucleotide substitution located in the center. Depending on the types of editing, the corresponding primers are designed and synthesized commercially.
RESOURCE AVAILABILITY
Requests for resources should be directed to and will be fulfilled by the Lead Contact, Aixin Yan (ayan8@hku.hk).
MATERIALS AND EQUIPMENT
Centrifuge ( Note: Any alternative commercial kits for PCR, enzymatic digestion, ligation etc. may be used as well, as long as the DNA products are obtained with sufficient quantity and quality as described in the steps below.
STEP-BY-STEP METHOD DETAILS
This protocol provides detailed procedures for one-step genome editing (Gene deletion, Gene insertion and Single nucleotide substitution) in the native type I-F CRISPR-Cas-containing MDR Step 1: An 88-bp mini-CRISPR which encompasses a 32-bp CC-preceding sequence (Dashed Box) within the target gene flanked by two 28-bp repeat sequences (Orange diamond) is submitted for commercial synthesis. Mini-CRISPR flanked by restriction sites KpnI and BamHI is supplied in the plasmid pUC57-mini-CRISPR.
Step 2: The mini-CRISPR is amplified by PCR using primers pUC57-F/R.
Step 3: After digestion with KpnI and BamHI, the purified mini-CRISPR is inserted into the plasmid pAY5211 which is treated with the same restriction enzymes, generating the targeting plasmid pTargeting. pTargeting is verified by sequencing using primers pMS-402-F/R.
Step 4: pTargeting is linearized by enzyme digestion with XhoI.
Step 5: 500-bp Up donor (Pink), containing a 15-bp 5 0 -end homology (Green) with the 3 0 end of the linearized pTargeting and a 15-bp 3 0 -end homology (Magenta) with the Down donor, is amplified by PCR using primers Donor-UF/UR. 500-bp Down donor (Magenta), containing a 15-bp 5 0 -end homology with the 3 0 -end of the Up donor and a 15bp 3 0 -end homology (Dark green) with the 5 0 end of the linearized pTargeting, is amplified by PCR using primers Donor-DF/DR.
OPEN ACCESS
STAR Protocols 1, 100039, June 19, 2020 P. aeruginosa isolate PA154197 ( Figure 2). A platform plasmid (pAY5211, Figure 4A) for constructing the customized targeting plasmid and editing plasmid is available upon request. pAY5211 contains a mini-CRISPR insertion site which is flanked by the KpnI and BamHI sites, a donor insertion site at the XhoI site, and a kanamycin-luminescence dual selection marker. Sequence information of pAY5211 is presented in Figure 4A.
Construction of the Targeting Plasmid (pTargeting)
Timing: 2-3 days 2. Purify the 236-bp PCR product using the MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa, Japan) following the manufacture's instruction.
Alternatives: If the KpnI-mini-CRISPR-BamHI fragment is synthesized and supplied in the linear double-stranded DNA form, it can be directly proceeded to Step 3.
Purified PCR product from
Step 2 and the plasmid pAY5211 are digested with KpnI and BamHI (NEB, USA). Digestion reaction is set up as below:
Figure 2. Continued
Step 6: Up Donor and Down donor are assembled into the linearized pTtargeting by Gibson assembly, generating the editing plasmid pEditing. pEditing is verified by sequencing using primers pMS-402-F/R. Detailed donor design for various types of genetic editing are presented in Figure 3. 6. Ligate the enzyme-digested pAY5211 (0.5 mg) and mini-CRISPR (0.1 mg) using the Quick Ligation Kit (NEB, USA) to generate pTargeting. Reaction mixture without addition of digested mini-CRISPR serves as the negative control. Ligation reaction is set up as below:
Component
7. Incubate the reaction at 25 C for 45 min. 8. Mix the ligation mixture with 100 mL cold CaCl 2 (0.1 mM) and 200 mL DH5a component cells gently and thoroughly. Chill on ice for 10 min. 9. Heat shock the mixture at 42 C for 90 s and quickly chill it on ice for 5 min.
Component
Volume ( 10. Add 1 mL fresh LB broth into the mixture and recover the cells at 37 C with 220-rpm agitation for 1 h. 11. Harvest the cells by centrifugation with 16,000 3g for 1 min and resuspend them in 100 mL LB broth. Spread 100 mL cell suspension onto a LB agar plate containing 20 mg/mL kanamycin and incubate the plate at 37 C overnight (15-16 h) to obtain colonies. No colony should be recovered in the negative control (without digested mini-CRISPR).
Note: Overnight incubation in this protocol is approximately 15-16 h.
12. Perform colony PCR to verify the construction of pTargeting (Figure 2, Step 3). Colony PCR reaction using the platform plasmid pAY5211 as the template serves as the negative control. PCR reaction (a) and cycles (b) are set up as below: a) Colony PCR Reaction setup: b) Colony PCR cycle: 13. Run an 1% agarose gel to examine the PCR product. 14. Visualize bands with UV transillumination after the gel is stained by GelRed. PCR product of the expected pTargeting is 468 bp due to the replacement of 2-bp sequence flanked by KpnI and BamHI in pAY5211 by the 88-bp mini-CRISPR in pTargeting. PCR product of the negative control pAY5211 is 382 bp. 15. Inoculate a validated colony into 5 mL LB broth supplemented with 20 mg/mL kanamycin and propagate the plasmid by growing the inoculum at 37 C with 220-rpm agitation overnight. Extract pTargeting from the overnight culture using the QIAprepTM Spin Miniprep Kit (Qiagen, Germany).
Pause Point: Plasmids can be stored at -20 C for 3 months Note: This step is conducted to examine the self-targeting DNA interference activity of the native CRISPR-Cas system and to further validate the PAM sequence. This step only needs to be conducted once prior to the first genome-editing exploitation in the strain of interest.
Self-Targeting Activity Assay
pTargeting and the control plasmid pAY5211 are delivered into PA154197 cells, respectively, by electroporation following the steps from 24 to 31 (below). Since pTargeting contains a mini-CRISPR guide which targets a genomic locus in PA154197 and is expressed by a strong promoter Ptat, if the CRISPR-Cas system in PA154197 is active, the crRNA expressed from the mini-CRISPR will form a Cascade with the Cas complex and elicit site-specific DNA breakage in the genome, resulting in cell death. Hence, no colony should be recovered following pTargeting transformation relative to the high colony recovery of the pAY5211 transformation. As shown in Figure 5, expected colony recovery pattern was observed. The result showed that the native type I-F CRISPR-Cas system in PA154197 is active to interfere genomic DNA and confirmed the authenticity of the predicted PAM sequence. Moreover, the fact that no colony was recovered upon introduction of the pTargeting indicated that as in most bacteria, non-homologous end joining (NHEJ) does not occur in this strain. Once the self-targeting activity of the native CRISPR-Cas system is confirmed and the PAM sequence is validated, direct genome editing in this strain can be performed followingly (Steps 16 to 36).
Construction of the Editing Plasmid (pEditing)
Timing: 2-3 days 16. Amplify the donor DNA fragments using the genomic DNA of PA154197 as template and primer pairs (Donor-UF/UR and Donor-DF/DR) as indicated in Figure 2. Amplify and purify the donor DNA fragments following the procedures as described in Steps 1 and 2. Figure 2). Assembly reaction without addition of donor fragments serves as the negative control. Assembly reaction is set up as below: 21. Incubate reactions at 37 C for 30 min. 22. Transform the reaction mixture into E. coli DH5a component cells as described in Steps 8 to 11. Following overnight incubation at 37 C on LB agar plates containing 20 mg/mL kanamycin, perform colony PCR using the primer pairs pMS402-F/R to verify the construct (pEditing) as described from Step 12 to 15. No colony should be recovered in the negative control (without addition of donor fragments).
Note: Expected size of the PCR product should be 1,468 bp if the upstream and downstream donors are 500 bp, respectively. The negative control colony PCR reaction using pTargeting as template will yield a product size of 468 bp.
23. Sequence the plasmid (pEditing) using primer pairs (pMS402-F/R) to confirm the desired incorporation of mini-CRISPR and repair donor. An example of sequence information of the pEditing (pAY5235) to construct mexB-deletion was shown in Figure 4B.
CRITICAL: Concentration of pEditing should be >500 ng/mL. Lower concentration will lead to increased volume of plasmid for electroporation at Step 27, which reduces electroporation and editing efficiencies. 24. Overnight PA154197 cell culture is diluted 1:100 into 50 mL fresh LB broth and grow at 37 C with 220-rpm agitation for 4-5 h till OD 600 is 0.6-0.8.
Fast Preparation of Electro-component Cells and Electroporation of pEditing
CRITICAL: Harvest cells at OD 600 as 0.6-0.8 (mid-late log phase). Early log phase cells will result in low editing efficiency and stationary phase cells such as overnight cultured cells will increase the false positive transformants.
25. Cell culture is chilled on ice for at least 30 min. Cells are collected by centrifugation at 4 C with 5,000 3g for 15 min and washed three times with 25 mL cold Milli-Q H 2 O.
CRITICAL: Wash the cells gently and carefully to avoid the loss of cells during the process.
CRITICAL: Do not incubate over 90 min, as prolonged growth will lead to cell lysis presumably due to over-expression of the CRISPR RNA (crRNA) in PA154197, as only the transformants of pEditing, but not the control plasmid pAY5211, displayed this phenomenon ( Figure 6).
30. Harvest the cells by centrifugation and resuspend them in 100 mL LB broth. Spread the suspension onto LB agar plates containing 500 mg/mL kanamycin.
Note: Concentration of kanamycin for selection is dependent on the kanamycin susceptibility of the P. aeruginosa isolates subjected to editing. For example, 800 mg/mL is used for selection of another clinical isolate PA150567 and 500 mg/mL for an environmental isolate Ocean-100 (Kumagai et al., 2017).
31. Incubate the plates at 37 C for 24 h.
Inoculate the colonies recovered from
Step 31 in 100 mL LB broth supplemented with 100 mg/mL kanamycin in a 96-well white plate (SPL, Korea). 33. Incubate the 96-well white plate at 37 C with 220-rpm agitation for 3 h. 34. Measure the luminescence intensity using the Synergy HTX Plate Reader (Bio Tek, USA). 35. Select cell cultures with high luminescence value (>1000) for further colony PCR verification using primers located upstream and downstream of the donor regions (Veri-F/R, Figure 2).
Note: Luminescence reading of cultures containing pEditing is usually more than 1,000 after incubation, while the false positive colonies have undetectable luminescence intensity. For colony PCR verification, include a negative control reaction using the genomic DNA of PA154197 as template.
36. Verify the desired genome editing by DNA sequencing of the PCR product.
OPEN ACCESS
Curing of pEditing for the Next Round of Editing Note: In some cases, multiple (2 to 3) rounds of curing are required for complete loss of the plasmid.
EXPECTED OUTCOMES
Representative plates resulting from the electroporation of pAY5211 (control plasmid), pTargeting (negative control), or pEditing are shown in Figure 5A. Compared with the transformation of pTargeting which resulted in no colony recovery due to self-targeting, provision of a repair donor in the pEditing resumed transformants recovery, suggesting the occurrence of DNA repair at the self-targeted site through homologous recombination (HR) and consequently the desired genome editing. An example of PCR verification of the desired gene deletion is shown in Figure 5B. The fact that the PCR products of these colonies displayed either the size of the WT or the desired full-length gene deletion suggested that microhomology-mediated end joining (MMEJ) did not occur or was not frequent.
LIMITATIONS
This native type I-F CRISPR-Cas-mediated genome-editing protocol is limited to P. aeruginosa isolates that express active type I-F CRISPR-Cas system. Currently, strains that do not contain such a native CRISPR-Cas system or those containing a degenerated CRISPR-Cas system are not editable by this method.
Similar to the limitations encountered in other CRISPR-based genetic applications, constructing gene insertion and single nucleotide substitution is dependent on the presence of a PAM sequence within a strict range of the desired editing site. For gene insertion, presence of a PAM sequence Figure 6. Prolonged Growth during Recovery of PA154197 Cells Electroporated with pEditing Resulted in Undesirable Cell Lysis Suspension of cells electroporated with the control plasmid pAY5211 or pEditing following recovery for 120 min. Cell lysis and subsequent precipitation (red arrow) was observed in the cell suspension electroporated with pEditing but not with the control plasmid pAY5211.
within the 32-bp upstream of the desired insertion site is required. For single nucleotide substitution, presence of a PAM sequence at the desired editing site or within 2-bp upstream of the site is required. The editing efficiency is strictly dependent on the distance between the PAM and the desired editing site in these applications (Figure 7).
Potential Solution
If the online alignment using CRISPRTarget fails to predict PAM sequences, an alternative experimental PAM screening strategy, PAM depletion assay, can be considered. PAM depletion assay employs a plasmid library of all possible 3-6-nt PAM sequences to screen for the effective PAM. Detailed description for PAM depletion assay was reported by Walker et al. (Walker et al., 2020).
Problem
Native type I-F CRISPR-Cas system is inactivated by anti-CRISPR elements in the strains of interest.
Potential Solution
To exploit the native CRISPR-Cas-mediated genome editing in these strains, one potential solution is to delete the anti-CRISPR element first using other genetic approaches. Alternatively, overexpression of the common anti-CRISPR repressor Aca can be considered to inhibit the expression of anti-CRISPR (Stanley et al., 2019). Success of single nucleotide substitution (N to A) was examined in the PAM and PAM proximal region in a selected region in mexB. Editing plasmids containing a repair donor (U and D) for site specific point mutation were introduced into PA154197. For each of these mutagenesis, five randomly selected transformants with high luminescence intensity were subjected to colony PCR and DNA sequencing using primers Seq-F/Seq-R. Sequencing results demonstrated that mutations only in the PAM and PAM proximal 2 nucleotides can be efficiently achieved.
Non-effective targeting
Potential Solution 1. For deleting a gene which contains multiple available targets (PAM-Protospacer) to select, try an alternative target and assemble a corresponding new mini-CRISPR to achieve targeting. Alternatively, we recommend designing a mini-CRISPR cassette simultaneously incorporating 2-3 spacers (e.g. Repeat+Spacer 1 + Repeat + Spacer 2 + Repeat) to avoid potentially non-effective targeting by one spacer. 2. For other editing which requires the presence of a PAM sequence within a strict range of the desired editing site, we recommend a two-step In-Del method as described in our Cell Reports Resource article .
Problem
Low editing efficiency to delete genes with large sizes (i.e. >3 kb)
Potential Solution
Increase the donor size can help to increase the editing efficiency.
Low yield of electroporation
Potential Solution pAY5211 contains an origin of transfer (oriT). Try to use conjugation to deliver the editing plasmid into the isolate of interest. Detailed procedures for conjugation were reported by Withers et al. (Withers et al., 2014).
Problem
Genome editing in P. aeruginosa isolates with other types of CRISPR-Cas systems or in other prokaryotic/eukaryotic cells lacking a native CRISPR-Cas system.
Potential Solution
For genome editing based on the native type I-F CRISPR-Cas system in other species, consider using species-compatible plasmid and promoters to express the mini-CRISPR. For species containing native CRISPR-Cas system belonging to other subtypes, the similar workflow described here is still applicable except that plasmid vector, PAM sequence, repeat sequence implemented should be compatible with the corresponding CRISPR-Cas subtypes and the host strains. For example, type I-B and I-E systems have been harnessed for genome editing in Clostridium and Lactobacillus, respectively, by applying plasmids pCParray-delcpaAIR and pTRK1183 and PAM sequences of 5 0 -AATTG-3 0 and 5 0 -AAA-3 0 , respectively (Pyne et al., 2016;Hidalgo-Cantabrana et al., 2019).
DECLARATION OF INTERESTS
The authors declare no competing interests. | 2020-06-04T09:06:23.773Z | 2020-06-01T00:00:00.000 | {
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155001338 | pes2o/s2orc | v3-fos-license | Job satisfaction and turnover under the effect of person-organization fit in Albanian public organizations*
This paper presents the results of research conducted on public organizations employees for testing a model that relates person-organization fit, job satisfaction and turnover. Using a sample of 100 employees from 17 Albanian public organizations, this study brings out that there is a significant relationship between person-organization fit and job satisfaction in the public organizations in Albania. The implications of the study are discussed. Dieser Artikel stellt die Resultate einer Untersuchung dar, deren Gegenstand war, an Angestellte von öffentlichen Organisationen ein Modell zu erproben, welches das Harmonieren der Person mit der Organisation mit der Arbeitszufriedenheit und der Fluktuation verknüpft. Durch die Benutzung einer Stichprobe von 100 Angestellten von 17 albanischen öffentlichen Organisationen, kommt diese Studie zu dem Ergebnis, dass es eine signifikante Verbindung zwischen dem Harmonieren einer Person mit der Organisation und der Arbeitszufriedenheit in öffentlichen Organisationen in Albanien gibt. Die Konsequenzen der Studie werden diskutiert.
Rationale
In the book Democracy and the Public Service, Mosher (1982) explains that Government decisions and behaviour are tremendously influential in our society; these decisions and behaviour are heavily influenced by non-elected administrative officials; the kinds of decisions and behaviours taken depend upon the officials' capabilities, orientations and values; these attributes depend upon their background, training, education etc.
After the communist period, public service was one of the things that had to be totally "invented" in Albania.Differently from other countries with a tradition of public service, that actually are facing certain heterogeneity and suffering a certain loosening of the ties with their traditions (Perry 2007), Albania is creating a public service from scratch.Coming from the experience of a partystate country, with an original way of understanding and practising, the terms public administration, public servant, civil servant, did not exist at all in the vocabulary of the Albanian language up to the beginning of the 1990's.In these last 20 years, a full set of adjustments has been necessary to build and bring together democracy with the public service, going from legislation to practical rules, form structures to mentality.The first Civil Service law in Albania was adopted in 1996, but it had a limited implementation due to the lack of institutions and detailed procedures.Although there has been a continuous advancement in the reform of public administration, the level of its achievements during the last period of 6-7 years is yet far off pre-established objectives (Shapo et al. 2008).
Public servant and civil servant are two terms that are sometimes used interchangeably.In the Western Balkans region, the term "public servant" has often a wider meaning than the term "civil servant".It is frequently used to denominate employees in all public services, including such specific specialised sectors like education, health protection, and the like.Two basic criteria are used to define civil servant (Respa 2009): Where does the public servant work?
Or at which level of government is the public administration body positioned; the group of 4000 employees are centrally managed by the Department of Public Administration, established in 1999 (Shapo et al. 2008).
Civil servants are employed permanently (for life) and termination of employment can occur only in situations which are strictly specified by law.This service is considered to be the safest employer.As a rule, job security is significantly higher than in the private sector.This relative advantage offered by the legislation is assumed to constitute one of the main attractiveness factors of public administration jobs (Respa 2009).Other elements like: paid leave, stable working hours, strict observance of national holidays which are non-working days, equal treatment for women with regard to men, intergrated in the civil service legislation provide incentives to attract talent in the public administration in Albania.
Constructive efforts in the public sector are, at the same time, challenged by the development of the private market that is as young as the public service (there was no private sector at all before 1990 in Albania).Being without a tradition, and facing the competition with the private, the public service in Albania is challenged by a set of forces that are comparable with that of other countries, but differently from the others, the lack of the tradition makes this more challenging and with more opportunities at the same time.
The Albanian civil service is organised to be a mixed system, based mainly on the job/position, but including elements of career systems.The dominance of the position system can be observed in the recruitment criteria, the rules governing appraisal, transfer, etc.The actual law on civil service establishes an open system, allowing at the same time mobility and career development inside the system.But when it comes to career development instruments like lateral transfer and promotion, preference is rather given to attracting personnel from the private sector, who might in the current situation, presumably improve the quality of the civil service.A lateral transfer is given priority to fill in vacancies.But when the number of internal candidates is insufficient (at least four in-house candidates), the competition is opened to external candidates.
The uncertainty about the interpretation of the relevant provisions is another reason for the partially not existing application of the rules of career development (Shapo et al. 2008).The system does not allow promotion of two or three steps in the career scale (no fast track).
At the 2008 Regional Workshop on the attractiveness of Civil Service, it was briefly summarised in the Albanian presentation: "Seniority is more important than merit, and promotions are not clearly linked to performance.Career paths can be unclear and little emphasis is placed on staff development".
Besides the promotion to a higher rank/position, the international practice also aknowledges the possibility of promotion through the salary grid, linked to performance and appraisal results, combined with seniority.In the Albanian system, a civil servant cannot advance without obtaining a higher rank/position, which requires competition.This means that a civil servant cannot obtain a permanent increase of salary based on performance.Only annual (one-off) bonuses are awarded to the civil servants with the best marks in the annual appraisal, depending on budget.On the other side, performance appraisal is not yet playing the designated role as a human resources management instrument.Evaluators are still lacking the necessary skills and experience with regard to this task (Shapo et al. 2008).
Given the budgetary constraints and the expanded public services expected, the state faces a complex challenge of managing an effective specialised workforce.Government has created a special job classification and compensation system in an effort to attract qualified specialists, but as the system of position stimulates a higher inflow of professional candidates from outside the public administration, the urge to excel and seek professional advancement is not very high in those already in the system.We can say that "the links between performance and promotion are weak in Albania" (RESPA 2008).
Although the state has made important changes in job classification and compensation, average private sector salaries are still higher than the salaries that the government can offer.One young graduate may begin the first employment at the lowest possible level of payment no higher than about 30 thousand leke per month in the public service, while the basic salary in the private sector can be as much as 60 thousand leke per month.
One can arrive at the conclusion that although the public service is by law a safe place of employment, with some valuable incentives, being employed there is also a big challenge.It is not very "safe" in career possibilities, it does not remunerate in terms of effort required by the employee (everything at the end is a matter of budget availabilities), it is subject to subjectivity in performance evaluation and it is subject to change following political elections every four or eight years.Under the circumstances of high competition from the private sector in terms of high payment and fast career possibilities, employment by the public administration seems to be not a very lucky choice for young educated aspirants.Although in the public service they have a great chance of learning the 'ropes' of the profession, getting trained without payment (in Albania training of the public employmees is considered as a right), after a while, they find themselves with experience, but no real advancement opportunities, with a payment that is always constrained within rigid limits.
But still, the number of job applications is high compared to the job vacancy, and the turnover is low.The data shows that the average number of applicant per position is 5 applicants, where as for the positions at the level of Director the average is at around 4.5 (Shapo et al. 2008).According to an interview conducted by the author with the Head of the Department of Public Administration of Albania (DoPA/DAP in Albanian) for the purpose of this paper, the last four years data show a voluntary turnover not higher than 3-4%.Probably the individual finds in public institution values that can not be found in private institutions, and probably the match of individual values with those of the institution makes the employment a satisfying experience that affects his decisions of employment and turnover.Probably the knowledge that he is working for something "big" and important gives individual reasons to be satisfied with being employed by the public administration, or the similarity of certain values and standards in perceiving the reality gives him the feeling of fitting in.So, what can be some non economic factors that link the individual with public organization and make him satisfied?Is this satisfaction that bonds individual with the public institution?Or is it the fit with the institution that makes the individual more satisfied and reluctant to leave ?Is the fit a question of age, or/and education, or/and tenure, or/and gender?Do these variables moderate the presumed effect of fit over satisfaction?Is turnover in public administration in Albania a matter of age, or education, or gender?Some of these questions were in the base of this inquiry and what was discovered, is broadly explained in the following parts of this paper.
Literature review
For the purpose of this study, a literature review was considered, with the main concern on: understanding what may be some reasons that may satisfy an educated employee beyond monetary incentives; understanding if there are reasons to believe that these satisfactory factors can be enough to keep someone employed for long; understanding what can be some ways/factors that make an individual feel like he belongs in an organization and doesn't want to leave as he is getting what is expecting (excluding here the monetary incentives).So the concepts of job satisfaction and person-organization fit were explored and some reasoning based on these concepts understanding was used to lay down the study hypotheses.
Person-Organization Fit
One of the reasons one likes to work for an organization is finding it a satisfying experience, among others, probably because the individual feels he fits within the organization.Person-organization fit is one of the most popular areas of research in the field of organizational behaviour and management.This domain of research concentrates on conceptualizing the levels of possible fit, the elements included in the fit, the ways of assessing the congruence, the effects it has on a number of variables within the organization, etc. Literature review reveals different levels of possible fit studied.Among them we can distinguish: a) Congruence between the individual and organizational values.This value congruence reflects the similarity between an individual's personal values and the cultural value system of an organization.When individual's personal values are similar or aligned with organizational values, positive outcomes such as satisfaction, commitment, performance, career success, reduced stress, and lower turnover intentions are realized (e.g. C. Ostroff, et al. 2004).As values are components of organizational culture that guide an employee's behaviour (Schein 1992), and they are "fundamental and relatively enduring" (Chatman 1991), this individual-culture fit can be considered as an individual-organization fit, too.b) Congruence between individual's goals and organization's goals (B. Schneider 1987).c) Congruence between the individual preferences and needs and organizational systems and structures (Bretz et al. 1989;Cable/Judge 1994).In most of cases this conceptualization is used to explain person-vocation fit, but it is also used as an explanation for person-organization fit (Bretz/Judge 1994). d) Congruence between the characteristics of individual personality and the organizational climate-labelled even as organizational personality (Burke/Deszca 1982).
It is not easy to differentiate among these different fit perspectives.Kristof (1996) defines person-organization fit as the compatibility between people and organizations that occurs when: (a) at least one entity provides what the other needs, or (b) they share similar fundamental characteristics, or (c) both.As such, it can be spoken about supplementary and/or complementary congruence.Supplementary congruence is achieved when the fundamental characteristics of an individual and those of the organization are similar to each other, i.e. when personality, values, goals, and attitudes of the individual are related with the culture, climate, values, goals and norms of the organization.Complementary congruence is achieved when the characteristics of individuals and organizations add something that is missing to make each other whole (Kristof 1996;Muchinsky/Monahan 1987).For example, from a supplementary standpoint, congruence is achieved when organizations attract individuals who have similar goals and values, whereas from a complementary standpoint, congruence is achieved when the unmet needs of individuals are satisfied by the resources and tasks that are provided within organization.In either case, there is strong evidence that person-organization fit has a positive benefit on a range of employee attitudes and behaviors, particularly job satisfaction and turnover intentions (Bretz/Judge 1994;Kristof 1996, Kristof-Brown et al. 2005;Vancouver/Schmitt 1991).
Based on some facts presented above, there is some reason to believe that probably the low turnover in Albanian Public Administration is a result of supplementary or complementary congruence of the individual employed with this organization.
Job satisfaction
Job satisfaction essentially reflects the extent to which an individual likes his job.Formally defined, job satisfaction is an affective or emotional response toward various facets of one's job.Herzberg found that people are motivated when their needs for achievement, recognition, stimulating work, and advancement are satisfied.When an employee has the opportunity to experience achievement, recognition, stimulating work, responsibility and advancement, he feels an intrinsic motivation (Hackman/Oldham 1976).Research overwhelmingly demonstrates a moderately strong relationship between these job characteristics and satisfaction (Ambrose/Kulik 1999).
One early debate in the public administration literature centred on whether public employees were satisfied with the characteristics of public organizations (DeSantis/Durst 1996; Steel/Warner 1990).Some believed the bureaucratic nature of public organizations coupled with low salary levels inhibited high levels of job satisfaction among public employees.Contrary to these expectations, many researchers found job satisfaction to be high among public employees at all levels of government, whereas other studies reached an opposite conclusion (Bright 2008).The work conditions found to be the most influential on the job satisfaction and turnover intentions of public employees were the intrinsic non-monetary characteristics of their work, such as good social relationships with co-workers and supervisors, promotion opportunities, professional development opportunities, and participatory management strategies (Borzaga/Tortia 2006;DeLeon/Taher 1996;Ellickson 2002;Emmert/Taher 1992;Kim 2002Kim , 2004;;Wright/Davis 2003).
Person-organization fit and job satisfaction
Causes of job satisfaction are broadly explained by five predominant models (Brief).They are need fulfilment, discrepancies, value attainment, equity, and dispositional/genetic components.In brief, we can say the need fulfilment model proposes that satisfaction is determined by the extent to which characteristics of a job allow an individual to fulfil his needs.The unmet needs can affect satisfaction and turnover (Karr 1999).Although need fulfilment models generated controversy, it is generally accepted that need fulfilment is correlated with job satisfaction (Stone 1992).The discrepancies models propose that satisfaction is the result of difference between what an individual expects to receive from a job, such as good pay, promotional opportunities, and what he actually receives.Studies have revealed that met expectations are significantly related to job satisfaction (Wanous, et al. 1992).The value attainment model presents the idea that satisfaction results from the perception that a job allows for fulfilment of an individual's important work values (Locke 1984).In general, research consistently supports the prediction that value fulfilment is positively related to job satisfaction (Chatzky 2005).The equity model considers satisfaction as a function of how fairly an individual is treated at work.Employee's perceptions of being treated fairly at work are related to overall job satisfaction (Cohen-Charash/Spector 2001).The genetic model implies that stable individual differences are as important in explaining job satisfaction as are characteristics of the work environment.
Considering all the models of job satisfaction, and the different levels of individual -organization congruence commented above, we can suppose a strong relation among these two variables.The relation is materialized either in supplementary, or complementary, or both ways.
First study hypothesis: Person-organization fit appears to be a very important cause of the job satisfaction As the Person-organization fit literature strongly suggests, individuals who are compatible with the characteristics of their organizations will have higher job satisfaction when compared with individuals who are less compatible.There are many kinds of public organizations with different missions, goals, cultures, resources, and job tasks.Similarly, public employees have different characteristics from each other.
Hypothesis 1: Person-organization fit is significantly related to job satisfaction.As the compatibility between employees and organizations increase, their job satisfaction will increase.
Job satisfaction, person-organization fit and turnover intentions
Job satisfaction and turnover intentions are reflections of the outlook that employees have about their employment.This outlook is influenced by the degree to which employees' salient needs are satisfied by their work (Bright 2008).Employees display higher levels of job satisfaction, and subsequently lower turnover intentions, when the characteristics of their working environment satisfy their needs (Bright 2008).Schneider (1987) includes turnover as a long term outcome attributed to P-O fit.Individuals with lower levels of value congruence with their organizations are more likely to leave their organizations than those of higher congruence levels (Chatman 1991;O'Reilly et al. 1991).O'Reilly's analysis (1991) indicated that value congruence was a significant determinant of actual employee turnover within two years of initial assessment of fit.Chatman (1991) reported that levels of value congruence measured both at entry and after one year of employment and socialization significantly predicted turnover.Utilizing multiple conceptualizations of person-organization fit, Bretz and Judge (1994) found that person-organization fit had a strong direct effect on organizational tenure.
Although scholars found public employees to have acceptable levels of job satisfaction, burnout was found to be a major threat in public organizations (Bright 2008).There is evidence that tenure is negatively related to the job satisfaction of public employees (Kamdron 2005;Naff/Crum 1999).In other words, the longer employees worked in public organizations, their job satisfaction decreased.Similarly, other studies have found burnout and exhaustion to be two of the most cited reasons individuals left public sector jobs (Kim 2004;Samantrai 1992).
Second study hypothesis: Person-organization fit appears to be a very important cause of the turnover intentions.As the person-organization fit literature strongly suggests, individuals who are compatible with the characteristics of their organizations will have higher job satisfaction and lower turnover intentions when compared with individuals who are less compatible.
Hypothesis 2: Person-organization fit is significantly related to turnover intentions.As the compatibility between employees and organizations increase, their turnover intentions will decrease.
Deducing from what has been said so far, this was an effort to understand the relation among person-organization fit, job satisfaction and turnover intentions for the public servants in Albania.This relation was supposed to be moderated by some biographical characteristics like: age, tenure, level of education, and gender.The model used, is as in the figure 1.
Methodology
Study settings 100 public employees took part in this study.The participants were randomly selected from the Ministry of Interior, the Ministry of Finance, the Ministry of Labour and Social Affairs, the Ministry of Education and Sciences, the Regional Education Directorate of Tirana, Durres and Shkoder, the University of Shkoder, the University of Durres, the Financial supervision authority, the Bank of Albania, the University Hospital Centre, the Central Elections Commission, the Albanian Electricity Power Corporation, the Parliament of Albania, the Tirana Library and the Albanian Army.These organizations were chosen to create a diverse sample of participants who represented a broad range of governmental occupations.As expected, the respondents represented a diverse mix of public sector occupations, some of which were: finance and budgeting specialists, specialist lawyers, HRM specialists, R&D specialists, academic staff, medical doctors, maintenance staff and security guards.The respondents were also demographically diverse by their age, gender, and education.Table 1 makes a description of the control variables in the sample.
There was no direct contact between the author and the respondents.As the research was not commissioned by the institutions observed, no identifying information was collected.As such, it was not possible to generate an accurate response rate for each participating organization.However, after reviewing the respondents' job titles, the author was confident that each participating organization was evenly represented.
Education level
High school diploma
Person-organization fit
There are different strategies that can be used to assess the fit, based on perceived fit, or on the assessed characteristics of the organization and the individual.Direct strategies assess such a fit by asking the respondents for their perceptions of their fit in their organizations.Indirect strategies, in contrast, assess fit by comparing separate assessments of the respondents' characteristics and the characteristics of their organizations (Kristof-Brown et al. 2005).
Empirical research has shown that direct measures are stronger and better predictors of employee outcomes than indirect measures (Kristof-Brown et al. 2005;Verquer et al. 2003).
This study took a supplementary and direct approach to measuring the congruence between the respondents and their organizations.The participants were asked to indicate their agreement with the following four statements from 1 (strongly disagree) to 7 (strongly agree): My values and goals are very similar to the values and goals of my organization; I am not very comfortable within the culture of my organization (reverse scored); I feel a strong sense of belonging to my organization; and what this organization stands for is very important to me.These statements were developed from a review of existing research (Kristof 1996; O'Reilly/ Chatman 1986).
Job satisfaction
This study used a multi-item scale to measure the job satisfaction of the respondents.This scale was developed from a review of the public administration job satisfaction literature.As discussed, existing research shows that the job satisfaction of public employees is mainly influenced by the intrinsic non-monetary characteristics of their work, such as advancement opportunities, professional development, and meaningful work (Borzaga/Tortia 2006;Deleon/Taher 1996;Ellickson 2002;Emmert/Taher 1992;Kim 2002;Wright/Davis 2003).The participants of this study were asked five questions about their satisfaction with opportunities for achievement, recognition, responsibility, meaningfulness, and advancement in their jobs (one for each).A seven scale measure was used for the answers, from 1(very dissatisfied) to 7 (very satisfied).The questions about Job satisfaction are as in the table 2.
The responses to these questions were used as five observed indicators of job satisfaction.
Turnover intentions
One question was used to measure turnover intentions.The respondents were asked to answer the following question using a scale from 1 (very unlikely) to 6 (very likely): Within the next 2 years, how likely are you to leave your current organization for a job in another organization?
Control variables
Based on the supposition that job satisfaction and turnover intentions are somehow related with biographical characteristics of the individual, some control variables were used.These were: age, gender, education level, and years of public sector experience.The age of the respondents was measured using the open-ended question: When were you born?The current age was then calculated by subtracting the year of birth from the year 2009 which is the year of the study.The gender of the participants was collected with the direct multiplechoice question: What is your gender?The 0/1 coding was used for that.The level of education of the participants was collected from the open question: What is the highest level of your education?These responses were coded from 1 (high school) to 4 (PhD).The tenure in public sector was collected from the open question: For how many years have you been working in the public sector?
Analysis process
After the data collection process, the data were analyzed in two stages.First, the data were examined to ensure the normality.The data were found to be inacceptable.Only one questionnaire had no data on control variables.According to Curran, West, and Finch (1996), skewness ranges should be fewer than 2 and kurtosis ranges fewer than 7.As shown in Table 3, almost of the variables used in this study are between these suggested ranges.Second, the data were analyzed using SPSS program.The structuring equation modelling should have been more appropriate, but the author lacked the ability to deepen analysis in this direction.Figure 1 displays the conceptual model that was tested in this study.This model tested the relationships among personorganization fit, job satisfaction, and the turnover intentions of the respondents.
The results of this study are displayed in the following Tables and will be discussed later.Statistical significance was set at .001, two-tailed.Total 100 Count and % values are equivalent, as N=100.
Description of the Survey Responses
This study collected the respondents' level of person-organization fit, job satisfaction, and turnover intentions.It intended to understand the supposed relation among the Person-Organization fit and the satisfaction; as well as the relation among the person-organization fit and turnover in the public organization employees.Four control variables were used for their supposed modification effect.The results are summarized in Tables 5 through 11, and these results will be the focus of the following discussion.
The majority of the respondents suggested that they were congruent with their respective organizations.Each of the components of congruence considered, were significantly connected with the concept of fit.Values congruence (.816), the culture (.665), belongingness (.821) and liking the contribution of the organization in the general welfare of the country (.770) all came out to very important components of the individual-organization fit.In total, these components explain 51% of the variance of this variable.The vast majority of the respondents (from 39% to 65%) appeared to have high levels of job satisfaction.The meaningfulness is the most appreciated element of job satisfaction.65% of respondents were satisfied or highly satisfied by this element.Even the possibility of perceiving responsibility doing the job makes 60% of the respondents satisfied or highly satisfied.Following on from that, comes the recognition (52%) and achievement (41%).Only the perceived promotion possibility has a lower level of respondents satisfied or very satisfied (39%).This probably is correlated with the open career systems applied in the Albanian Public Administration.The Job satisfaction concept is 57% explained by the intrinsic non-monetary characteristics of the respondents work, such as achievement, recognition, responsibility, meaningfulness and advancement.Finally, 64% of the respondents suggested that they had few intentions of leaving their organizations.The author believes the results over turnover may have a more complex interpretation under the present supply-demand conditions of the labour market.
It is important to note that none of the control variables was significantly related to the study variables.The first hypothesis focussed on the relationship between person-organization fit and the job satisfaction of the respondents.It was hypothesized that personorganization fit would be significantly related to the job satisfaction of public employees.This hypothesis was strongly supported by the findings of this study.Person-organization fit was found to be significantly and positively related to the job satisfaction of the respondents.The respondents that reported being highly congruent with their organizations also reported being significantly more satisfied with their jobs when compared with their counterparts.It is also important to highlight the magnitude of the relationship between personorganization fit and job satisfaction.That is, person-organization fit accounted for most of the variance in job satisfaction (0.7).This finding suggests that person-organization fit was the most important predictor of job satisfaction in this study.The second hypothesis focussed on the relationship between person-organization fit and the turnover intentions of the respondents.It was hypothesized that person-organization fit would be significantly related to the turnover intentions of the respondents.The findings of this study do not support this hypothesis in a significant level (-0.3).
Discussion of empirical findings
The purpose of this study was to explore the extent to which person-organization fit is related with job satisfaction and the turnover intentions of public employees in Albanian environment.A possible correlation was assumed, with person-organization fit as a possible cause of job satisfaction and turnover intentions.Person-organization fit was evaluated by the self reporting of the perceptions held by public employees.It came out that only 51% of the "fit" was explained by the questions included.
As was stated above, a supplementary congruence was assumed.What about a possible complementary congruence?Although public administration is now performing in the environment of the Albanian market economy, it follows the well known way of a bureaucracy, in the classical meaning of the word.
Excluding the political factor that interferes with employment once in four or eight years following the political changes in Government, the public administration is a more stable place to work.One of the values one may seek is that of continuity.But no item addressed this in the questionnaire (Part of person-organization fit).Also some important facets of one's job within public organization institutions like achievement, recognition, responsibility, etc were not addressed.The sense of responsibility was evaluated in the out-of-theinstitution way of thinking (It is very important for me what this organization makes for my country) but not evaluated the feeling of being responsible within the institution, with colleagues and within organizational structures.These facets of one's job were only considered as factors of job satisfaction (Achievement, Recognition, Responsibility, Meaning, and Advancement).Under this construction, the correlation person-organization and job satisfaction, is considerable (0.7), but probably the cause-effect direction is not as clear as it was thought in the beginning.
Job satisfaction on the other side was considered as a construct of perceptions about some non-monetary benefits one may have from the job.The vast majority of the respondents (from 39% to 65%) appeared to have high levels of job satisfaction.The meaningfulness is the most appreciated element of job satisfaction.65% of respondents were satisfied or highly satisfied by this element.Even the possibility of perceiving responsibility doing the job makes 60% of the respondents satisfied or highly satisfied.Then there follows recognition with 52% and achievement with 41%.Only the perceived promotion possibilities have a lower level of respondents satisfied or very satisfied (39%).This probably is correlated with the mixed career systems applied in the Albanian Public Administration (Kasimati/Mitllari 2009), as well as non quantified effect of political interference in decision making.In year 2008, there were 307 individuals employed; only two of them were from inside the institution (DAP/DoPA, 2009).
These five benefits / components of job satisfaction explain 57% of the variance of this variable.There are of course other non-monetary and monetary aspects of one's job that make it satisfactory, especially in the actual situation of the labour market in Albania.Although not published, informality is calculated to cover up to 20% of the country's GDP.Although not included in this data, this informality is certainly to be found in the labour market as well.Individuals employed in the private market suffer this informality in many ways: social insurance payment under the legal provision level; the individual is forced to fill the labour week with many small works out of the field of specialization, thanks to the small company that can't fully use a specialist, but has to keep in the payroll; the employment is never secure, as the culture within the companies, for many reasons, is not consolidated yet, but mostly because of the mentality of the private Albanian companies who have not yet understood the importance of regulations within organization.
In public administration, payment may be lower, but there are not so many other worries.Institutional performance is regulated by law.Public organizations are much more matured than most of the Albanian small and medium private companies, and things are more settled.In these organizations, the individual is granted any necessary time to learn, and a lot of effort and money are spent every year by the Government for training, and improvement in procedures.
Only in 2009 there were spent about 100 thousand EUR on training (ITAP/TIPA 2010).
These circumstances might account for the 43% of the unexplained variance of job satisfaction.Not only this, but some of these factors may be reasons for a perceived better person-organization fit, probably in the complementary side of the congruence.
There was assumed a possible cause-effect relation between person-organization fit and turnover intention.Although 64% of the respondents suggested that they had few intentions of leaving their organizations, the relation is not significant (-0.3), i.e. they may have no intentions to move on, but this doesn't come about because they fit well within the public administration, but for other reasons which were not included in the study.The author believes the results in turnover may have a more complex interpretation under the present supply-demand conditions of the labour market in Albania.
In general, we may say based on the results of this study, that there is a considerable relationship between person-organization fit of the public employee and his/her job satisfaction, in terms specified here.There is no significant relation of person-organization fit of the public employee and his/her intention to leave the public administration, in terms specified here.For this sample, this is true independently from age, education, tenure, or gender.
It appears not normal and is not based theoretically the non relation of turnover intention and job satisfaction with the tenure in public administration for this sample.
Conclusion
Albania is a country committed to the objective of becoming a candidate country for membership of the European Union.In pursuit of this objective, one of the medium-term priorities is the reform of the administrative system towards standards that ensure an effective support for the adoption and for the implementation of the acquis communitare.The reinforcement of administrative capacities, as identified in the document of European Partnership with Albania (2007), is a key priority for the implementation of SAA.
As a matter of fact, in last ten years, Public Administration in Albania is following a way of reforms that intends to increase the efficiency of services.
All the human resources are managed in the function of achieving the highest quality possible.The rules and regulations of recruitment and promotion are adjusted aiming at the inclusion of the most motivated individuals and development of organizational commitment (Shundi, 2002).The employee in this administration is promoted according to personal and group achievement, as well as according to demonstrated personal characteristics (Albanian Law of Civil Service 1999, and changes).The selection process intends the best fit of the individual with the public institution, for the benefit of both.
In this research, the fit of the individual (as an employee) with the public institution was assumed to be the influencer of his job satisfaction and turnover intentions.However, as it comes out from the results of this research, it is very possible that the job satisfaction and low turnover intentions of the public employees influenced their perceptions of fit.As a result, the findings of this study only suggest that a causal relationship exists among the study variables, while recognizing the possibility of two-direction relationships.
Based on this experience, and upon the knowledge that monetary incentives are only part of one individual's satisfaction at work, more effort has to be dedicated on the identification of job-related variables that affect retention of qualified individuals in the public administration.That was the primary goal of this study, but as the sample was not very high, extensive research is needed.It is also important to broaden the understanding of the public managers on the variety of subordinates' job expectations.Certain aspects of the concept of job satisfaction may be included in the public managers' soft skills training.
Figure 1 .
Figure 1.The conceptual model tested in this study
Table 2 :
Description of questionnaire questions
Table 3 .
Study variables
Table 9 :
Correlation coefficients of control variables
Table 10 :
Correlation between person-organization fit and job satisfaction.
Table 11 :
Correlation between person-organization fit and turnover intentions | 2018-12-18T09:06:03.381Z | 2011-10-01T00:00:00.000 | {
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15368517 | pes2o/s2orc | v3-fos-license | TRAIL: Topology Authentication in RPL
The IPv6 Routing Protocol for Low-Power and Lossy Networks was recently introduced as the new routing standard for the Internet of Things. Although RPL defines basic security modes, it remains vulnerable to topological attacks which facilitate blackholing, interception, and resource exhaustion. We are concerned with analyzing the corresponding threats and protecting future RPL deployments from such attacks. Our contributions are twofold. First, we analyze the state of the art, in particular the protective scheme VeRA and present two new rank order attacks as well as extensions to mitigate them. Second, we derive and evaluate TRAIL, a generic scheme for topology authentication in RPL. TRAIL solely relies on the basic assumptions of RPL that (1) the root node serves as a trust anchor and (2) each node interconnects to the root in a straight hierarchy. Using proper reachability tests, TRAIL scalably and reliably identifies any topological attacker without strong cryptographic efforts.
I. INTRODUCTION RPL [1] has been designed as an efficient and scalable routing protocol for low-power and lossy networks (LLN). It promises to reduce the overall power consumption by minimizing the control traffic, which is a major requirement for the energy constrained devices envisioned in the future Internet of Things (IoT). Such tiny intercommunicating devices like sensor nodes used in (home) automation, smart grids or surveillance systems are expected to massively populate our environment soon.
RPL constructs one or several tree topologies oriented towards a single root node. Each node in the RPL routing graph has a rank derived from its parent relationship that describes the topological distance to the root. Every node joining the topology calculates a higher rank than its parent, lower ranks are used for default upstream. This proactive organization leads to a Destination Oriented Directed Acyclic Graph (DODAG) topology, from which RPL is able to detect and remove inconsistencies reactively.
Control traffic in this topology consists of DODAG Information Objects (DIOs). A DIO advertises parameters and constraints for a specific DODAG that is uniquely identified by a version number. A node uses the information obtained from a DIO to select a parent node, compute its rank and join the DODAG, from which it inherits an upward route towards the root node. An optional upwards advertisement of Destination Advertisement Objects (DAO) generates downward-oriented routes to children of a subtree. Depending on the mode of operation, these routes are either maintained and stored at each node (storing mode), or forwarded to the root node and collected there (non-storing mode). The integrity of distribution trees is essential for RPL, as an inconsistent hierarchy will lead to traffic redirections and a loss of routes to the root. In addition, RPL will attempt to cure tree deficiencies by reorganization, and a node that will hold up failures of the routing hierarchy may trigger repeated reconfigurations that drain resources of the network.
RPL offers basic protection against external attackers breaking into the topology [1]. However, as nodes may be captured and security keys can be extracted from them, the RPL topology is threatened by various attacks from inside the network. The rank of a node and the DODAG version number are focal attributes in the topology. Known attacks are foremost based on them. A false rank of a node forges the relative topological distance to the root and thus disarranges the hierarchy. An inconsistent version breaks the reference to the topological graph and causes the network to rebuild its routing graph. Corresponding protections are not part of the current RPL specifications.
Recently, VeRA [2] has been proposed to fix these two classes of vulnerabilities by adding reverse hash chaining to DIO messages. Receivers shall be enabled to verify the advertised hierarchy. However, in the following we can show that VeRA remains vulnerable to rank attacks by forgery and replay. We analyze its incompleteness of message-rankauthentication, and present enhancements to VeRA for repair. Leaving aside the complexity of VeRA, our remaining work concentrates on a generic scheme for verifying RPL topologies. We design and evaluate TRAIL (Trust Anchor Interconnection Loop) that can discover and isolate bogus nodes while they attack the RPL routing hierarchy. TRAIL is derived of first hand principles and shall resolve the issues of topological infringements. It has been implemented and made openly available on the RIOT platform [3].
The remainder of this work is structured as follows. Section II discusses the problem of securing RPL, common attacks and related work. The incompleteness of VeRA is examined in Section III. Countermeasures for fixing VeRA are presented in Section IV. Section V introduces TRAIL, our generic solution for topology authentication. Finally, we conclude in Section VI and look out on future work.
II. RPL SECURITY CHALLENGES & RELATED WORK
RPL constructs a reverse path forwarding hierarchy by announcing tree parameters in the downward direction, starting from the root node. A node that successfully joined the tree advertises its rank towards its potential children in so called DIO messages, while unconnected nodes select as parent the neighbor of lowest rank, i.e., in closest position to the root. Following this algorithm, a fully connected graph of unambiguous hierarchical relations is created in compliance to wireless reachability. Each of such DODAGs is associated with a unique version number to survey consistency.
RPL specifies secured control plane messages for authenticity, integrity, and optional confidentiality [1]. Even though these basic security features defend against external attackers [4], RPL remains unprotected against adversaries from inside the network [2], [5]. Capturing a node and extracting security credentials enables an attacker to gain access to the control plane and to modify the routing topology. The rank and the version number are the key information for defining the structure of the routing system. The essential challenge for securing the routing topology thus is to protect rank and version number from any unwanted modification. Next, we introduce the core attacks against the RPL topology and the assumptions made on the attacker.
A. Attacker Model
We assume the presence of one or multiple attackers that physically captured and compromised multiple, arbitrary nodes on the network. The attacker has access to all available keys on the captured nodes, which include all information for joining and participating in the DODAG without restrictions. The compromised nodes are successfully integrated in the network and are thus authorized to transmit authenticated messages. Furthermore, the attacker is limited by the resources and constraints of the captured nodes. Hence, we assume that the attacker cannot install directed antennas or create multiple identities [6] to seemingly use several malicious nodes with one physical interface or to establish out-of-band channels. The attacker aims at maximizing his impact on the network, for example by attracting as much traffic as possible for eavesdropping or sink-holing, or by affecting the operational conditions of as many nodes as possible.
B. Topology Attacks 1) Version Number Attacks: The version number of the DODAG is increased by the root node, whenever a global repair is needed. This occurs, if inconsistencies cannot be repaired locally. In a version number attack [2], an attacker illegally increases the version number of the DODAG. Publishing a higher version number will lead to a reconstruction of the RPL topology. This either serves as a preparation for a following attack such as on the rank, or can be repeatedly executed to disturb the network and drain the resources of nodes.
2) Rank Spoofing Attack: In a rank spoofing attack [2], a malicious node propagates an incorrect rank to change its position in the routing tree. Commonly, an attacker will choose a lower rank to improve its position in the hierarchy and achieve larger impact on the network. In response to forged (a) Topology after a rank spoofing (b) rank advertisements, neighboring nodes select the attacker as parent and forward traffic towards it. Fig. 1(a) visualises the topological manipulations caused by a strict rank decrease. The attacker M propagates the lowest rank of the vicinity and attracts all its neighbors. In this example, the parent node H is also attracted by the malicious node M , which creates a sinkhole. Node 3 correctly selects M as parent, but unknowingly propagates the illegal rank downtree. Thus, 3 and its parents potentially attract even more children and increase the number of nodes that forward traffic towards the attacker M .
3) Rank Replay Attack: An attacker who learned a valid rank from a (potential) parent may replay this value in its own advertisements and pretend to run at one hierarchy level above the proper value. This special case of a rank spoofing will not disconnect the attacker from the root as visualised in Fig. 1 (b). In contrast to arbitrary rank forgery, the replay allows a malicious node to re-use a proper rank, even if rank verification schemes apply. We will show in the following section that present protection schemes are vulnerable to rank replay attack.
C. Related Work
Up until now, only limited work has addressed the security of the RPL routing system. A security threat analysis for LLNs by the IETF [4] focuses on potential threats and attacks. However, the analysis solely proposes generic countermeasures to the described attacks. Some attempts have been made to deal with topology attacks [7], [2], [8], [9]. The authors in [7] propose an Intrusion Detection System to mitigate the rank attack and a local repair mechanism by installing additional monitoring nodes. VeRA addresses the rank and version number attacks by adding a rank and version control obtained from hash chaining [2], [10] mitigating a version number attack, the VeRA approach is still subject to two topology attacks [11]. As we will work out in the following section, the first attack is a general rank spoofing, which allows an attacker to pretend any rank and therefore any position in the DODAG. The second attack is a rank reply attack, which allows an attacker to claim one level closer to the root by replaying its parent's rank. Weekly and Pister [8] concentrate on the evaluation of sinkhole attacks and their impact on the data throughput in RPL. They utilize a rank authentication based on VeRA and introduce a parent failover technique to blacklist sinkhole nodes. The root maintains a list of nodes that go below a threshold, which defines the minimum expected data receptions for each node. A node that finds itself on the list, blacklists its default next-hop towards the root, since some node on the path seems to not forward traffic. The authors observe that an adversary can attack VeRA by replaying the rank of his parent. Similarly, Wallgren et al. [9] propose to maintain a whitelist in combination with a heartbeat protocol in which the root periodically sends echo requests to each node to check the connectivity. A node that does not respond is considered malicious and is thus removed from the whitelist.
Our work goes beyond mitigating sinkhole attacks. By performing generic topological tests, we inquire on the integrity of the routing hierarchy, identify and isolate individual attackers. The rank protection approaches discussed above rely on the authentication of digital signatures. Authentication in RPL is implemented either by an asymmetric signature scheme like RSA [12], or a symmetric message authentication code like CBC-MAC [13] with a pre-shared key. Even though the complex use of asymmetric cryptography in wireless LLNs is not per se feasible [14], [15], [16], it adds the advantage to unambiguously authenticate the sender of a message. Conversely, when a MAC is created from a group key, it suffers the disadvantage of authenticating any sender from that group. Recent work on pre-computation techniques [17] strengthened the case for standard signature deployment on sensor nodes. In this work, we rely on the RPL root node as a trust anchor, since it is commonly deployed as a more powerful gateway node. Signature creation remains bound to this RPL root.
III. ATTACKING VERA A. VeRA in a nutshell
VeRA is performed in two steps: initialization and version number update. The scheme assumes that the nodes are given a public key pk for a public key signature scheme like RSA, and the corresponding secret key sk is known only to the DODAG root.
Initialization: The DODAG root generates the hash chains to be used for securing version number and rank updates. For n version updates, the root picks a random number r, a secure hash function h, and computes a hash chain, . Additionally, the root generates a rank hash chain of size l + 1 for each version V i . Let R i,0 , · · · , R i,l denote the rank hash chain for V i . Then, its elements are computed as where x i is a random number. Subsequently, for bootstrapping the security, the root broadcast an initialization message , σ} to all nodes in a DIO message. Thereby, V N 0 denotes an initial version number chosen by the root and σ = Sig sk (V 0 , V N 0 , M AC V1 (R 1,l )) the signature. Each node stores this message after verifying the signature using the public key pk.
Version number update: To update the version of a DODAG from Rank sender is its new rank 1 . Each intermediate node receiving this message checks first whether the new version number is higher than the current one, i.e., if If this is the case, it continues to verify that the version update was indeed initialized by the root by checking if If any of these verifications fails, the node terminates the version update operation. Otherwise, it proceeds with verifying the rank of its parent by checking the hash chain consistency, i.e., Note that M AC Vi (R i,l ) was received in the previous update, while V i is received in the current update. Finally, the child node calculates its own rank τ using the objective function and forwards the received DIO message to nodes lower in the topology with the corresponding rank chain element
B. (In)Security of VeRA
The security of VeRA relies on the assumption that increasing the version number or decreasing the rank value requires an attacker to compute the pre-image of a hash chain element. However, due to the stateful nature of the VeRA protocol, the pre-image resistance of the hash chains alone are not sufficient for security. VeRA is a stateful protocol, since the security of each version update relies on the parameters revealed in a previous update. Although, the initialization message is signed, as shown in Section III-B1, it is not sufficient to mitigate rank chain forgery performed by malicious insiders or when jamming attacks are considered. Hence, additional methods preserving backward secrecy of the rank hash chains are needed to mitigate VeRA against such attacks 2 . Within the scope of the VeRA protocol, we give the following definitions: Definition Perfect-backward-secure version update protocol: A version update protocol is perfect-backward-secure if an adversary cannot efficiently calculate a valid rank hash chain {x i , R i,l } i∈{n,···,1} with x i = x i even if it is given all elements of the version hash chain, i.e., V n 3 , the corresponding rank hash chains {x i , R i,l } i=n,···,1 , and the signature σ. A hash chain {x i , R i,l } is valid, if its verification in the ith version update, i.e., {V N i } i∈{n,···,1} , at any receiving node returns a success.
Definition λ-backward-secure version update protocol: A version update protocol is λ-backward-secure if an adversary cannot efficiently calculate a valid rank hash chain {x i , R i,l } i∈{n,···,1} with x i = x i even if it is given up to λ < n elements of the version hash chain, i.e., V λ , the corresponding rank hash chains {x i , R i,l } i=λ,···,1 , and the signature σ. A hash chain {x i , R i,l } is valid, if its verification in the ith version update, i.e., {V N i } i∈{n,···,1} , at any receiving node returns a success.
Lemma: The VeRA protocol is a λ-backward-secure version update protocol.
Proof (Sketch): Consider a VeRA setting for n = 3. Furthermore, consider the version hash chain {r, Given the version hash chain element V 2 , the adversary can calculate a valid hash chain {x 2 , R 2,l } by simply picking a random number x 2 = x 2 and subsequently authenticating R 2,l with the MAC using V 2 as the key. This rank hash chain would be verified as valid in a version update V 2 . Hence, the VeRA protocol is 2-backward-secure version update protocol. That is, it remains secure against rank hash chain forgery as long as no version hash chain element V λ≥2 is compromised.
Remark Praxis relevance of backward-secrecy: RPL is a routing protocol for LLNs. A typical characteristic of such networks, such as WSNs, is that they are often deployed in public and even in hostile environments. Hence, they are typically easy to access by attackers. Wireless communication used in such networks allows an attacker to disrupt and even entirely block the communication between nodes. For instance, (selective) jamming attacks [18] allow to partition a network. Similarly, selective-forwarding attacks [19] allow the attacker to drop selected packets during routing. Such attacks allow the adversary for decreasing its own rank and, hence, the rank of those nodes located in its sub-DODAG if the version update protocol used is only λ-backward-secure like VeRA. In the following, we describe a practical rank chain forgery attack in existence of e.g., selective-forwarding or jamming attacks.
1) Rank hash chain forgery attack: VeRA is only 2backward-secure. Hence, it is vulnerable to rank chain forgery. Such an attack might be performed as follows. The DIO messages for two subsequent version updates V N i and V N i+1 2 Solutions based on time synchronization are not considered in this work. 3 All other elements V n−1 , · · · , V 0 , can be calculated from Vn are prevented from being received by all or some of the nodes within the network. This can be achieved e.g., through a selective-forwarding or (selective-)jamming attack on the DIO messages of the version updates. After receiving the hash chain element V i+1 in the version update V N i+1 , the attacker calculates a bogus hash chain {x i+1 , R i+1,l } by simply picking a random number x i+1 and subsequently authenticating it with the MAC using V i+1 as the key. Subsequently, the blocked version update V N i is resumed by forwarding the DIO message containing the MAC of the forged rank hash chain M AC Vi+1 (R i+1,l ). Finally, once the version update V N i is completed, the version update V N i+1 is initiated, in which the attacker can claim an arbitrary rank value.
2) Rank replay attack: In each rank update, VeRA discloses the cryptographic credentials needed for verifying the advertisements from parents to each node. These credentials are not bound to any sender-specific attributes. Hence, a malicious node can transparently forward them down the tree to decrease its rank. As visualized in Fig. 1(b), a malicious node M receives valid rank announcements from the honest node H. This includes the version hash V i , its rank j, and the associated hash element R i,j . It can simply re-use them in its own rank advertisements to nodes 1 . . . 4 for gaining one hierarchy level. In consequence, the honest nodes 1, 2, and 4 can verify the bogus rank announcements and prefer M over H due to better connectivity. Node 3 correctly selects M as parent, but calculates a falsely improved rank. All children of M propagate maliciously lowered ranks down the sub-DODAG.
IV. FIXING VERA
We introduce two countermeasures to fix VeRA against the described attacks. Our first countermeasure makes the VeRA approach perfect-backward-secure and, hence, it mitigates the rank hash chain forgery attacks. Our second countermeasure is a simple challenge-response procedure proposed for mitigating the rank replay attacks.
A. VeRA++: Perfect-backward-secure VeRA
VeRA authenticates a rank hash chain for a version V i using a MAC keyed with V i . In each version number update a MAC key is revealed. Hence, VeRA provides only the λ-backwardsecrecy. To achieve the perfect-backward-secrecy, we propose to authenticate the rank hash chains using an encryption chain instead of MACs. In the following, we first describe the construction of the proposed encryption chain. Subsequently, we describe the VeRA++ approach, i.e. an extension of VeRA with the proposed encryption chain. Finally, we show that VeRA++ provides the perfect-backward-secrecy.
1) Construction of the encryption chain: After generating the version number hash chain and the rank hash chains {x i , R i,l } i=n,...,1 as described in Section III-A, the root node computes the (rank) encryption chain {c i } i=n,...,1 as follows: c n is set to the last element of the rank hash chain for V n , i.e., c n = R n,l . Subsequent elements of the encryption chain c i are calculated by encrypting the last element of the corresponding rank hash chain R i,l using c i+1 as the encryption key. That is, {c i = enc ci+1 (R i,l )} i=n−1,...,1 , where enc is a symmetric key encryption scheme such as AES.
2) Extension of VeRA with the encryption chain: In VeRA++, the initialization and version number update steps are performed slightly different than in VeRA. In the initialization step, the root broadcast the initialization message {V 0 , V N 0 , c 1 , c n , σ} to all nodes in a DIO message. Thereby, σ = Sig sk (V 0 , V N 0 , c 1 , c n ). As in the VeRA, each node stores this message after verifying the signature with pk.
In the version number update step, to update the version of DODAG from V N i−1 to V N i , the root sends a DIO mes- If this is the case, it continues to verify that the version update was indeed initialized by the root by checking if If one of these verifications fail, the node terminates the version update operation. Otherwise, it proceeds with verifying the rank of its parent.
Assume that the parent node claims to have the rank value Rank parent . The child node verifies its validity by checking if h l−Rankparent = dec ci (c i−1 ). Note that c i−1 was received in the previous update. A successful verification implies that the rank of its parent is increasing monotonically 4 . Subsequently, the child node calculates its own rank τ using the objective function and forwards the received DIO message to the nodes lower in the topology as in VeRA.
3) Security of VeRA++: We show that the VeRA++ approach is a perfect-backward-secure version update protocol.
Proposition: The VeRA++ approach described above is a perfect-backward-secure version update protocol if the underlying encryption function enc, the signature scheme Sig, and the hash function h are cryptographically secure.
Proof (Sketch): Assume that the VeRA++ approach a λbackward-secure version update protocol. Then, according to our definition, given a hash chain element V λ with λ < n and the encryption chain {c i } i=λ,···,1 and the rank hash chains {x i , R i,l } i=λ,···,1 and the signature σ, there exist an efficient algorithm that calculates a forged rank hash chain • i = 1: c 1 is signed. Thus, there is only one possibility for calculating a forged rank hash chain {x 1 , R 1,l } with x 1 = x 1 . The adversary needs to find a c 2 such that R 1,l = dec c 2 (c 1 ) for an arbitrarily chosen x 1 . The probability of finding such inputs (c 2 , c 1 , x 1 ) is negligible if the underlying encryption function enc is secure and the hash function h is pre-image resistant and the signature scheme is secure.
• i = λ: For calculating a forged rank hash chain {x λ , R λ,l } with x λ = x λ , the adversary needs to find a c λ+1 such that R λ,l = dec c λ+1 (c λ ) for an arbitrarily chosen x λ . However, the probability of such inputs (c λ+1 , c λ , x λ ) is negligible if the underlying encryption function enc is secure and the hash 4 In the last version update, no decryption is required since cn = R n,l . function h is pre-image resistant and the signature scheme is secure 5 .
• i = n: c n = R n,l is signed. Thus, there is only one possibility for calculating a forged rank hash chain{x n , R n,l } with x n = x n . The adversary needs to forge the signature message σ. However, the probability of a signature forgery is negligible if the underlying signature scheme is secure.
This shows that a rank hash chain forgery for λ ≤ n requires to break the security of either h or enc or Sig. Hence, given the secure instances of algorithms h, enc, and Sig, according to our definition, the VeRA++ protocol is a perfect-backwardsecure version update protocol.
B. Countermeasure against Rank Replay Attacks
To mitigate rank replay attacks, we introduce a challengeresponse scheme bASED on the rank hierarchy implemented by RPL. A malicious node, claiming a lower rank value than its actual value, is challanged to prove that it has a parent node of lower rank than the claimed.
1) General Idea: Each RPL node receives the rank hash chain element of its parent to verify the parent's rank as well as to calculate its own rank. A parent node, claiming to have the rank j − 1 in a version update i, sends the hash chain element R i,j−1 to its children. Each child receiving this message verifies it by checking if R i,l = h l−(j−1) (R i,j−1 ) holds. However, due to pre-image resistance of the hash function h, they cannot calculate R i,j−2 , i.e., the hash chain element valid for their grandparent nodes. Our scheme relies on this one-way property of the rank hash chains. That is, any node claiming to have a rank j knows (or can calculate) the hash chain element of their parents, their own, and their children only, i.e. {R i,k } k=j−1,...,l . The hash chain elements for lower ranks (e.g.., for their grandparents) {R i,k } k =1,...,j−2 remain unknown to them (or cannot be calculated by them). Hence, any node claiming to have a rank j must be able to encrypt a challenge message using R i,j−1 as the key, correctly. An attacker that incorrectly replays the rank hash element of its own parent cannot encrypt such a challenge, since it needs to know the key R i,j−2 in such a case.
2) Challenge-Response Scheme: Assume that the node M depicted in Fig. 2 is suspicious of replaying the rank j of its parent H to obtain an improved position in the DODAG topology. To verify the rank of M , the parent node H constructs a challenge message ID M , r , which includes the host ID of M ID M and a random number r. This message is to be encrypted by M with R i,j−1 . M shall reply with enc Ri,j−1 ( ID M , r ) to H. H can then check whether M holds the correct rank. If M cannot solve the challenge, it has no valid parent of the claimed rank and incorrectly announced j.
3) Applying the Challenge-Response Scheme: The challenge-response scheme is initiated by a RPL node that complies with two requirements: (a) it is at the same rank level as claimed by the attacker, (b) it is within the transmission range of the attacker. The first requirement allows for self-organization among RPL nodes according to correct and incorrect ranks (i.e., who initiates the challenge). The second requirement is necessary to react on suspicious rank announcements (i.e., observing rank upgrade). It is noteworthy that these requirements fully comply to the RPL protocol message design. Our approach detects the potential attack by requiring each node to multicast its rank to all neighbors. If a parent H detects an inconsistent routing state, which is not removed by a local repair, the suspicious node is challenged to proof its rank. If the node does not pass the challenge, either the sub-DODAG can simply be excluded from upward routing, or the root node can be included in the validation process. The root creates a validation packet for the children of the malicious node M . Finally, as many surrounding nodes as possible are informed about the rank state of node M . This gives each node the ability to independently react and for example discard M as a routing node. Note that the root node can trust H after the update is delivered successfully and the replay detection is applied recursively from H to the root.
Remark Our challenge-response scheme is not secure in existence of out-of-band channels or k directly connected attackers. An attacker, who can obtain the rank hash chain element for a rank j − ∆ through such a tunnel, can correctly respond any challenge issued for verifying the ranks ≤ j − ∆. Another limitation of this approach is that the children of a malicious cannot be reliably notified about an attack. Introducing a white-or blacklist containing benign or malicious nodes respectively, as proposed in [9] and [8], holds the drawback of straining the entire network with local information. Hence, in the next section we propose TRAIL which inverses the direction of rank validations.
V. TRAIL -TRUST ANCHOR INTERCONNECTION LOOP
We introduce TRAIL, our generic approach to detect and prevent topological inconsistencies. In contrast to the previous approaches, each node is enabled to validate its upward path to the root and to detect rank spoofing on it. Our test furthermore identifies the largest sub-DODAG(s) affected by nonmonotonous rank order. Having learned such inconsistency, the root of that sub-DODAG may either trigger a local repair, or disconnect its malicious sub-tree and rely on alternate paths.
A. TRAIL Idea: Path Validation
The key idea of TRAIL is to validate upward paths to the root using a round trip message. Without relying on encryption chains as in VeRA++, a node can conclude rank integrity from an recursively intact upward path.
A child node that received a rank advertisement from its parent initiates a positive attestation of the rank as follows. It sends a test message with a random nonce η upwards to its parent. The parent adds its rank j and forwards the test message j, η upstream towards the root. At each intermediate hop, the receiving upper node verifies that (a) the rank in the test message is higher than its own, and (b) the rank of the sending node lies in between the rank of the test message and its own. If a rank violation is observed, the test message is discarded and the sub-DODAG gets either disconnected or a local repair is started (see Fig. 3). The test message eventually arrives at the root, which adds the current version number to the test message and signs for its way back to the initiating client. Every forwarding node verifies if the signed message contains the scribed rank j > its own rank before forwarding it. A violated relation stops the propagation of the message. On reception, the client verifies the signature, matches its nonce, and obtains evidence of the current version number and the rank advertised by its parent. As the rank announcement had consistently travelled to the root, no honest node on the path had observed a rank violation and the upstream is valid. A child not receiving the reply, continues without positive attestation of its parent. It may choose another upstream, if available, or apply additional measures for transport security.
After all nodes have applied this test recursively down the hierarchy with success, it is assured that none of the nodes has a parent that illegally lowered its rank. The highest ranked node that unsuccessfully performs the test identifies the root of the largest sub-DODAG affected by rank spoofing. It should be noted, though, that a directly connected chain of k malicious nodes can secretly replay rank values k − 1 times so that they are counted in the test as one node. However, this costly attack does not decrease rank values of the attackers, but solely extends the wireless reach of the malicious group and cannot be observed without surveillance of the wireless geometry.
As every node in the network needs to inquire with the root individually, the overhead in messages and signature processing grows linearly with the network size. Hence, this simple scheme of path validation suffers the obvious drawback of scalability. In the following, we will present an aggregated scheme that keeps messages per node and signature computation constant.
1) Rank Attestation Scheme:
The path validation can be turned into a scalable procedure by aggregating all clientspecific inquiries into a single message exchange. Starting from the leaf nodes of a DODAG, we design a convergecast that reaches up to the root. The root node receives and signs a single, converged request that serves as a universal path attestation message when distributed downtree via multicast.
After a leaf node N l,k of the DODAG has received the rank advertisement of its parent (and discovered that it has no further children), it issues a nonce η l,k to its parent. The parent node collects the nonces {η l,k } k of all children and writes them into a single array element. For space efficiency, the parent combines the nonces in a Bloom filter [20]. Note that this Bloom filter can be very short, as the number of entries is limited by the number of children per node. This array element containing a single Bloom filter is sent upstream to the grandparent and saved by the node.
From each of its children, the grandparent receives such an array of Bloom filters together with an individual nonce. It should be noted that these arrays need not be of equal lengths, as the tree may be unbalanced. The grandparent aligns every array on the position below the child node rank and merges the entries of equal index using the scalable Bloom filter technique of Almeida et al. [21]. In detail, the grandparent node extracts all first index elements A i (1), merges them and writes the result to a new output array B at the index 2 (incremented by one). In general, {A i (k)} i are merged into B(k + 1), if existent. Finally, the node adds the Bloom filter that aggregates all nonces of its immediate children to the array element B(1) forwards the array B upwards together with its own nonce and saves both B and its nonce. Fig. 4, in proceeding this way stepwise towards the root, an array is created whose index represents the rank and whose values are merged Bloom filters of all nonces issued at a specific rank. Thereby array elements are of variable length, each accommodating the concatenated Bloom filters as generated according to the shape of the tree. Additionally every node on the path saves the array and nonce they forward for latter validation. The root node adds the current version number and signs the data structure consisting of the Bloom filter array and the version number. Thereafter, the signed data is distributed via multicast down the tree.
On the reception, each node can verify the version, and the rank of its parent. It accesses the corresponding array element to match its nonce in the Bloom filter and verifies that no further array element contains the same nonce. Finally, it verifies that the signed Bloom filter array does not contain less nonces than the previously saved array. Note that the probability of a false positive hit can be chosen sufficiently low when configuring the Bloom filter. A successful match testate that ranks have increased monotonically from the root downwards and that the array and contained nonces have not been manipulated or reordered. Whenever the matching fails, monotonic rank order has been violated on the upward path from the current node to the root. The highest ranked node detecting such violation forms the root of an inconsistently connected sub-DODAG. Any node experiencing such inconsistency may choose another upstream, if available, or apply additional measures for transport security.
2) Security Proof: We show that a malicious node cannot improve its rank by modifying the data structure, and that improper modifications are detected in the verification phase.
Assumptions: We rely on the attacker model specified in Section II-A. In particular, we refer to an attacker that has no means to establish an out-of-band communication channel. A chain of k malicious neighbors is considered as one attacker with an extended wireless reach. Distributed attackers scattered among different hierarchy levels communicating out-of-band channel cannot be detected and are not considered in our model. However, non-collaborating attackers distributed in the topology are considered. Finally, we ignore the false positive rates on queries to bloom filters as they can be made arbitrarily small by choosing appropriate parameters.
Proof: We consider the security of TRAIL in existence of i) multiple non-collaborating malicious nodes and ii) multiple malicious nodes with limited collaboration: i) Multiple non-collaborating malicious nodes: Since the nodes are not allowed to collaborate, they can be considered as multiple single attackers. For simplicity, we provide the analysis for a single malicious node. A malicious node receiving a topology test message η, A from its child(ren) has the option to (1) not include its child(ren) in the message array or to not merge-and-forward the array A at all. It may as well (2) rearrange the array, and in particular include the nonces of its child(ren) at a wrong array position. It may (3) attempt to exclude itself from the attestation hierarchy by not submitting its nonce value to its parent. These four choices of malicious nodes will lead to the following conditions: (1) By not forwarding the test nonces of its children or the attestation array, the malicious node causes its immediate detection. When receiving the signed attestation message of the root, the child(ren) of the malicious node will test for its nonces without success and detect the inconsistency.
(2) The best a malicious node can do to its children is writing nonces at the foreseen position. Any misplacement will move data of the children to a lower rank position and thus cannot be aligned with a malicious rank upgrade. Other rearrangements of the array will change the data positions for nodes lower in the tree. This implies that affected nodes are not within the wireless transmission range of the malicious node -they had chosen the better rank of the malicious node otherwise. As the malicious node cannot coordinate rank advertisements outside its wireless reach, nodes will remain unaware of their nonce moving to other rank positions. Nodes will thus search at the original rank position in the attestation message and corresponding tests will fail.
(3) If the malicious node withholds its own nonce, but cooperates in traversing the merged filter array, its honest parent will merge the data with data from its other children and insert at the proper position. Not delivering the nonce will simply lead to a Bloom filter that does not contain the nonce of the malicious node. Hence, an malicious node causes nothing but excluding itself from the verification process.
ii) Multiple malicious nodes with limited collaboration: We mean by a limited collaboration that multiple attackers know in advance their position in the topology and the desired rank which they want to claim during an attack. This can be realized by configuring them accordingly during their deployment. Limited indicates that once they are deployed, those malicious nodes, which are not within each other's communication range, cannot communicate anymore. TRAIL mitigates such attacks as follows: A malicious node close to the root merges array elements on behalf collaborating malicious nodes lower in the topology that claim a false rank. Consequently, nonces of honest nodes that are affected by the rank spoofing, are moved to the correct array element. However, due to the malicious merging of array elements, these nonces exist multiple times. Such a duplicate either denotes a fraud or a false positive. Given a false positive rate of f , we detect the attack with probability 1 − f . Deleting nonces from filters will cause that an honest node on the path will detect the attack by comparing the forwarded array with the signed one.
In any of the cases, forgery will not comply to a rank decrease and will be detected, whenever it affects third party nodes. All parents of a malicious node will always exclusively write to the lower rank-test positions, which is the obvious protection from rank spoofing in this procedure.
3) Details of the Bloom Filter: We use Bloom filters [20], a space-efficient random data structure, to reduce message lengths in our attestation scheme. A Bloom filter is defined as a bit-vector, v of m bit and represents a data set. By using k independent hash functions, each element of a set of A = {a 1 , . . . , a n } is mapped to k bits in v. By these means, the size of each input element is reduced to at most k bits. Due to randomized overlapping of bits from different elements, the size may be reduced even further, but this may return a false positive result of a query. Essentially, there is a linear relation between number of bits used for storing each element, and the false positive rate. Mitzenmacher [22] could show that properly designed Bloom filters can be compressed even further by about 30 % at a given false positive rate. Almeida et. al. [21] designed a scalable extension of Bloom filters that linearly add filter elements with increasing set sizes.
In TRAIL, we require tiny Bloom filters that store nonces from the children set of a single node. For a commonly small fanout of k nodes and a false positive rate below 1%, an appropriate bit-size m of the (compressed) Bloom filters can be estimated as m = 6 k [bits].
C. Evaluation
For the evaluation of our RPL security scheme, we have implemented TRAIL authentication as an extension to the RPL protocol on the RIOT platform [3] and performed experiments on the DES Mesh Testbed of FU Berlin [23]. For the sake of brevity, we concentrate on the overhead cost and the temporal performance of the routing protocol.
Critical cost metrics for wireless sensor nodes relate to over the air transmission, e.g., the number of messages sent, as well as message lengths. Hence, we first analyze the message characteristics of TRAIL as a function of the network size. The critical resource consumption of TRAIL is given by the sizes of the test messages. As nodes need to accumulate nonce values of their parent nodes, the attestation array grows with increasing network sizes. While messages are tiny at the leaf nodes, the array gets larger towards the root node. Fig. 5 visualizes the average message sizes for different fanout degrees k of the tree nodes as functions of the total network size. For simplicity, we assume balanced k-ary trees, but results are not strongly dependent on tree shapes. It is clearly visible that small message sizes compliant to 6LowPAN MTUs constrain network dimensions by about ≈ 250 nodes. The characteristic performance aspects of TRAIL for different network sizes and tree configurations are summarized in Table II. Our second evaluation targets at the temporal performance of route convergence. We deployed TRAIL on 25 MSBA2 nodes distributed in the sensor network testbed. RPL/TRAIL Fig. 6(a). Choosing an attacker (node 60) to announce a root rank led to a break up of the pure RPL network. Only seven nodes remained in the initial, upright network, while 17 nodes reconnected to the bogus tree. TRAIL could discover and isolate the attacker, and thus rearrange a connected tree of all honest neighbors. Routing convergence times for tree construction were measured node-wise during the experiments. Comparisons between the pure RPL and the overhead induced by TRAIL are plotted in Fig. 6(b). Naturally, the wireless ad-hoc regime produces large variations that are visible for both, RPL and TRAIL. Nevertheless, the additional times needed to join a DODAG while operating TRAIL remain below 20 % in most cases. Occasional authentication messages exceed this limit due to message loss and retransmissions. However, such performance fluctuations are characteristic for all mesh routing operations including RPL.
VI. CONCLUSIONS & OUTLOOK
This work focuses on routing security of RPL, a new routing protocol for the emerging Internet of Things. Intrinsically, RPL is vulnerable to topology attacks. Its rank and version number need particular protection, since by spoofing version and rank an attacker can obtain dominant impact on the network. The current state of the art leaves relevant security issues unresolved.
Our first contribution in this paper was to analyze and improve VeRA, a cryptographically centered protection scheme. We identified new attack vectors and modified VeRA to withstand them. While returning to the topological core of the problem, our second contribution introduces TRAIL. TRAIL defines a test procedure to inquire on the actual path properties of the routing system. This generic approach is built on firsthand principles and -different from VeRA -requires almost no cryptography. Its main cryptographic workload is carried out by the root node, which acts as a (stronger) gateway in typical RPL deployments. TRAIL is designed to minimize network message exchanges and node resource consumption. Our evaluations revealed that the transmissions of bits required by TRAIL remain feasible for typical challenged environments, and that a testbed of typical shape can well operate TRAIL with limited additional effort. Future directions of this work are twofold. First, we will further optimize our algorithms to reduce dependency on network sizes. Second, we intend to apply the TRAIL approach proposed for RPL to other routing protocols. | 2013-12-03T15:46:08.000Z | 2013-12-03T00:00:00.000 | {
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10070669 | pes2o/s2orc | v3-fos-license | Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora
Background Isepamicin is a weakly toxic but highly active aminoglycoside antibiotic derivative of gentamicin B. Gentamicin B is a naturally occurring minor component isolated from Micromonospora echinospora. 2ʹ-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2ʹ-OH-containing gentamicin B is produced as a minor component. However, the biosynthetic pathway of gentamicin B remains unclear. Considering that gentamicin B shares a unique C2ʹ hydroxyl group with kanamycin A, we aimed to design a new biosynthetic pathway of gentamicin B by combining twelve steps of gentamicin biosynthesis and two steps of kanamycin biosynthesis. Results We blocked the biosynthetic pathway of byproducts and generated a strain overproducing JI-20A, which was used as a precursor of gentamicin B biosynthesis, by disrupting genK and genP. The amount of JI-20A produced in M. echinospora ∆K∆P reached 911 μg/ml, which was 14-fold higher than that of M. echinospora ∆P. The enzymes KanJ and KanK necessary to convert 2ʹ-NH2 into 2ʹ-OH from the kanamycin biosynthetic pathway were heterologously expressed in M. echinospora ΔKΔP to transform JI-20A into gentamicin B. The strain with kanJK under PermE* could produce 80 μg/ml of gentamicin B, which was tenfold higher than that of the wild-type strain. To enhance gentamicin B production, we employed different promoters and gene integration combinations. When a PhrdB promoter was used and kanJ and kanK were integrated in the genome through gene replacement, gentamicin B was generated as the major product with a maximum yield of 880 μg/ml. Conclusion We constructed a new biosynthetic pathway of high-level gentamicin B production; in this pathway, most byproducts were removed. This method also provided novel insights into the biosynthesis of secondary metabolites via the combinatorial biosynthesis. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0402-6) contains supplementary material, which is available to authorized users.
Background
Aminoglycoside antibiotics have been widely used to treat severe bacterial infections; for instance, streptomycin was administered as the first effective anti-tuberculosis agent. Other aminoglycoside antibiotics include 2-deoxystreptamine (2-DOS) containing gentamicin, kanamycin, neomycin, and butirosin. However, the critical resistance mechanism of aminoglycoside antibiotics in pathogens is enzymatic inactivation. As such, semi-synthetic aminoglycosides have been created to overcome pathogen enzymatic inactivation. For example, isepamicin is developed by introducing (S)-3-amino-2-hydroxypropionyl side chains to the 1-amino group of gentamicin B. This side chain can block the modification by various aminoglycoside-modifying enzymes. Thus, isepamicin is highly stable against aminoglycoside-inactivating enzymes [1].
Isepamicin is manufactured from gentamicin B, which is co-produced in Micromonospora echinospora. Gentamicin C complex is produced by M. echinospora as its major product. By contrast, gentamicin A, B, and X are [2]. Gentamicin A and X are intermediates of gentamicin C biosynthesis. However, gentamicin B is not an intermediate of the gentamicin C biosynthetic pathway because gentamicin B cannot be biotransformed by M. echinospora into any gentamicin C component [2]. Thus far, the biosynthetic pathway of gentamicin B remains unclear.
The structure of gentamicin B resembles that of both gentamicin C and kanamycin A. Unlike most 2-DOScontaining aminoglycosides, kanamycin A contains a hydroxy group at C 2ʹ ; gentamicin B shares this unique structure with kanamycin A. The methylated pentose ring of gentamicin B is also similar to that of gentamicin C and is designated as garaosamine. Gentamicin B can be synthesized via a biosynthetic pathway similar to that of kanamycin A on the basis of molecular structure. In the kanamycin A biosynthetic pathway in Streptomyces kanamyceticus, 2ʹ-NH 2 is converted into 2ʹ-OH by using KanJ and KanK; in the process, 2-oxoglutarate, Fe 2+ , O 2 , and NADH are utilized [14]. We hypothesized that gentamicin B could be biosynthesized from JI-20A, a compound structurally similar to gentamicin B, except NH 2 group at C 2ʹ (Fig. 1). Thus, JI-20A could be converted into gentamicin B in JI-20A-overproducing strain via two steps catalyzed by KanJ and KanK. Using the assembly and metabolically engineered biosynthesis pathway of gentamicin B, we can eliminate byproducts and improve gentamicin B production.
Construction of JI-20A-overproducing strain by disrupting genP and genK genes
The original strain can potentially generate gentamicin B; as such, we determined whether gentamicin B is synthesized from the 2ʹ-amino-containing precursor by the KanJ and KanK homologs. However, the genes homologous to kanJ and kanK have yet to be detected in M. echinospora. Considering that the biosynthetic pathway of gentamicin B remains unclear, we aimed to construct an artificial pathway of gentamicin B biosynthesis. JI-20A and gentamicin B are similar in terms of chemical structure except at C2ʹ. JI-20A contains an amino group at C2ʹ, whereas gentamicin B comprises a hydroxyl group at the same position. The structural difference between JI-20A and gentamicin B is similar to the difference between kanamycin A and B. Considering that kanamycin B is converted into kanamycin A by KanJ and KanK, we determined whether KanJ and KanK can also transform JI-20A into gentamicin B.
To generate a JI-20A-producing strain, we simultaneously disrupted the 6ʹ-methyltransferase gene genK and the 3ʹ-phosphotransferase gene genP in this study (Fig. 2). We separately generated genP-and genK-disrupting strains in our previous work [6,7]. The genP-disrupting strain does not produce gentamicin C complex, which are the main products of the wild-type strain but are the main byproducts in gentamicin B production. In this study, the genK-disrupting plasmid was introduced to M. echinospora ∆P to obtain the double-gene-disrupting strain M. echinospora ∆K∆P (Additional file 1: Figure S1A). The disrupting strain was fermented and its products were analyzed through high-performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD). JI-20B and JI-20Ba were the main products of M. echinospora ∆P but were undetectable in M. echinospora ∆K∆P (Fig. 3). JI-20A was produced up to 911 μg/ml, which was 14-fold higher than that produced by M. echinospora ∆P. We blocked the biosynthesis of gentamicin C1, C1a, C2, C2a, JI-20B, and JI-20Ba and generated a JI-20A-overproducing strain by disrupting genP and genK.
Construction of a new gentamicin B biosynthetic pathway through the heterologous expression of kanJ and kanK
The kanJ and kanK genes under the control of the strong promoter PermE* were cloned into the site-specific integration plasmid pEAP1 to construct pSPUJK1. pSPUJK1 was introduced to M. echinospora ∆K∆P through conjugation and exconjugants were selected through erythromycin resistance; as a result, M. echinospora JK1 was generated (Additional file 1: Figure S1B). M. echinospora JK1 was fermented under the same condition used to ferment the wild-type strain. The fermentation products of M. echinospora JK1 were also analyzed through HPLC-ELSD. Compared with the wild-type strain, M. echinospora JK1 produced a new product exhibiting a retention time similar to that of gentamicin B (Fig. 3).
To determine the structure of the new product of M. echinospora JK1, we analyzed the purified product through mass spectrometry. The exact mass of the product was 482.26 (m/z 483.26), which corresponds to gentamicin B (Fig. 4). Furthermore, the protonated fragments were the same as those of the reported mass spectra of gentamicin B [15], that is, the glycoside bond of gentamicin B cleavage formed the fragment b + c (m/z 324.17). The glycoside found at the C 6 -O of 2-DOS decomposed; thus, the fragments b + c + x (m/z 366.18) were produced. Nevertheless, gentamicin B yields the same molecular weight as that of gentamicin X2, which is another minor component of gentamicin. To verify the structure of the new product, we recorded the corresponding 1 H and 13 C NMR data. The 1 H spectrum of the new product was similar to that of gentamicin B [16] (Additional file 2: Figure S2). Moreover, the 13 reported NMR data of gentamicin B [17]. MS and NMR data demonstrated that the product from M. echinospora JK1 is gentamicin B.
Although gentamicin B production is greatly improved in M. echinospora JK1 compared with that of the wildtype strain, the yield is only 85 μg/ml in M. echinospora JK1. A large quantity of JI-20A in M. echinospora JK1 was also found in the fermentation broth. Therefore, the expression levels of kanJ and kanK were genetically manipulated to improve gentamicin B production.
Enhancing gentamicin B production through the genetic manipulation of kanJK
The genetic stability of the strains applied in industrial fermentation is very important. However, gentamicin B production in M. echinospora JK1 decreased from 162 μg/ml to 80 μg/ml in five generations of unselected passages (Fig. 5a). We proposed that the instability of M. echinospora JK1 is caused by the homologous recombination between the PermE* upstream of kanJK and the native promoter of ermE, which was used as a selection marker gene in pSPUJK1. In addition, the instability may be caused by chromosomal rearrangements or plasmid elimination from the chromosome, which can occur when a φC31-derived plasmid is used in strains containing multiple pseudo attB sites [18].
To avoid genetic instability, we integrated kanJ and kanK into the chromosome through homologous recombination instead of site-specific insertion. Considering that genP has been disrupted in M. echinospora ∆K∆P, we designed kanJ and kanK to replace genP (Additional file 1: Figure S1C). To reduce JI-20A production and improve gentamicin B production, we placed kanJ and kanK under the control of the strong promoter PermE* (Fig. 2). Moreover, the fermentation broth of the gene replacement strain M. echinospora JK2 was analyzed through HPLC-ELSD. Figure 5a shows that the gentamicin B production by M. echinospora JK2 was more stable than that by M. echinospora JK1. Gentamicin B production increased to 342 μg/ml. However, M. echinospora JK2 produced a considerable quantity of JI-20A.
The native promoter of genP was used to improve gentamicin B production. To eliminate any possibility of polar effects on other genes, we designed kanJK as a replacement of the open reading frame of genP, and no other elements were introduced (Additional file 1: Figure S1D). As such, the native ribosome binding site (RBS) and promoter of genP (or promoter of its operon) were employed by kanJK. Although the regulatory elements of genP have yet to be determined, we proposed that all regulatory elements of genP can be used. In the fermentation of M. echinospora JK3, kanJK was expressed as gentamicin B was produced in JK3. Gentamicin B production also reached 436 μg/ml. PermE*, which exhibits a strong promoter activity, is widely used for the gene expression in Actinomyces. However, this work found that PermE* is weaker than the native promoter of genP.This phenomenon occurred possibly because PermE* originates from Saccharopolyspora erythraea; as such, PermE* cannot be as effective in M. echinospora as in S. erythraea. Another possible cause is the ineffectiveness of the RBS of the construction because the translation initiation of PermE*-kanJ-kanK is weak when analyzed with the RBS Calculator [19]. [20]. Therefore, a kanJK under the control of PhrdB was constructed and introduced to M. echinospora ∆K∆P; as a result, a mutant strain M. echinospora JK4 was generated (Additional file 1: Figure S1E).
The HPLC analysis of the fermentation broth of M. echinospora JK4 revealed that gentamicin B production reached 880 μg/ml; by contrast, JI-20A production decreased to 143 μg/ml. Figure 5b shows that gentamicin B production increased by 2.5-fold when the PhrdB promoter was used compared with that when PermE* was used. This result confirmed that the expression level of KanJK was enhanced by replacing the promoter PermE* with PhrdB.
The genome analysis of Actinomyces revealed the presence of numerous gene clusters encoding secondary metabolites [21]. Actinomyces can be metabolically engineered to overproduce native metabolites or analogs. With the development of synthetic biological tools, improved bioinformatics tools of metabolic engineering, and enhanced sensitivity and sophistication of analytical methods, secondary metabolite production is feasible in various cellular factories [22].
Conclusions
We successfully established an artificial biosynthetic pathway to achieve a high-level production of gentamicin B. The genes genK and genP were disrupted in M. echinospora to producethe JI-20A, which is the precursor of gentamicin B. JI-20Aproduction in the gene-disrupting strain M. echinospora ΔKΔP reached 911 μg/ml, which was 14-fold higher than that of M. echinospora ∆P. We disrupted the biosynthesis of gentamicin C1, C1a, C2, C2a, JI-20B, and JI-20Ba by disrupting genP and genK. The removal of byproducts in fermentation broth will be beneficial to purification because antibiotic purification is costly and time consuming.
An artificial pathway for the conversion of JI-20A to gentamicin B was constructed through the heterologous overexpression of kanJ and kanK in M. echinospora ΔKΔP. The kanJK-overexpressing strain under the control of PermE* can produce 80 μg/ml gentamicin B, which was tenfold higher than that of the wild-type strain. Different promoters and gene integration combinations were investigated to improve gentamicin B production. When the PhrdB promoter was used and kanJ and kanK were integrated in the genome through gene replacement, gentamicin B was produced as a major product with a maximum yield of 880 μg/ml.These results confirmed that microbiological strains can be engineered through the metabolic engineering of an intrinsic biosynthetic pathway and the introduction of exogenous genes to produce high yields of target products.
Bacterial strains, plasmids, media, and culture conditions
The strains and plasmids used in this work are listed in Table 2. Escherichia coli Top10 was used as the cloning host grown on Luria-Bertani (LB) liquid or solid medium. Liquid ATCC172 was used for the vegetative growth of M. echinospora. The conjugal transfer was performed on MS agar. Solid slanting medium was used for M. echinospora sporulation. The previously described media and culture conditions were used for gentamicin production [8].
Construction of kanJ and kanK expression plasmids
DNA isolation and manipulation were performed as described by Sambrook [23]. Additional file 3: Table S1 lists the primers used in this work. The kanJ and kanK genes were amplified from the genomic DNA of S. kanamyceticus. The primers were designed using the biosynthetic gene sequence of kanamycin (GenBank accession number: AJ628422.2) and gentamicin (GenBank accession number:AJ628149.4). The primers Pkanjk-up1 and Pkanjk-down1 were used to amplify a 1.95 kb fragment containing intact kanJ and kanK. The PCR product was digested with HindIII and BamHI and then ligated to pSPU241; thus, pJK241 was generated. The 2.6 kb insert containing PermE*-kanJK-To was recovered as a BglII fragment and then inserted into the same site of pEAP1 to generate pSPUJK1.
The primers Ph1 and Ph2 were used to amplify the downstream homologous arm and to generate a gene replacement vector with kanJK genes under PermE*. The primers Ph3 and Ph4 were utilized to amplify the upstream homologous arm. Pkanjk-up2 and Pkanjk-down2 were also employed to amplify the intact kanJK fragment. Perm-up and Perm-down were used to amplify PermE*. PermE*, kanJK, and the upstream homologous arm fragment were fused through overlap extension PCR in accordance with a previously described procedure [24]. The overlapping PCR product was ligated with pMD18-T (Takara) and digested with XbaI and SmaI; the downstream homologous arm was digested with XbaI and SmaI. Afterward, the digested fragments were ligated to pKC1139; thus, pSPUJK2 was generated.
The primers Ph1 and Ph2 were used to amplify the downstream homologous arm and the primers Ph33 and Ph4 were utilized to amplify the upstream homologous arm to generate a vector for gene replacement. Pkanjk-up3 and Pkanjk-down2 were also used to amplify kanJK. The overlap extension PCR was employed using the primers Ph4 and Pkanjk-down2 to fuse the kanJK fragment and the upstream homologous arm. The overlap PCR product was ligated using pMD18-T(Takara) and then digested with XbaI and SmaI. The downstream homologous arm was digested with XbaI and SmaI. The digested fragments were then ligated to pKC1139; thus, pSPUJK3 was generated.
The primers Ph34 and Ph4 were used to amplify the upstream homologous arm to generate a gene replacement vector with kanJK genes under the strong promoter PhrdB. Pkanjk-up4 and Pkanjk-down2 were utilized to amplify the intact kanJK fragment. Phrd-up and Phrddown were also used to amplify PhrdB. The upstream homologous arm fragment, PhrdB, and kanJK were then fused through overlap extension PCR. The overlap PCR product was then ligated with pMD18-T and then digested with XbaI and SmaI. The downstream homologous arm was digested with XbaI and SmaI. The digested fragments were ligated to pKC1139. Thus, pSPUJK4 was generated.
Construction of genK-disrupting and kanJK-expressing strains
genK-disrupting and kanJK-expressing plasmids were introduced to M. echinospora through conjugation on MS medium at 28 °C for 24 h. After the medium was spread with 50 mg of apramycin (erythromycin was used for pSPUJK1) and 100 mg of pipemidic acid per liter, incubation was performed at 28 °C for 7 days. Exconjugants were initially selected to determine the apramycin-resistance (the first crossover event) phenotype because pSPU503, pSPUJK2, pSPUJK3, and pSPUJK4 plasmids contain an apramycin-resistance gene; the apramycin-sensitive (the second crossover event) phenotype was then used to isolate the strains via the desired double-crossover homologous recombination event. Genomic DNA was extracted and used as a template DNA in PCR. The PCR products were subjected to DNA sequencing to demonstrate that kanJ and kanK exist in the kanJK-expressing strains.
Antibiotic isolation and analysis
The pH of the culture broth was adjusted to 2.0 by using H 2 SO 4 . The acidified broth was agitated for 30 min and then centrifuged at 11,378×g 10 min. The pH of the supernatant was readjusted to 7.0 with NaOH. The pretreated supernatant was centrifuged again at 11,378×g for 10 min. The supernatant was then applied to strongly acidic resin 001 × 7 (Shandong Lukang Record Pharmaceutical Co., Ltd).The bound substances were eluted with 2 mol/L NH 4 OH. Second cation-exchange chromatography was performed on weakly acidic resin D152 (Shandong Lukang Record Pharmaceutical Co., Ltd). The bound substances were eluted with the gradient elution of NH 4 OH (from 0.1 to 1.0 mol/L).
The elution from the acidic resin was used as the sample for the reversed-phase HPLC-ELSD analysis in a reverse C18 column at an evaporation temperature of 45 °C, nitrogen pressure of 3.5 bar, and a mobile phase of 0.2 mol/L trifluoroacetatic acid-methanol (97:3) at a flow rate of 0.6 ml/min. Authentic gentamicin B was used as standard. The purified products were analyzed using an LC/MS/MS instrument (Bruker micrOTOF-Q). The mass spectrometer was set in a positive mode. 1 H and 13 C NMR data were recorded on Bruker AV600 at 600 MHz frequency, and D 2 O was used as a solvent. | 2017-06-26T12:39:10.691Z | 2016-01-05T00:00:00.000 | {
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267785878 | pes2o/s2orc | v3-fos-license | SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP
The newly discovered zoonotic coronavirus swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute diarrhea, vomiting, dehydration, and high mortality rates in newborn piglets. Although SADS-CoV uses different strategies to evade the host’s innate immune system, the specific mechanism(s) by which it blocks the interferon (IFN) response remains unidentified. In this study, the potential of SADS-CoV nonstructural proteins (nsp) to inhibit the IFN response was detected. The results determined that nsp1 was a potent antagonist of IFN response. SADS-CoV nsp1 efficiently inhibited signal transducer and activator of transcription 1 (STAT1) phosphorylation by inducing Janus kinase 1 (JAK1) degradation. Subsequent research revealed that nsp1 induced JAK1 polyubiquitination through K11 and K48 linkages, leading to JAK1 degradation via the ubiquitin–proteasome pathway. Furthermore, SADS-CoV nsp1 induced CREB-binding protein degradation to inhibit IFN-stimulated gene production and STAT1 acetylation, thereby inhibiting STAT1 dephosphorylation and blocking STAT1 transport out of the nucleus to receive antiviral signaling. In summary, the results revealed the novel mechanisms by which SADS-CoV nsp1 blocks the JAK–STAT signaling pathway via the ubiquitin–proteasome pathway. This study yielded valuable findings on the specific mechanism of coronavirus nsp1 in inhibiting the JAK–STAT signaling pathway and the strategies of SADS-CoV in evading the host’s innate immune system.
The newly discovered zoonotic coronavirus swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute diarrhea, vomiting, dehydration, and high mortality rates in newborn piglets.Although SADS-CoV uses different strategies to evade the host's innate immune system, the specific mechanism(s) by which it blocks the interferon (IFN) response remains unidentified.In this study, the potential of SADS-CoV nonstructural proteins (nsp) to inhibit the IFN response was detected.The results determined that nsp1 was a potent antagonist of IFN response.SADS-CoV nsp1 efficiently inhibited signal transducer and activator of transcription 1 (STAT1) phosphorylation by inducing Janus kinase 1 (JAK1) degradation.Subsequent research revealed that nsp1 induced JAK1 polyubiquitination through K11 and K48 linkages, leading to JAK1 degradation via the ubiquitin-proteasome pathway.Furthermore, SADS-CoV nsp1 induced CREBbinding protein degradation to inhibit IFN-stimulated gene production and STAT1 acetylation, thereby inhibiting STAT1 dephosphorylation and blocking STAT1 transport out of the nucleus to receive antiviral signaling.In summary, the results revealed the novel mechanisms by which SADS-CoV nsp1 blocks the JAK-STAT signaling pathway via the ubiquitinproteasome pathway.This study yielded valuable findings on the specific mechanism of coronavirus nsp1 in inhibiting the JAK-STAT signaling pathway and the strategies of SADS-CoV in evading the host's innate immune system.
Coronaviruses have significantly challenged worldwide public health in the last 20 years.Coronaviruses are frequently present in animal and human groups and can cause economic disruption and catastrophic loss of life (1).In February 2017, the newly discovered swine acute diarrhea syndrome coronavirus (SADS-CoV) was first reported in southern China.SADS-CoV infection leads to acute diarrhea, vomiting, and high mortality rates among young piglets (particularly those <7 days old) but only causes mild or asymptomatic infections in adult swine (2,3).
SADS-CoV is a positive-sense, single-stranded enveloped RNA virus with a genome size of approximately 27 kb, which encodes 16 nonstructural proteins (nsp), four structural proteins, and three accessory proteins (4,5).SADS-CoV is believed to have originated from bats before crossing species to infect swine (6,7).Earlier studies demonstrated that SADS-CoV displays a wide range of cell tropisms and replicates in different vertebrate cell types, indicating its potential zoonotic transmission risk (8,9).
Then, the acetylated STAT1 interacts with T cell protein tyrosine phosphatase (TCPTP) to trigger dephosphorylation, ultimately leading to STAT1 losing its DNA-binding activity and relocating to the cytoplasm (28).The STAT1 phosphorylation-acetylation-dephosphorylation cycle regulates JAK-STAT signaling pathway activation or silencing to maintain the balance of IFN signaling.
Cell cycle control is largely dependent on ubiquitination, which is an essential regulatory process (29)(30)(31).It is believed that ubiquitin (Ub) chain linkage differences result in different Ub protein functions (31).Among the eight potential homogeneous Ub chains, the canonical K48-linked Ub is critical in inducing the proteasomal degradation of cell cycle regulators (32).Furthermore, K11-linked and K29-linked Ub chains promote proteasomal degradation (33,34).Additionally, only K63-linked Ub chains do not target proteins for proteasomal degradation (31).
Several studies provided evidence of the capacity of different coronaviruses to block IFN production and response by specifically targeting and degrading the host's antiviral elements via the Ub-proteasome pathway.The severe actuate respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF6 promotes the degradation of CHK1, a kinase involved in responding to DNA damage, through the proteasome pathway (35).SADS-CoV N protein interacts with RIG-I and induces its degradation through the Ub-proteasome pathway (36).No research to date has indicated the inhibition of IFN response by SADS-CoV nonstructural proteins through hijacking the Ubproteasome system (UPS).
Previously, our study demonstrated that SADS-CoV nsp1 significantly inhibits IFN production (37).This inhibition was primarily attributed to nsp1 blocking of TBK1 Ub-like modification, which suppressed TBK1 phosphorylation.Furthermore, nsp1 induces CBP degradation through the Ub-proteasome pathway, thereby blocking IFN transcription enhancer formation.In this study, the potential of SADS-CoV nonstructural proteins to inhibit the IFN response was detected.SADS-CoV nsp1 was eventually identified as a potent antagonist in the IFN response.Nsp1 was important in IFN inhibition by SADS-CoV.This inhibition promoted SADS-CoV replication during the early stages of viral infection.
Despite earlier studies also demonstrated that coronavirus nsp1 blocks the JAK-STAT signaling pathway, the potential mechanism remains unclear.Our research indicated that SADS-CoV nsp1 inhibited the JAK-STAT signaling pathway by blocking STAT1 phosphorylation and acetylation.This blocking was attributed to the nsp1-induced JAK1 and CBP degradation.Overall, the investigation revealed new mechanisms by which SADS-CoV nsp1 blocked the IFN response.The findings contribute to comprehending the strategies utilized by coronavirus nsp1 to evade the host innate immune system.
Nsp1 was crucial in SADS-CoV replication
Previously, the authors demonstrated that the Phe39 and Leu98 of SADS-CoV nsp1 were critical amino acids in antagonizing IFN production.These SADS-CoV nsp1 amino acids were mutated, ultimately generating a mutant virus (SADS-CoV-mutant) (Fig. 1A).Then, whether SADS-CoVmutant infection inhibited IFN production and response was investigated.LLC-PK1 cells were infected with SADS-CoV-WT (wildtype) or SADS-CoV-mutant, and the cells were collected at 4, 6, and 8 h postinfection (hpi).The IFN-β and ISG15 mRNA levels were significantly increased in cells infected with SADS-CoV-mutant compared to the cells infected with SADS-CoV-WT.This result indicated that the SADS-CoV-mutant was not able to effectively inhibit IFN production and response (Fig. 1, B and C).
To determine if the ability of nsp1 to block the IFN system is essential for SADS-CoV replication, the SADS-CoV-WT and SADS-CoV-mutant growth kinetics were compared via a growth curve assay.In ST cells and Vero cells, the SADS-CoVmutant produced lower quantities of progeny virions compared to SADS-CoV-WT from 8 hpi to 24 hpi.Due to the absence of IFN genes in Vero cells, this difference is more significant in ST cells than in Vero cells.(Fig. 1, D and E).The immunofluorescence assay result demonstrated that the proportion of cells infected with SADS-CoV-mutant was significantly reduced compared to cells infected with SADS-CoV-WT at 12 hpi (Fig. 1F).These results suggested that nsp1 was critical in SADS-CoV antagonism of IFN production and response.Additionally, the nsp1 mutations reduced the SADS-CoV replication capacity.
Nsp1 blocked the IFN-β response pathway STAT1 and STAT2 combine to form ISGF3 with IRF9 and subsequently induce ISRE-driven ISG transcription in the nucleus.Whether nsp1 inhibited the ISRE promoter activity stimulated by STAT1, STAT2, IRF9, or ISGF3 was detected to explore the potential regulatory role of SADS-CoV nsp1 in the IFN response.The ISRE promoter activity was examined using a dual-luciferase reporter assay, whereas the exogenous protein expression was detected via Western blotting.Compared to the negative control, STAT1, STAT2, IRF9, ISGF3, and human IFN-β significantly induced ISRE promoter activation.Contrastingly, nsp1 significantly inhibited ISRE promoter activity in cells stimulated by human IFN-β, STAT1, STAT2, or IRF9 (Fig. 2, A-C and E).However, this inhibition was attenuated in the ISFG3-stimulated cells, suggesting that nsp1 blocked the IFN response by targeting ISGF3 complex formation (Fig. 2D).Furthermore, nsp1 had no negative effect on exogenous STAT1, STAT2, IRF9, and ISGF3 expression levels (Fig. 2F).
Nsp1 inhibited STAT1 phosphorylation STAT1 and STAT2 are crucial transcription activators in the JAK-STAT pathway.Endogenous STAT1 and STAT2 expression and phosphorylation were detected by Western blotting to investigate the effect of nsp1 and nsp1-mutant on these proteins.The result demonstrated that nsp1 decreased STAT1 phosphorylation in human embryonic kidney cell line (HEK)-293T cells stimulated by human IFN-β and LLC-PK1 cells induced by Sendai virus (SeV).The nsp1-mutant did not show a similar function (Fig. 3, A-C).Nevertheless, nsp1 did not block STAT2 phosphorylation.Nsp1 expression did not influence STAT1 and STAT2 endogenous expression, suggesting that nsp1 specifically inhibited STAT1 phosphorylation (Fig. 3, A and C).Furthermore, nsp1 induced IRF9 degradation (data not shown).These results indicated that STAT1 and IRF9 are the primary targets of nsp1-mediated inhibition of the IFN response.
Nsp1 degraded JAK1
After observing the nsp1-induced inhibition of STAT1 phosphorylation, the investigation was subsequently focused on the endogenous expression and phosphorylation of JAK1 and TYK2, which are crucial kinases for inducing STAT1 activation.Nsp1 efficiently induced JAK1 degradation in HEK-293 cells and LLC-PK1 cells, whereas the nsp1-mutant did not induce the JAK1 degradation (Fig. 4, A-D).Contrastingly, endogenous TYK2 expression and phosphorylation were not inhibited in the nsp1-expressing cells (Fig. 4, A and B).The results demonstrated that nsp1 might degrade JAK1 to inhibit STAT1 phosphorylation, thereby blocking the JAK-STAT signaling pathway.
Nsp1 degraded JAK1 via the proteasome pathway
The pathway of JAK1 degradation by nsp1 was analyzed to investigate the mechanism through which nsp1 downregulated JAK1 expression.Specifically, HEK-293T cells were treated with Dulbecco's modified Eagle's medium (DMEM, the negative control), 10 μM MG132 (a proteasome pathway inhibitor), 5 μM Z-VAD-FMK (an apoptosis pathway inhibitor), or 10 μg/ ml NH 4 Cl (an autophagy pathway inhibitor).Nsp1 persistently induced the degradation of endogenous JAK1 in all treated cells (Fig. 5, A-C).However, this degradation was effectively attenuated in cells treated with 5 μM MG132, suggesting that MG132 is an effective antagonist against nsp1-induced JAK1 degradation (Fig. 5D).These results indicated that nsp1 degraded JAK1 through the proteasome pathway.
Subsequently, it was detected that nsp1 inhibited the STAT1 phosphorylation level in MG132-treated cells.Compared to the negative control, nsp1 did not induce JAK1 degradation and inhibit STAT1 phosphorylation (Fig. 5E).Overall, the results indicated that nsp1 inhibited STAT1 phosphorylation by inducing JAK1 degradation via the proteasome pathway.
stimulated by human IFN-β to detect whether nsp1 inhibited STAT1 acetylation and dephosphorylation.The cells were collected to detect STAT1 acetylation and the interaction between STAT1 and TCPTP.Nsp1 significantly inhibited STAT1 acetylation (Fig. 8A).Based on previous findings on nsp1-induced CBP degradation, it was concluded that nsp1 inhibited STAT1 acetylation by degrading CBP.Furthermore, nsp1 did not affect TCPTP expression.However, it slightly inhibited the interaction between STAT1 and TCPTP, suggesting that nsp1 blocked STAT1 dephosphorylation by inhibiting STAT1 acetylation (Fig. 8A).To verify these results, the cells were treated with MG132 to eliminate the nsp1 function in degrading CBP, and the STAT1 acetylation and dephosphorylation were detected.The nsp1 inhibition of STAT1 acetylation and dephosphorylation was significantly attenuated when CBP was no longer degraded (Fig. 8B).A and B, HEK-293T cells were plated onto 6-well plates and transfected with pCAGGS-3×Flag-nsp1.Then, the cells were incubated with DMEM or MG132 (5 μM) for 6 h.The cells were collected and incubated with STAT1-tagged beads.The interaction between STAT1 and TCPTP/Ace was detected using Western blotting.C, HEK-293T cells were plated onto 6-well plates and transfected with pCAGGS-3×Flag-nsp1-mutant. The cells were incubated with DMEM for 6 h.Then, the cells were collected and incubated with STAT1tagged beads.The interaction between STAT1 and TCPTP/Ace was detected using Western blotting.D, schematic presentation of the STAT1 phosphorylation-acetylation-dephosphorylation cycle created using BioRender.com.E and F, HEK-293T cells were plated in 60-mm glass-bottom dishes and transfected with pCAGGS-3×Flag-nsp1 (5000 ng per dish).The cells were collected after 24 or 36 h.Nuclear and cytoplasmic proteins were extracted using a protein extraction kit.Then, the CBP and STAT1 expression levels and STAT1 phosphorylation level were detected by Western blotting.CBP, CREB-binding protein; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney cell line; IFN-β, interferon-β; JAK1, Janus kinase 1; nsp1, nonstructure protein 1; STAT, signal transducer and activator of transcription; TCPTP, T cell protein tyrosine phosphatase; TYK2, tyrosine kinase 2.
Nsp1 inhibition of STAT1 acetylation might block STAT1 export from the nucleus to the cytoplasm.HEK-293T cells were transfected with pCAGGS-3×Flag-nsp1 to verify this finding.At 24 h or 36 h post-transfection, the cells underwent nucleocytoplasmic fractionation.Nsp1 significantly degraded CBP in both the 24-h and 36-h groups (Fig. 8, D and E).In the 24-h group, nsp1 increased STAT1 content in the cytoplasm and decreased p-STAT1 content in the nucleus by inhibiting STAT1 phosphorylation.In the 36-h group, STAT1 content in the cytoplasm was decreased, and p-STAT1 content in the nucleus was increased, which was due to nsp1 blocking STAT1 acetylation.These results suggested that nsp1 blocked STAT1 nuclear translocation by inhibiting STAT1 phosphorylation and prevented STAT1 translocation out of the nucleus by inhibiting STAT1 acetylation.
Discussion
Host cells adopt multiple strategies to defend against coronaviruses, including the IFN response.Simultaneously, coronaviruses have developed various methods to evade the host's innate immune response by blocking the IFN response.Increasing evidence indicates that coronavirus nonstructural proteins are important in suppressing the IFN response by targeting the JAK-STAT signaling pathway.For example, porcine deltacoronavirus nsp5 cleaves STAT2 to block ISGF3 formation (38).SARS-CoV-2 nsp13 and ORF6 block STAT1 nuclear translocation to inhibit ISG transcription (39).However, the mechanism of SADS-CoV nonstructural proteins in inhibiting the IFN response remains unknown.In this study, SADS-CoV nonstructural proteins that inhibit the IFN response were identified.The results demonstrated that SADS-CoV nsp1 was the most potent antagonistic influence on the IFN response by blocking the JAK-STAT signaling pathway.Additionally, the study yielded novel findings that demonstrated the mechanisms of SADS-CoV nsp1 in IFN response inhibition, which provided valuable insights into SADS-CoV evasion of the host's innate immune response.
It is generally believed that coronavirus nsp1 induces host mRNA degradation (40)(41)(42).Coronavirus nsp1 also inhibits host protein translation by interacting with the 40S ribosome subunit, ultimately inhibiting the IFN system (41,42).However, accumulating evidence suggests that coronavirus nsp1 also inhibits the IFN system, which is separate from its ability to inhibit host protein translation and mRNA degradation (43)(44)(45)(46)(47)(48)(49)(50).In the present study, mutation of SADS-CoV nsp1 critical amino acids (Phe39 and Lys98) significantly diminished its ability to inhibit the IFN response.Consequently, the nsp1 mutations reduced the replicative capacity of SADS-CoV during the early stages of viral infection.Further research demonstrated that SADS-CoV nsp1 significantly inhibited ISRE promoter activity and ISG mRNA levels, suggesting that SADS-CoV nsp1 was a potent antagonist in blocking the IFN response induced by antiviral molecules (STAT1, STAT2, and IRF9).These results indicated that SADS-CoV nsp1 antagonized the IFN response by targeting the ISGF3 complex, which acts as an important virulence factor for SADS-CoV.
IFN signal transduction relies on the normal phosphorylation and nuclear translocation of STAT1 (50,51).STAT1 can form homodimers that mediate a noncanonical JAK-STAT signaling pathway (STAT1/STAT1-driven), while STAT2 does not have a similar function (52).These studies indicated that STAT1 is dominant in the JAK-STAT signaling pathway.Thus, coronavirus nsp1 inhibition of STAT1 phosphorylation instead of STAT2 was more effective in suppressing the IFN response.Although STAT1 is considered an important target for coronavirus nsp1, it remains unclear how SADS-CoV nsp1 inhibits STAT1 phosphorylation.In this study, it was determined that SADS-CoV nsp1 induced JAK1 degradation to inhibit STAT1 phosphorylation.Further investigation demonstrated that nsp1 promoted K11-and K48-linked JAK1 polyubiquitination, resulting in JAK1 degradation through the Ub-proteasome pathway.
Prolonged activation of the IFN response can damage the host's innate immune system (53).The balance between STAT1 phosphorylation and dephosphorylation is important for the IFN response (54).STAT1 activation is rapid but transient to maintain the normal IFN response.JAK1 induces STAT1 activation, while CBP and TCPTP negatively regulate STAT1 activity by promoting STAT1 acetylation and dephosphorylation (27,28).Multiple coronavirus proteins block the IFN response by inhibiting STAT1 phosphorylation and nuclear translocation.However, it has not been determined that coronavirus proteins inhibit the IFN response by targeting STAT1 acetylation and phosphorylation.In the present study, SADS-CoV nsp1 inhibited the CBP-mediated SADS-CoV nsp1 blocks the JAK-STAT signaling pathway STAT1 acetylation by inducing CBP degradation, then blocked the interaction between TCPTP and STAT1 to suppress STAT1 dephosphorylation.This effect inhibited STAT1 transport to the cytoplasm and prevented STAT1 from receiving antiviral signaling through JAK1 and TYK2, delaying the IFN response.Although sustaining nuclear STAT1 levels is critical for the IFN response, the CBP degradation induced by SADS-CoV nsp1 inhibited STAT1 formation of the STAT1-CBP complex and prevented ISG production.For SADS-CoV, it would be a more efficient and powerful immune evasion strategy that inhibits the upstream process (STAT1 phosphorylation) and downstream process (STAT1 acetylation and dephosphorylation) of the JAK-STAT signaling pathway.
Much research has demonstrated that regulating ubiquitination is critical in innate immunity.However, many viral proteins manipulate the UPS to inhibit the IFN system.Influenza A virus (IAV) NS1 inhibits RIG-I K63-linked ubiquitination by interacting with TRIM25 (55).Rotavirus nsp1 degrades the host antiviral proteins by hijacking the cullin-RING E3 Ub ligases, which inhibits the IFN system (56).Coronaviruses also have evolved mechanisms of manipulating the UPS to degrade immune-related proteins (57,58).This immune evasion strategy can provide more time for coronaviruses to replicate, thereby increasing their survival and spreading ability (58).The present study demonstrated that SADS-CoV nsp1 induced JAK1 and CBP degradation through the Ub-proteasome pathway, suggesting that nsp1 blocked the host IFN system by hijacking the UPS (Fig. 9).Nevertheless, the specific mechanisms underlying this interesting finding remain unclear.It is believed that there are two potential mechanisms: SADS-CoV nsp1 might interact with E3 Ub ligase to manipulate UPS (as influenza A virus NS1 does) or directly act as an E3 Ub ligase to induce the ubiquitination of target proteins (as rotavirus nsp1 does).Resolving this question would improve understanding of the molecular mechanisms of SADS-CoV infection.
Overall, this study indicated that SADS-CoV nsp1 inhibited the phosphorylation of STAT1 by inducing JAK1 degradation through K11/K48-linked JAK1 polyubiquitination.Furthermore, nsp1 blocked p-STAT1 acetylation and dephosphorylation by inducing CBP degradation.These findings increase our knowledge of SADS-CoV immune evasion strategies and enhance comprehension of the mechanism implicated in the coronavirus nsp1's capacity to inhibit the IFN response.
Virus growth kinetics
Vero cells and ST cells were cultured in 12-well plates.The cells were infected with SADS-CoV-WT or SADS-CoVmutant at a multiplicity of infection of 0.1.The medium was changed after 2 hpi.Then, the infected cell supernatants were collected at 6, 8, 12, 16, 24, and 32 hpi and titrated on Vero cells.The virus titers were determined by calculating the log 10 TCID 50 (median tissue culture infective dose)/ml using the Reed-Muench method.
Immunofluorescence assay
Vero cells were infected with SADS-CoV-WT or SADS-CoV-mutant at a multiplicity of infection of 0.1 for 12 h.The cells were washed three times using phosphate-buffered saline, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 2% bovine serum albumin.The cells were incubated with primary antibody overnight at 4 C, then incubated with secondary antibody for 2 h at 37 C.The nucleus was stained with DAPI.The percentage of the number of infected cells (green) to the total number of cells (blue) was determined using ImageJ.The statistical experiments were repeated three times.
Western blotting
HEK-293T cells and LLC-PK1 cells were transfected with pCAGGS-3×Flag-nsp1.After 24 h, the HEK-293T cells were incubated with human IFN-β for 4 h, and the LLC-PK1 cells were stimulated by SeV.Then, the cells were collected using a lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, and 0.5% sodium deoxycholate.Subsequently, the samples were treated with phosphorylation inhibitors for 30 min.The samples were mixed with loading buffer containing β-mercaptoethanol and heated at 100 C for 10 min.The samples were separated by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to PVDF membranes for immunoblotting.The SADS-CoV nsp1 blocks the JAK-STAT signaling pathway membranes were examined with the primary antibodies at 4 C overnight and then exposed to the secondary antibodies for 1 h at room temperature.The bands were visualized using the Tanon imaging system and examined with ImageJ.
Co-immunoprecipitation assay
HEK-293T cells were transfected with pCAGGS-2×HA-Ub or a plasmid panel expressing Ub mutants.After 12 h, the cells were transfected with pCAGGS-3×Flag-nsp1.The cells were collected using IP buffer [containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% TritonX-100] for 30 min.The cell lysates were incubated overnight at 4 C with anti-HA beads.After three washes, the beads were supplemented with the SDS loading buffer and incubated at 95 C for 5 min.The protein expression levels were analyzed using Western blotting.
Statistical analysis
Statistical analyses were conducted using Student's t test to analyze two groups of data.All differences were considered statistically significant when p < 0.05, p < 0.01, and p < 0.001.
Figure 1 .
Figure 1.Nsp1 was critical for virus replication.A, schematic diagram depicting the key amino acid sites in SADS-CoV nsp1.The alignment of nsp1 amino acids was conducted using the websites https://espript.ibcp.frand https://www.genome.jp.B and C, LLC-PK1 cells were infected with SADS-CoV-WT or SADS-CoV-mutant at an MOI of 0.1.Subsequently, the cells were collected to detect the IFN-β and ISG15 mRNA levels using RT-qPCR.The data are the means ± SD.The p-value was calculated using the t test.*p < 0.05.D and E, Vero cells and ST cells were infected with SADS-CoV-WT or SADS-CoV-mutant at an MOI of 0.1.The infected cell supernatants were collected and titrated on Vero cells.The virus titers were determined by calculating the log 10 TCID 50 /ml.F, Vero cells were infected with SADS-CoV-WT or SADS-CoV-mutant at an MOI of 0.1 for 12 and 24 h.After 12 or 24 hpi, the cells were fixed, permeabilized, and blocked.The cells were then stained with SADS-CoV N mouse-pAb, followed by Alexa Fluor 488-conjugated goat anti-mouse staining (green).The nuclei were stained with DAPI (blue).Scale bar represents 200 μm.IFN-β, interferon-β; ISG, interferon-stimulated gene; MOI, multiplicity of infection; nsp1, nonstructure protein 1; SADS-CoV, swine acute diarrhea syndrome coronavirus.
Figure 3 .
Figure 3. Nsp1 inhibited STAT1 phosphorylation.A and B, HEK-293T cells and LLC-PK1 cells were transfected with pCAGGS-3×Flag-nsp1.After 24 h, HEK-293T cells were incubated with human IFN-β for 4 h, and LLC-PK1 cells were stimulated by SeV for 8 h.STAT1, p-STAT1, STAT2, and p-STAT2 expression was detected by Western blotting.All protein levels were analyzed using ImageJ.Western blotting assay was repeated in two independent experiments.C, HEK-293T cells were transfected with pCAGGS-3×Flag-nsp1-mutant. After 24 h, HEK-293T cells were incubated with human IFN-β for 4 h.STAT1 and p-STAT1 expression was detected by Western blotting.All protein levels were analyzed using ImageJ.Western blotting assay was repeated in two independent experiments.The data are the means ± SD.The p-value was calculated using the t test.***p < 0.001.HEK, human embryonic kidney cell line; IFN-β, interferon-β; nsp1, nonstructure protein 1; SeV, Sendai virus; STAT, signal transducer and activator of transcription.
Figure 8 .
Figure 8. Nsp1 inhibited STAT1 acetylation and dephosphorylation.A and B, HEK-293T cells were plated onto 6-well plates and transfected with pCAGGS-3×Flag-nsp1.Then, the cells were incubated with DMEM or MG132 (5 μM) for 6 h.The cells were collected and incubated with STAT1-tagged beads.The interaction between STAT1 and TCPTP/Ace was detected using Western blotting.C, HEK-293T cells were plated onto 6-well plates and transfected with pCAGGS-3×Flag-nsp1-mutant. The cells were incubated with DMEM for 6 h.Then, the cells were collected and incubated with STAT1tagged beads.The interaction between STAT1 and TCPTP/Ace was detected using Western blotting.D, schematic presentation of the STAT1 phosphorylation-acetylation-dephosphorylation cycle created using BioRender.com.E and F, HEK-293T cells were plated in 60-mm glass-bottom dishes and transfected with pCAGGS-3×Flag-nsp1 (5000 ng per dish).The cells were collected after 24 or 36 h.Nuclear and cytoplasmic proteins were extracted using a protein extraction kit.Then, the CBP and STAT1 expression levels and STAT1 phosphorylation level were detected by Western blotting.CBP, CREB-binding protein; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney cell line; IFN-β, interferon-β; JAK1, Janus kinase 1; nsp1, nonstructure protein 1; STAT, signal transducer and activator of transcription; TCPTP, T cell protein tyrosine phosphatase; TYK2, tyrosine kinase 2. | 2024-02-23T16:04:16.379Z | 2024-02-01T00:00:00.000 | {
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3775294 | pes2o/s2orc | v3-fos-license | Hepatic oxidative stress in an animal model of sleep apnoea: effects of different duration of exposure
Background Repeated apnoea events cause intermittent hypoxia (IH), which alters the function of various systems and produces free radicals and oxidative stress. Methods We investigated hepatic oxidative stress in adult mice subjected to intermittent hypoxia, simulating sleep apnoea. Three groups were submitted to 21 days of IH (IH-21), 35 days of IH (IH-35), or 35 days of sham IH. We assessed the oxidative damage to lipids by TBARS and to DNA by comet assay; hepatic tissue inflammation was assessed in HE-stained slides. Antioxidants were gauged by catalase, superoxide dismutase, glutathione peroxidase activity and by total glutathione. Results After IH-21, no significant change was observed in hepatic oxidative stress. After IH-35, significant oxidative stress, lipid peroxidation, DNA damage and reduction of endogenous antioxidants were detected. Conclusions In an animal model of sleep apnoea, intermittent hypoxia causes liver damage due to oxidative stress after 35 days, but not after 21 days.
Background
In obstructive sleep apnoea (OSA), pharyngeal occlusion occurs, typically for 10 to 40 seconds, causing a decrease of PaO 2 and an increase in PaCO 2 , ending with an arousal [1]. Intermittent hypoxia due to OSA causes oxidative stress, a recognized mechanism in the nonalcoholic fatty liver disease (NAFLD), which may progress to nonalcoholic steatohepatitis (NASH) [2].
The formation of ROS in OSA is similar to what occurs in ischemia-reperfusion [18]. Oxidative stress leads to inflammation, recognised as a mechanism of the pathophysiology of OSA [19]. Excessive formation of ROS leads to lipid peroxidation in cell membranes, protein oxidation and DNA damage [20][21][22]. Several ROS are formed in hepatocytes through the activation of Kupffer cells and inflammatory cells [23].
Another group has exposed mice to IH and to a highcholesterol diet for 6 months, revealing the involvement of OSA in non-alcoholic steatohepatitis (NASH) [3]. IH aggravates paracetamol-induced liver damage after 21 days [24]. To understand the mechanisms leading to NAFLD and NASH it may relevant to identify the time frame in which these phenomena occur. There are, however, no studies specifically investigating the duration of IH exposure that causes liver damage in an animal model of sleep apnoea. This knowledge will be relevant to help design future studies.
The aim of the present study was to establish the duration of exposure to intermittent hypoxia necessary and sufficient to trigger liver damage and oxidative stress in mice.
Methods
The experimental procedures complied with the rules established by the "Research in Health and Animal Rights" according to the Commission of Research and Ethics in Health of the Research and Postgraduate Group of the Hospital de Clínicas de Porto Alegre.
Thirty-six male CF-1 mice (8-11 weeks old) from Fundação Estadual de Produção e Pesquisa (FEPPS) were employed. They were kept at the Animal Experimentation Unit of the Research Center of the Hospital de Clínicas of Porto Alegre in plastic boxes measuring 30 × 19 × 13 cm lined with wood chips, in a 12-hour dark/ light cycle (light from 7 a.m. to 7 p.m.) at a temperature of 22 4°C. The mice were given food (Purina-Nutripal, Porto Alegre, RS, Brazil) and water ad libitum.
The animals were randomly divided into three groupings (n = 12): group SIH, sham intermittent hypoxia, which underwent the simulated procedure; group IH-21, exposed to hypoxia for 21 days; and group IH-35, exposed hypoxia for 35 days.
IH procedures were described in detail before [25]. In brief, during five weeks, 7 days per week, 8 hours a day, from 9 a.m. to 5 p.m., in the lights-on period, the rodents were placed in the cages (Figure 1). A mixture with 90% nitrogen and 10% CO 2 was released in the hypoxia chamber, for 30 seconds. The gas mixture reduced the oxygen fraction from 21% to approximately 8% and the CO 2 fraction to 6%. Subsequently, a fan insufflated room air in the chamber for 30 seconds, restoring the oxygen fraction to 21%. Each hypoxia/normoxia cycle lasted for 60 seconds; in 8 hours, 480 IH periods occurred, equivalent to an apnea index of 60 per hour.
The SIH group was housed in an adjacent cage and underwent the same fan activity as the IH group, but no gas was introduced in the cage during the hypoxia cycle ( Figure 1).
On the 21st or 35th day, the animals were killed. They were first anaesthetised with ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (50 mg/kg ip). Blood was collected from the retro-orbital vein with the aid of a heparinised glass capillary [26] to complete the hepatic integrity (AST, ALT and ALP) test and comet assay. We removed the liver of animals for histological analysis; the rest were frozen -80°C for later biochemical analysis. The animals were euthanized by exsanguination under deep anaesthesia [27,28].
Nine millilitres of phosphate buffer (140 mM KCL, 20 mM phosphate, pH 7.4) per tissue gram was added, and tissue was homogenised in an Ultra Turrax at 4°C. Next, it was centrifuged for 10 minutes at 4,000 rpm (2150.4 g). The samples were stored again at -80°C for posterior analyses.
We used the Bradford method to quantify protein, with bovine albumin as the standard (Sigma ® ). The samples were measured spectrophotometrically at 595 nm, and values expressed in mg/g liver [29] were used to calculate values of TBARS (thiobarbituric acid-reactive substances) and antioxidant enzymes.
The amount of aldehydes generated by lipid peroxidation is measured by the TBARS method, which measures the amount of substances reacting with thiobarbituric acid. The samples were incubated at 100°C for 30 minutes after addition of 0.37% thiobarbituric acid in 15% trichloroacetic acid and centrifuged at 3000 rpm (1612.8 g) for 10 minutes at 4°C. Absorbance was determined spectrophotometrically at 535 nm [30].
The analysis of SOD is based on the inhibition of the reaction of the superoxide radical with adrenaline [31]. The auto-oxidation rate of epinephrine, which is progressively inhibited by increasing amounts of SOD in the homogenate, was monitored spectrophotometrically at 480 nm. The amount of enzyme that inhibited 50% of epinephrine auto-oxidation was defined as 1 U of SOD activity.
The analysis of CAT activity is based on measuring the decrease in hydrogen peroxide [32]. Catalase activity was determined by measuring the decrease in absorption at 240 nm in a reaction medium containing 50 mM phosphate buffer saline (pH 7.2) and 0.3 M hydrogen peroxide. The enzyme activity was assayed spectrophotometrically at 240 nm.
The activity of GPx is based on the consumption of NADPH in the reduction of oxidised glutathione [33]. The glutathione peroxidase activity was determined by the oxidation rate of NADPH in the presence of reduced glutathione and glutathione reductase. Sodium azide was added to inhibit catalase activity. The GPx activity was measured with a spectrophotometer at 340 nm.
Total glutathione (GSH), a water soluble non-enzymatic antioxidant, [34] was measured as described previously [35], in a reaction medium consisting of a solution of 300 mM phosphate buffer (Na2HPO4·1H2O) and a solution of dithionitrobenzoic acid (DTNB). The reaction products were read at 412 nm.
The alkaline comet assay was carried out as described in [36], with minor modifications [37]. The liver tissue samples (200-250 mg) were placed in 0.5 mL of cold phosphate-buffered saline (PBS) and finely minced in order to obtain a cell suspension; the blood samples (50 μL) were placed in 5 μL of anti-coagulant (heparin sodium 25.000 UI-Liquemine ® ). Liver and blood cell suspensions (5 μL) were embedded in 95 μL of 0.75% low melting point agarose (Gilco BRL) and spread on agarose-precoated microated microscope slides. After solidification, slides were placed in lysis buffer (2.5 M NaCl, 100 mM EDTA an 10 mM Tris, pH 10.0), with freshly added 1% Triton X-100 (Sigma) and 10% DMSO for 48 h at 4°C. The slides were subsequently incubated in freshly prepared alkaline buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 20 min, at 4°C. An electric current of 300 mA and 25 V (0.90 V/cm) was applied for 15 min to perform DNA electrophoresis. The slides were then neutralized (0.4 M Tris, pH 7.5), stained with silver and analyzed using microscope. Images of 100 randomly select cells (50 cells from each of two replicate slides) were analyzed from each animal. Cells were also visually scored according to tail size into five classes ranging from undamaged (0) to maximally damage (4), resulting in a single DNA damage score to each animal, and consequently to each studied group. Therefore, the damage index (DI) can range from 0 (completely undamaged, 100 cells × 0) to 400 (with maximum damage, 100 × 4). Damage frequency (%) was calculated based on the number of tailed versus tailless cells.
The levels of nitrates and nitrites were measured by the reaction of the samples with Griess reagent. Aliquots of 50 μL were incubated with enzyme cofactors and nitrate reductase for 30 minutes at room temperature for the conversion of nitrate to nitrite. The nitrite formed was then analysed by reaction with the Griess reagent, forming a coloured compound that was measured by spectrophotometer at a wavelength of 540 nm [38].
For histological evaluation, part of the liver was preserved in 10% formalin for 24 hours, embedded in paraffin, and cut into 6-μm thick sections with a microtome. Sections were stained with hematoxylin and eosin.
The results are expressed as mean ± standard error. We used ANOVA and the Student-Newmann-Keuls or Student's t-test for comparing groups. The significance level was 5% (p < 0.05).
Results
The circulating levels of the liver enzymes aspartate aminotransferase (AST), alanine amino transferase (ALT), and alkaline phosphatase (ALP), parameters of liver damage, showed no significant difference between the IH-21 group and the SIH. The IH-35 group showed significantly increased levels (p < 0.05) compared to the sham intermittent hypoxia group (Table 1).
Lipid peroxidation measured by the TBARS technique showed no oxidative damage in group IH-21 compared to SIH. However, there was significant damage in the lipid peroxidation in liver subjected to hypoxia for 35 days (Figure 2). Evaluation of the antioxidant enzymes showed a significant decrease in the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in liver tissue with intermittent hypoxia for 35 days ( Table 2). The quantification of total endogenous glutathione in the liver showed a significant decrease in the 35-day hypoxia group compared with the sham intermittent hypoxia (Figure 3). These results demonstrate that IH induced a decrease in the endogenous antioxidant defence. The assessment of DNA damage by the comet assay showed that the damage in blood did not differ between groups, but the liver tissue exhibited a significant increase in DNA damage in group IH-35 compared with SIH (Table 3).
In the assessment of metabolites of nitric oxide in liver tissue of mice subjected to IH for 35 days, we noted a significant increase in NO in these animals compared with SIH (Table 4).
Several histological liver changes were also observed in animals of the IH-35 group -ballooning, steatosis, necrosis and the presence of neutrophils -when compared with mice under sham intermittent hypoxia (Figures 4 and 5).
Discussion
We report for the first time that 35 but not 21 days of exposure to IH, simulating an OSA of 60 events per hour, reducing for 6% the concentration of oxygen, causes hepatic damage. This is also the first report to combine the description of enzyme, lipid, DNA, oxidative, and nitrosative hepatic damage. We used an experimental model that produces levels of hypoxia comparable to those observed in patients with severe OSA [24,39]. Although our findings cannot be immediately translated to the clinical setting, they are in agreement with the literature indicating an OSA-NASH association [40,41].
Two mechanisms are proposed for the morbidity caused by OSA: the activation of inflammatory factors and oxidative stress [42,43], which also can be modulated by genetic, lifestyle and environmental factors [43,44]. Oxidative stress plays an important role in various diseases as well as in OSA, which causes an effect similar to ischemia-reperfusion [18] in which there is activation of xanthine oxidase, leading to the formation free radicals and further imbalance between oxidants and antioxidants [4][5][6].
The analysis of liver integrity showed that the liver tissue of mice subjected to intermittent hypoxia was damaged, but only after 35 days, as demonstrated by the significant increase in circulating AST, ALT and alkaline phosphatase. The present results demonstrate damage both at cytoplasmic and mitochondrial level, confirmed by the presence in the histological examination of ballooning, steatosis, necrosis and the presence of neutrophils in the liver, similar to what is observed in NASH [45].
In the evaluation of hepatic lipid peroxidation, we observed a significant increase in lipid oxidative damage in animals that were subjected to hypoxia for 35 days, as indicated by the TBARS test, but not in group IH-21. This damage can be caused by the increase of free radicals in the liver tissue. Similar data have been reported in other studies of intermittent hypoxia [46][47][48] and by our laboratory in other experimental models of hepatic oxidative damage [49][50][51][52][53][54].
As we did not observe liver damage in animals exposed to IH for 21 days, by the liver enzyme, histological, or lipid peroxidation assays, we concluded that this duration of IH causes no damage to the organ. Therefore, dosages of antioxidant enzymes, comet assay and nitrites metabolites were not conducted in the IH 21 group.
Comet assay in liver tissue revealed a significant increase in DNA damage in the IH-35 group in comparison to the SIH group. No evidence of damage was observed in blood tissue. The rate of DNA damage detected by the comet assay depends on the tissue or organ analyzed [55]. Here, the DNA damage was observed only in the tissue most susceptible to lesions produced by IH. In the alkaline version used, the comet assay detects a broad spectrum of DNA lesions, including single strand breaks [56,57].
Previous comet assay and TBARS data have demonstrated increased formation of free radicals in sleep apnoea patients [11]. Possibly, the formation of superoxide radical (O 2 -• ) and hydrogen peroxide (H 2 O 2 ), which appear to be increased in individuals with OSA, is due to the conversion of xanthine dehydrogenase (type D) into its oxidase (type O) form in hypoxia, followed by the activation of the oxidase form during reoxygenation (normoxia) by the hypoxanthine formed during hypoxia. This xanthine oxidase activity generates O 2 -• , H 2 O 2 , and uric acid [4,11].
Our evaluation of the endogenous antioxidant liver enzymes SOD, GPx and CAT showed that their activities were significantly decreased in mice after 35 days under intermittent hypoxia. Quantification of total glutathione revealed significant decreases in the group exposed to intermittent hypoxia compared to SIH, demonstrating a reduced hepatic antioxidant defence in these animals.
The increase in TBARS and decrease in endogenous antioxidants observed in the present study further promotes oxidative stress, contributing to aggravation of the liver tissue injury. This kind of pathological synergy is evidenced in experimental models of liver damage induced by xenobiotic agents that cause oxidative stress such as carbon tetrachloride and toluene [49,50,52,54,58], by surgical procedures such as ligation of the common bile duct [51,53] or by thymoquinone [59].
The increased nitric oxide metabolites nitrite and nitrate in the livers of IH-35 mice confirms findings by other authors, who demonstrated a significant increase of nitric oxide in animals exposed to IH simulating OSA (6 min/6 min) during 120 days [48], and to hypobaric hypoxia during 32 days [60]. The increase of NO, along with increased free radicals, may generate nitrosative stress caused by the reaction products of these two substances, such as peroxide nitrite (OONO • ) formed by the reaction between NO and O 2 -• [11]. Much evidence indicates that oxidative and nitrosative stress have important roles in the complication of hypoxia [61]. OSA is usually accompanied by arterial hypertension, pulmonary hypertension, myocardial infarction and stroke, which may be due to changes in nitric oxide production [62]. Veasey et al. had demonstrated irreversible basal forebrain nitrosative damage as a possible cause for residual sleepiness in OSA [63].
It is increasingly clear that IH is capable of causing liver tissue damage. This was here demonstrated by several lines of evidence: elevated circulating levels of liver enzymes, NO increase, damage to lipids and DNA, and reduced endogenous antioxidant defences. Further translational research is necessary to completely correlate these findings with the NASH pathology.
Conclusions
The present results suggest that a model of intermittent hypoxia for 35 days, simulating sleep apnoea, is useful to investigate liver injury by oxidative and nitrosative stress. Exposure to intermittent hypoxia during 21 days may be insufficient to produce hepatic damage. | 2014-10-01T00:00:00.000Z | 2011-07-05T00:00:00.000 | {
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257796587 | pes2o/s2orc | v3-fos-license | Zinc/Cerium-Substituted Magnetite Nanoparticles for Biomedical Applications
Numerous studies have reported the possibility of enhancing the properties of materials by incorporating foreign elements within their crystal lattice. In this context, while magnetite has widely known properties that have been used for various biomedical applications, the introduction of other metals within its structure could prospectively enhance its effectiveness. Specifically, zinc and cerium have demonstrated their biomedical potential through significant antioxidant, anticancer, and antimicrobial features. Therefore, the aim of the present study was to develop a series of zinc and/or cerium-substituted magnetite nanoparticles that could further be used in the medical sector. The nanostructures were synthesized through the co-precipitation method and their morpho-structural characteristics were evaluated through X-ray diffraction (XRD), inductively coupled plasma mass spectrometry (ICP-MS), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), zeta potential, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) analyses. Furthermore, the nanostructures were subjected to a ROS-Glo H2O2 assay for assessing their antioxidant potential, MTT assay for determining their anticancer effects, and antimicrobial testing against S. aureus, P. aeruginosa, and C. albicans strains. Results have proven promising for future biomedical applications, as the nanostructures inhibit oxidative stress in normal cells, with between two- and three-fold reduction and cell proliferation in tumor cells; a two-fold decrease in cell viability and microbial growth; an inhibition zone diameter of 4–6 mm and minimum inhibitory concentration (MIC) of 1–2 mg/mL.
Introduction
Iron oxides are a group of naturally occurring materials with immense potential within the biomedical area owing to the iron component, which is an essential element within the organism [1]. Among them, magnetite nanoparticles are the first generation of clinically approved nanomaterials that have been intensively used for their superparamagnetic properties in novel and efficient biomedical applications [1][2][3][4][5][6][7][8][9]. Besides the possibility to adsorb and immobilize large quantities of bioactive compounds [1], magnetite nanoparticles are widely known for their intrinsic antioxidant [10], antimicrobial [11], and anticancer [12] properties, which can be potentiated through hyperthermia effects [11].
Results
The present study aimed to obtain a series of zinc-and/or cerium-substituted magnetite nanoparticles for biomedical applications. The results of the morpho-structural characterization and biological evaluations are described in the following paragraphs.
X-ray Diffraction (XRD) Coupled with Rietveld Refinement
The XRD analysis coupled with Rietveld refinement was employed to qualitatively and quantitatively determine the mineral phases present within the obtained samples ( Figure 1; Table 1). The diffractograms reveal the formation of magnetite in the Fd-3m cubic crystal system as the single mineral phase within samples Fe 3 O 4 , Zn 0.1 Fe 2.9 O 4 , and Ce 0.1 Fe 2.9 O 4 , (according to JCPDS 01-084-2782 [33,34]). By contrast, the increase in the substitution degree to 16.66% leads to the occurrence of secondary mineral phases, namely ZnO and Ce 2 O 3 in the hexagonal crystal system (according to JCPDS 00-005-0664 [35] and JCPDS 00-023-1048 [36], respectively). Additionally, higher substitution degrees lead to the shift of the Fe 3 O 4 main diffraction peak at lower values, especially for the Zn 0.5 Ce 0.5 Fe 2 O 4 sample, thus confirming the presence of zinc/cerium within the lattice structure of magnetite due to larger atomic radii of zinc and cerium [37][38][39].
cubic crystal system as the single mineral phase within samples Fe3O4, Zn0.1Fe2.9O4, and Ce0.1Fe2.9O4, (according to JCPDS 01-084-2782 [33,34]). By contrast, the increase in the substitution degree to 16.66% leads to the occurrence of secondary mineral phases, namely ZnO and Ce2O3 in the hexagonal crystal system (according to JCPDS 00-005-0664 [35] and JCPDS 00-023-1048 [36], respectively). Additionally, higher substitution degrees lead to the shift of the Fe3O4 main diffraction peak at lower values, especially for the Zn0.5Ce0.5Fe2O4 sample, thus confirming the presence of zinc/cerium within the lattice structure of magnetite due to larger atomic radii of zinc and cerium [37][38][39]. Furthermore, the Rietveld refinement demonstrated the formation of a significantly lower percentage of the secondary mineral phase as compared to the stoichiometrically calculated amount that would be obtained at a substitution rate of 0%. In this context, it could be assumed that the substitution yield is the highest for the co-substituted samples. Moreover, the substitution of the iron ions with zinc and/or cerium is also demonstrated through the dimensions of the unit cell parameters and the average crystallite size. Furthermore, the Rietveld refinement demonstrated the formation of a significantly lower percentage of the secondary mineral phase as compared to the stoichiometrically calculated amount that would be obtained at a substitution rate of 0%. In this context, it could be assumed that the substitution yield is the highest for the co-substituted samples. Moreover, the substitution of the iron ions with zinc and/or cerium is also demonstrated through the dimensions of the unit cell parameters and the average crystallite size. Specifically, when (co-)adding zinc, the size of the unit cell increases proportionally, but the crystallite size significantly decreases at the higher concentration; in the case of cerium, the effects are the opposite, most likely due to the formation of vacancies within the unit cell. In terms of the nanoparticle crystallinity, it seems that the inclusion of the zinc and cerium ions results in the deformation of the magnetite unit cell and consequently to a decrease in crystallinity. However, the Zn 0.5 Fe 2.5 O 4 sample seems to have a higher crystallinity than the Fe 3 O 4 sample, which could be attributed to the presence of a secondary zinc oxide phase that is highly crystalline [40].
Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
Subsequently, samples were subjected to ICP-MS analysis to quantify the contents of the iron, zinc, and cerium ions to further estimate the stoichiometry of the nanosystems ( Table 2). It can be observed that for all samples, the amount of zinc and cerium determined through ICP-MS are similar to those added in the synthesis step. However, the iron amount is lower, which could be caused by the formation of the secondary phases. In this context, considering both the mass% of the secondary phases formed and the amounts of each ion present, the stoichiometry of the nanosystems were estimated in Table 3. Additionally, for the samples obtained at lower substitution degrees, the 5 wt% limit of detection of the diffractometer was considered as the maximum %mass of the secondary phase. Generally, the estimated stoichiometries are similar to the theoretical ones, thus indicating the successful incorporation of the zinc and/or cerium ions within the structure of magnetite.
X-ray Photoelectron Spectroscopy (XPS)
The XPS analysis was used for assessing the Fe 2+ :Fe 3+ ratio in order to determine the amount of zinc and/or cerium incorporated within the unit cell of magnetite and their affinity to substitute either the Fe 2+ or the Fe 3+ ions ( Figure 2). The Fe 3 O 4 sample is characterized by a 1:2 ratio of the Fe 2+ :Fe 3+ ions, thus proving the formation of magnetite as the unique mineral phase. In the case of the Zn 0.5 Fe 2.5 O 4 sample, the Fe 2+ :Fe 3+ ratio is 1:1.7, which demonstrates a higher affinity of zinc to substitute the Fe 3+ ion. Since the 1:2 molar ratio is maintained within the Ce 0.5 Fe 2.5 O 4 sample, it could be assumed that the cerium substitution did not occur. However, there are many literature studies reporting several possibilities. First, Ce 3+ is oxidized to Ce 4+ through the reduction of Fe 3+ to Fe 2+ , followed by the replacement of an octahedral Fe 3+ ion and, subsequently, the reducing of another Fe 3+ ion to Fe 2+ to maintain charge neutrality [38]. However, this mechanism would increase the Fe 2+ content, which is not the case for this sample. Second, another possibility involves the same initial oxidation of Ce 3+ to Ce 4+ and the replacement of an Fe 2+ ion with the Ce 4+ ion; nonetheless, the 1:2 ratio would not be maintained. Third, Ce 3+ could replace the Fe 3+ ion, which would also increase the Fe 2+ amount. Considering the Zn 0.5 Ce 0.5 Fe 3 O 4 sample, the Fe 2+ :Fe 3+ ratio is 1:2.25. Since zinc tends to replace the Fe 3+ ions, it could be assumed that the increase in Fe 3+ is caused by the replacement of the Fe 2+ with Ce 4+ . In this context, it could further be concluded that in the case of the Ce 0.5 Fe 2.5 O 4 sample, there is a balance between the substitution of Fe 2+ and the oxidation-reduction reaction of Ce 3+ /Ce 4+ , which maintains the 1:2 ratio. affinity to substitute either the Fe 2+ or the Fe 3+ ions ( Figure 2). The Fe3O4 sample is characterized by a 1:2 ratio of the Fe 2+ :Fe 3+ ions, thus proving the formation of magnetite as the unique mineral phase. In the case of the Zn0.5Fe2.5O4 sample, the Fe 2+ :Fe 3+ ratio is 1:1.7, which demonstrates a higher affinity of zinc to substitute the Fe 3+ ion. Since the 1:2 molar ratio is maintained within the Ce0.5Fe2.5O4 sample, it could be assumed that the cerium substitution did not occur. However, there are many literature studies reporting several possibilities. First, Ce 3+ is oxidized to Ce 4+ through the reduction of Fe 3+ to Fe 2+ , followed by the replacement of an octahedral Fe 3+ ion and, subsequently, the reducing of another Fe 3+ ion to Fe 2+ to maintain charge neutrality [38]. However, this mechanism would increase the Fe 2+ content, which is not the case for this sample. Second, another possibility involves the same initial oxidation of Ce 3+ to Ce 4+ and the replacement of an Fe 2+ ion with the Ce 4+ ion; nonetheless, the 1:2 ratio would not be maintained. Third, Ce 3+ could replace the Fe 3+ ion, which would also increase the Fe 2+ amount. Considering the Zn0.5Ce0.5Fe3O4 sample, the Fe 2+ :Fe 3+ ratio is 1:2.25. Since zinc tends to replace the Fe 3+ ions, it could be assumed that the increase in Fe 3+ is caused by the replacement of the Fe 2+ with Ce 4+ . In this context, it could further be concluded that in the case of the Ce0.5Fe2.5O4 sample, there is a balance between the substitution of Fe 2+ and the oxidation-reduction reaction of Ce 3+ /Ce 4+ , which maintains the 1:2 ratio.
Dynamic Light Scattering (DLS) and Zeta Potential
The subsequent characterization step consisted of assessing the stability of zinc/cerium-substituted nanostructures in terms of the agglomeration tendency in the suspension through a hydrodynamic diameter correlated with polydispersity index (Figure 3a) and zeta potential (Figure 3b) measurements. Results demonstrate that lower mono-
Dynamic Light Scattering (DLS) and Zeta Potential
The subsequent characterization step consisted of assessing the stability of zinc/ceriumsubstituted nanostructures in terms of the agglomeration tendency in the suspension through a hydrodynamic diameter correlated with polydispersity index (Figure 3a) and zeta potential (Figure 3b) measurements. Results demonstrate that lower mono-substitution degrees increase the hydrodynamic diameter, with the highest value registered for the cerium-containing sample. Increasing the substitution degree leads to considerably reduced hydrodynamic diameters and more monodispersed size distributions [1,41]. However, the Ce 0.5 Zn 0.5 Fe 2 O 4 sample behaves oppositely, probably caused by the heterogenous substitution with the two types of ions. Nonetheless, the high error bar calculated for the Ce 0.1 Fe 2.9 O 4 and Ce 0.5 Zn 0.5 Fe 2 O 4 samples could be attributed to polydisperse nanoparticles due to a non-homogenous substitution. All polydispersity index values are in the range of 0.25-0.6, which are values generally accepted for nanoparticles [42]. Since the zeta potential reflects the overall nanoparticle surface charge, it is generally regarded as a reference for the stability of the nanoparticle dispersions, i.e., values between -25 and 25 mV indicate an increased tendency of the nanoparticles to form aggregates [11,[43][44][45][46]. Therefore, it can be observed that the Ce 0.5 Fe 2.5 O 4 sample is the most stable, with a zeta potential value of +25 mV, which confirms the previous observations regarding the higher stability of the cerium-containing samples. Additionally, samples obtained at lower substitution degrees are characterized by zeta potential values close to 0 mV, which further denotes an increased agglomeration tendency. substitution degrees increase the hydrodynamic diameter, with the highest value registered for the cerium-containing sample. Increasing the substitution degree leads to considerably reduced hydrodynamic diameters and more monodispersed size distributions [1,41]. However, the Ce0.5Zn0.5Fe2O4 sample behaves oppositely, probably caused by the heterogenous substitution with the two types of ions. Nonetheless, the high error bar calculated for the Ce0.1Fe2.9O4 and Ce0.5Zn0.5Fe2O4 samples could be attributed to polydisperse nanoparticles due to a non-homogenous substitution. All polydispersity index values are in the range of 0.25-0.6, which are values generally accepted for nanoparticles [42]. Since the zeta potential reflects the overall nanoparticle surface charge, it is generally regarded as a reference for the stability of the nanoparticle dispersions, i.e., values between -25 and 25 mV indicate an increased tendency of the nanoparticles to form aggregates [11,[43][44][45][46]. Therefore, it can be observed that the Ce0.5Fe2.5O4 sample is the most stable, with a zeta potential value of +25 mV, which confirms the previous observations regarding the higher stability of the cerium-containing samples. Additionally, samples obtained at lower substitution degrees are characterized by zeta potential values close to 0 mV, which further denotes an increased agglomeration tendency.
Scanning Electron Microscopy (SEM); Energy Dispersive X-ray Spectroscopy (EDX)
The morpho-structural properties of the nanostructured systems were investigated through SEM. Micrographs reveal an increased agglomeration tendency and a quasi-spherical morphology within all the obtained nanoparticles ( Figure 4). Furthermore, the obtained micrographs were utilized for assessing the size distribution of the nanoparticles using the ImageJ software (version 1.8.0) to measure 100 nanoparticles within each sample ( Figure 5). The histograms demonstrate monomodal size distributions for all types of nanoparticles, with average sizes increasing proportionally with the substitution degree. Furthermore, one can deduce that the addition of cerium within the nanostructured systems leads to the formation of larger nanoparticles than the zinc-substituted ones, with the largest nanoparticles being registered for the nanoparticles containing both types of ions. The EDX semi-quantitative measurements demonstrate a proportional increase in the zinc/cerium amount with the substitution degree and a decrease in the iron amount ( Figure 6).
Scanning Electron Microscopy (SEM); Energy Dispersive X-ray Spectroscopy (EDX)
The morpho-structural properties of the nanostructured systems were investigated through SEM. Micrographs reveal an increased agglomeration tendency and a quasispherical morphology within all the obtained nanoparticles ( Figure 4). Furthermore, the obtained micrographs were utilized for assessing the size distribution of the nanoparticles using the ImageJ software (version 1.8.0) to measure 100 nanoparticles within each sample ( Figure 5). The histograms demonstrate monomodal size distributions for all types of nanoparticles, with average sizes increasing proportionally with the substitution degree. Furthermore, one can deduce that the addition of cerium within the nanostructured systems leads to the formation of larger nanoparticles than the zinc-substituted ones, with the largest nanoparticles being registered for the nanoparticles containing both types of ions. The EDX semi-quantitative measurements demonstrate a proportional increase in the zinc/cerium amount with the substitution degree and a decrease in the iron amount ( Figure 6).
Antioxidant Properties
After the physicochemical characterization, the zinc/cerium-substituted nanostructures were biologically evaluated through in vitro ROS-Glo H2O2 (Figure 7) and MTT (Fig-Figure 6. EDX spectra of the zinc/cerium-substituted magnetite nanoparticles.
Antioxidant Properties
After the physicochemical characterization, the zinc/cerium-substituted nanostructures were biologically evaluated through in vitro ROS-Glo H 2 O 2 ( Figure 7) and MTT ( Figure 8) assays in order to assess their antioxidant and anticancer effects, respectively. As it can be observed, the 24-h timepoint witnesses increased levels of oxidative stress within the BHK cells, which is generally caused by an initial shock suffered by the cells after the introduction of a novel compound into the culture media [47]. However, the 72-h mark shows a decrease by more than a half for almost all types of nanoparticles. The most significant reductions were registered for the zinc-containing nanostructures, with no differences between the two concentrations used for substitution, and for the cerium-containing nanoparticles substituted at the lower concentration. Increasing the amount of cerium added leads to higher oxidative stress levels, which is also the case for the double substitutions.
Antioxidant Properties
After the physicochemical characterization, the zinc/cerium-substituted nanostructures were biologically evaluated through in vitro ROS-Glo H2O2 ( Figure 7) and MTT (Figure 8) assays in order to assess their antioxidant and anticancer effects, respectively. As it can be observed, the 24-h timepoint witnesses increased levels of oxidative stress within the BHK cells, which is generally caused by an initial shock suffered by the cells after the introduction of a novel compound into the culture media [47]. However, the 72-h mark shows a decrease by more than a half for almost all types of nanoparticles. The most significant reductions were registered for the zinc-containing nanostructures, with no differences between the two concentrations used for substitution, and for the cerium-containing nanoparticles substituted at the lower concentration. Increasing the amount of cerium added leads to higher oxidative stress levels, which is also the case for the double substitutions.
Antitumoral Properties
Furthermore, the MTT assay was employed for assessing the anticancer potential of the substituted nanostructures. It is showcased that cell viability of the HepG2 line is decreased by half in almost all cases, both at 24 and 72-h time intervals. However, there is no significant difference between the control and the pristine or zinc/cerium-substituted Figure 8. Cell viability results for the zinc/cerium-substituted magnetite nanoparticles (HepG2 cell line; values are expressed as mean ± SD, n = 2; different signs indicate significant differences between the control and each sample; * and +-lower significance; ++-higher significance).
Antitumoral Properties
Furthermore, the MTT assay was employed for assessing the anticancer potential of the substituted nanostructures. It is showcased that cell viability of the HepG2 line is decreased by half in almost all cases, both at 24 and 72-h time intervals. However, there is no significant difference between the control and the pristine or zinc/cerium-substituted magnetite nanoparticles after 24 h. Still, the 72-h timepoint demonstrates significant differences between the control and the samples containing zinc and/or cerium at higher concentrations, denoting differentiated anticancer potential for both zinc and cerium oxides, along with zinc and cerium ions resulted from their release.
Antimicrobial Properties
The subsequent evaluation employed involved assessing the antimicrobial properties of the zinc/cerium-substituted magnetite nanoparticles. Regarding the inhibition zone (Table 4), it seems that the most efficient nanostructures are the ones containing lower amounts of the substitution agents, possibly due to a more facile ion dissolution and, consequently, an enhanced antibacterial effect. Specifically, zinc ions are the most efficient towards Gram-negative bacteria, cerium ions, and against fungi, and Gram-positive strains are the most efficiently inhibited by the presence of both zinc and cerium ions. Subsequently, the MIC values (Table 5) show higher concentrations necessary for inhibiting the growth of C. albicans for all types of nanoparticles. Similarly, the P. aeruginosa strain is more sensitive towards the action of both zinc ions and zinc oxide. In the case of S. aureus, a higher concentration of the nanoparticles containing higher amounts of both substitution agents is necessary.
Discussion
The current study targeted the development and evaluation of zinc-and/or ceriumsubstituted magnetite nanoparticles for potential biomedical applications. As results have demonstrated, lower substitution degrees only lead to the replacement of the iron ions with the substitution agents, while higher degrees cause the formation of secondary phases of zinc and cerium oxide. In this context, the present study investigated the effects of both zinc/cerium-substituted magnetite nanoparticles and the synergistic activity of the substituted nanoparticles, as well as the associated secondary oxides.
In regard to the generation of reactive oxygen species, all types of nanoparticles managed to significantly reduce the oxidative stress within the BHK cells. However, it was demonstrated that the presence of cerium oxide slightly reduces the antioxidant properties of the nanostructures. This behavior is in accordance with the previously published literature, where the depletion of intracellular glutathione and increased levels of reactive oxygen species, lipid peroxidation, and superoxide dismutase were reported after cell treatment with cerium oxide nanoparticles [48][49][50]. By contrast, numerous studies demonstrated the antioxidant properties of zinc oxide by scavenging 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis; 3-ethylbenzothiazoline-6-sulphonic acid, hydroxyl, and superoxide-free radicals [51][52][53][54].
Furthermore, the MTT assay showed that the most pronounced anticancer activity was registered for the nanostructures with the higher substitution degrees. In the case of zinc, results confirm previously published papers reporting cytotoxicity levels dependent upon the content of zinc added due to the formation of zinc oxide [55,56]. Moreover, studies have demonstrated different cytotoxic effects on normal and cancerous cells, with a cell viability decreasing to 80% for the BJ-human foreskin fibroblast cell line, as compared to 50% for the A549-human pulmonary cancer cells [57]. The cerium-substituted nanoparticles behave similarly, with a lower cell viability registered for the Ce 0.5 Fe 2.5 O 4 . While to the best of our knowledge there is no available literature investigating the anticancer properties of ceriumsubstituted magnetite nanoparticles, cerium oxide nanoparticles are known to normalize the tumor microenvironment through the redox switching mediation between Ce 3+ and Ce 4+ [58]. Such as in the case of zinc oxide, previously published results demonstrate increased levels of reactive oxygen species, and induced apoptosis in cancerous WEHI164 cell line and low toxicity levels in normal cells, i.e., L929 line [28]. The differences in cell viability could be explained by the enhanced permeability and retention (EPR) effect and the selective cytotoxicity towards cancer cells [28,59]. In the context of the present study, the antitumoral activity is owed to the synergistic effect between zinc/cerium oxide and the dissolution of zinc and cerium ions.
Hence, the suitability of zinc/cerium-substituted magnetite nanoparticles for anticancer applications was demonstrated both through the antioxidant properties proven towards normal cells and through the antitumoral effects exhibited upon the treatment of cancer cells.
In terms of antimicrobial activity, the available literature demonstrates the potential of both zinc/cerium-substituted magnetite nanoparticles and zinc/cerium oxide nanoparticles against a wide variety of microbial species, e.g., B. cereus [19], B. subtilis [24,60], S. aureus [20,32,[60][61][62], S. enterica [63], E. coli [19,20,24,[60][61][62][63][64], P. aeruginosa [60,65], C. jejuni [63], and C. albicans [19]. While the mechanistic pathway behind the activity of zinc/cerium-substituted magnetite nanoparticles has not been precisely determined, it could be assumed that it is based on the targeting of the microbial cell wall, followed by the release of the zinc and cerium ions within the microbial environment and the consequent generation of intracellular reactive oxygen species [31,60]. Considering the results of this study, the improved antimicrobial activity of the nanoparticles obtained at lower substitution degrees could be owed to a more facile dissolution of the zinc and cerium ions compared to the associated oxides. Moreover, the nanoparticle size could also play a key role in inhibiting the microbial growth [61], since higher substitution degrees resulted in the increase in the average nanoparticle size, as shown in SEM images.
Therefore, the present study successfully demonstrated the potential of zinc and cerium substitutions for tuning the antioxidant, antitumoral, and antimicrobial properties of magnetite nanoparticles. In this manner, zinc/cerium-substituted magnetite nanoparticles could act as strong candidates for drug delivery systems in a plethora of biomedical applications, such as cancer treatment, wound dressings, or implant coatings.
For the ICP-MS analysis, calibration solutions were prepared from multi-element calibration standards purchased from Agilent Technologies (Santa Clara, CA, USA, p.n: 8500-6944 and 8500-6940). The sample preparation was conducted using trace analysis grade nitric acid (HNO 3 ) purchased from Honeywell (Charlotte, NC, USA), while all dilutions were performed with Milli-Q water (18 MΩ·cm).
The Baby Hamster Kidney fibroblasts (BHK) and human hepatocarcinoma (HepG2) cell lines were provided by the Institute for Diagnosis and Animal Health (I. D.A.H.).
The microbial strains (S. aureus ATCC 25923, P. aeruginosa ATCC 15442, and C. albicans ATCC 10231) used for the antimicrobial assays were provided by the Microbiology and Immunology Department from the Faculty of Biology, University of Bucharest.
Synthesis of Zinc/Cerium-Substituted Magnetite Nanoparticles
All nanoparticle samples were obtained through the co-precipitation method [11,66]. The iron ion precursors were dissolved in ultrapure water in a molar ratio of Fe 2+ :Fe 3+ = 1:2. For the zinc and/or cerium-substituted nanoparticles, the zinc and/or cerium precursors were dissolved in the same solutions in appropriate amounts. The obtained solutions were dripped into the 1M NaOH solution using a peristaltic pump, which allowed for the precipitation of the zinc/cerium-substituted magnetite nanoparticles. Subsequently, the precipitate was separated by applying a high-power NdFeB magnet under the reaction beaker and afterward washed with deionized water until pH = 7. Table 6 summarizes all the obtained nanoparticle samples. The compositional and structural evaluation of the samples was performed through the XRD analysis, using a PANalytical Empyrean diffractometer (PANalytical, Almelo, The Netherlands), equipped with a CuKα radiation source. The patterns were recorded in a Bragg-Brentano configuration, with a 2θ angle varying from 10 • to 80 • , a step size of 0.0256 • , and time per step of 1 s. The Rietveld refinement method was afterward per-formed using the HighScore Plus software (version 3.0.5, PANalytical, Almelo, The Netherlands), to determine the average crystallite size and the unit cell parameters. Diffractogram fittings were considered acceptable if the goodness of fit < 4.
Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
The content of cerium and zinc ions from the substituted magnetite nanoparticles was evaluated in triplicate with an Agilent 8800 Triple Quadrupole ICP-MS instrument (Agilent Technologies, Tokyo, Japan). Both elements were quantified using the calibration curve method. The instrumental parameters were set to 1550 W radiofrequency power, 1 L/min of argon gas as carrier, 0.7 mL/min of helium, and 0.1 rpm for the peristaltic pump. The method was linear in the range of 1-25 µg/L, with correlation coefficients of 0.9992, 0.9997, and 0.9991 for iron, cerium, and zinc, respectively.
Using an Ethos UP microwave system (Milestone Inc., Sorisole, Italy), approximately 10 mg of each sample powder was weighed in special TFM digestion vessels and further digested in 8 mL of concentrated HNO 3 . The digested solutions were transferred quantitatively to 50-mL volumetric flasks and filled to capacity with ultrapure water. Subsequently, a 1000-fold dilution was performed to ensure that the actual values of the samples are within the calibration range of the instrument. The measured concentrations were achieved for isotopes 56 Fe, 66 Zn, and 140 Ce, and are expressed in µg/L. Finally, the final concentrations for each element are expressed in µg/mg, being calculated by applying the dilution factors used in the sample preparation step and the exact mass of the sample taken into analysis.
X-ray Photoelectron Spectroscopy (XPS)
The analysis of the material's surface chemistry was performed on an ASPECT photoelectron spectrometer (Scienta Omicron Technology GmbH, Taunusstein, Germany), equipped with a 160-mm hemispherical energy analyzer with a 1D detector. All measurements were made in a vacuum (~2 × 10 −9 mbar), employing a polychromatic Mg Kα X-ray source at 1253.6 eV with a power of 300 W. The diameter of the analysis spot was 1.3 mm. The XPS survey wide scan spectra were recorded from −5 to 1200 eV (binding energy), with a pass energy of 200 eV, while the high-resolution spectra were recorded with a pass energy of 20 eV and step of 0.1 eV. The obtained XPS spectra were post-processed to eliminate satellite peaks and evaluated with CasaXPS software (version 2.3.25). For the determination of background lines, the Shirley fitting function was used. The binding energy scale was calibrated based on the C-C component in the C 1s signal (by default at 248.8 eV) associated with the adventitious carbon.
Dynamic Light Scattering (DLS) and Zeta Potential
The particle size distribution and particle zeta potential were determined in triplicate with a DelsaMax Pro equipment (Backman Coulter, Brea, CA, USA). The nanoparticles were first dispersed in deionized water (0.3 mg/mL, pH~6.9), then homogenized for 10 min using an ultrasonic bath. Subsequently, a small amount was introduced inside the measurement cell and each sample was measured in triplicate.
Scanning Electron Microscopy (SEM): Energy Dispersive X-ray Spectroscopy (EDX)
The morphological and elemental aspects of the samples were investigated using the Inspect F50 scanning electron microscope (Thermo Fisher, Eindhoven, The Netherlands), equipped with an energy dispersive spectrometer. SEM images were acquired at different magnifications using the Everhart-Thornley secondary electrons detector and an accelerating voltage of 30 KeV energy.
ROS-Glo H 2 O 2 Assay
The evaluation of oxidative stress induced by the zinc/cerium-substituted nanoparticles on the BHK cell line was made through the ROS-Glo assay (Promega, Madison, WI, United States) [1]. A total of 10 mg of each sample was dispersed in 200 µL cell suspension, followed by the addition of the cell medium until a final concentration of 5 mg/mL. The H 2 O 2 levels were measured after 24 and 72 h of incubation based on the manufacturer instructions with a SpectraMax i3x Multi-Mode microplate reader. The results were collected with the Soft-Max Pro 6 software and presented as relative light units (RLU).
MTT Assay
The cell viability was determined through the MTT assay on a HepG2 cell line [1]. All powders were UV-sterilized for 1 h. A total of 10 mg of each sample was dispersed in 200 µL cell suspension, followed by the addition of the cell medium until a final concentration of 5 mg/mL, and maintained for 24 h and 72 h. After removing the media, the cells were thoroughly washed with Phosphate-buffered saline (PBS) and incubated at 37 • C and 5% CO 2 in the absence of light for 2 h with a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution. After reduction of the MTT and removal of the resulting solution, the precipitated formazan crystals were solubilized in isopropanol. The optical density (OD) was spectrophotometrically measured at 595 nm using a SpectraMax i3x Mul-ti-Mode microplate reader. The results were collected with the SoftMax Pro 6 software, and the cell viability of the samples was calculated using Equation (1): Cell viability(%) = OD595 sample OD595 control × 100 (1)
Antimicrobial Activity
The antimicrobial protocols were performed according to previously published studies [47,67,68]. The antimicrobial activity was assessed for all pristine and zinc/ceriumsubstituted nanoparticles after sterilization with UV radiation for 20 min. The nanoparticles were dispersed in sterile deionized water at a concentration of 1 mg/mL to obtain a stock solution. All measurements were performed in duplicate.
The inhibition zone diameter (qualitative antimicrobial assessment) was determined based on the adapted diffusion test from the Clinical & Laboratory Standards Institute (CLSI) guidelines. In brief, a microbial suspension of 1-3 × 10 8 CFU/mL (equivalent of 0.5 McFarland density standard) was swab-inoculated in Petri dishes containing the Mueller-Hinton agar medium for S. aureus and P. aeruginosa bacteria and Sabouraud Dextrose broth for C. albicans yeast. Stock solutions of the obtained nanoparticles were added dropwise (10 µL) on the inoculated plates and incubated at 37 • C for 24 h. The resulting inhibition zone diameter was measured, and the results were expressed in mm.
The minimum inhibitory concentration-MIC (quantitative antimicrobial assessment) was determined using the microdilution technique in 96-well plates. Specifically, each of the tested nanoparticles were consecutively diluted from 2 mg/mL to 0.015625 mg/mL in each type of nutritive broth and a microbial suspension of~10 6 CFU/mL was utilized to inoculate the plates. The cultures were incubated at 37 • C for 24 h. The MIC was assessed by a naked-eye analysis, where the lowest concentration of the nanoparticles that inhibited the visible growth of the microbial strains was considered [69].
Statistical Analysis
The biological evaluations were performed in duplicate, and the data are represented as mean ± standard deviation. The obtaining results were statistically analyzed using the one-way analysis of variance (ANOVA) followed by the two-tailed t-test with Bonferroni post hoc correction (p < 0.05). Data comparison was made with the GraphPad Prism 9 software (San Diego, CA, USA).
Conclusions
The hypothesis behind the design of the current study was based on the possibility of enhancing the properties of magnetite nanoparticles by incorporating zinc and/or cerium ions within its crystal lattice. The physicochemical characterization of the obtained nanostructures revealed the successful substitution of the iron ions with zinc and/or cerium by modifications of the magnetite unit cell, as well as the formation of zinc and/or cerium oxides as secondary mineral phases at higher substitution degrees. Furthermore, the substitution process resulted in the increase in the average nanoparticle size and, consequently, in more stable nanoparticles in terms of the agglomeration tendency. The biological evaluation confirmed the antioxidant, anticancer, and antimicrobial properties of the nanostructures, with the efficiency dependent upon the type and concentration of the ions used for the substitution. To summarize, as demonstrated through this study, | 2023-03-29T15:18:22.003Z | 2023-03-26T00:00:00.000 | {
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117706828 | pes2o/s2orc | v3-fos-license | AC Dielectric Strength of Mineral Oil-Based Fe3O4 and Al2O3 Nanofluids
: This paper deals with an experimental study of the influence of conductive (Fe 3 O 4 ) and insulating (Al 2 O 3 ) nanoparticles at various concentrations on the dielectric strength of transformer mineral oil. The method of preparation and characterization of these nanofluids (NFs) through the measurements of zeta potential, the real and imaginary parts of dielectric permittivity as well as the concentration and size of nanoparticles using scanning electron microscope images of nanoparticles powders and energy dispersive x-ray spectroscopy analysis are presented. Experimental findings reveal that these two types of nanoparticles materials significantly improve AC breakdown voltage and the magnitude of this enhancement depends on the nanoparticle concentration, and the size and nature (material) of nanoparticles. For a given type of nanoparticle, the effect is more marked with the smallest nanoparticles. The conductive nanoparticles offer higher enhancement of dielectric strength compared with insulating nanoparticle based nanofluids. With Fe 3 O 4 , the breakdown voltage (BDV) can exceed twice that of mineral oil and it increases by more than 76% with Al 2 O 3 . The physicochemical mechanisms implicated in this improvement are discussed.
Introduction
The search for increasingly efficient materials of integrated components in electric power transmission and distribution systems to improve the dielectric strength, and reduce their size, weight, and cost is a permanent task. Also, nanotechnologies and, more particularly, dielectric nanofluids (NFs) constitute an innovative line of research with a promising potential and future.
The effect of nanoparticles on the electrical properties of dielectric materials, in particular, on their ability to record the initiation voltage of partial discharges and to slow the propagation of electrical discharges, trees in polymers and streamers in liquids, leading to breakdown is the subject of many studies around the world for the past 20 years. It appears from the results reported in the literature that some polymers' nano-composites are promising materials for high voltage applications [1][2][3][4][5][6]. The fact that nano-materials present interesting dielectric characteristics results from the large volume fraction of interfaces in the bulk of the material and the ensuing interactions between the surface of the charged nanomaterial and the molecular structure of the hosting material. However, if the solid dielectrics provide a function mainly of insulation and mechanical support (equipment envelope, support isolators, bushing, etc.), the liquid dielectrics must ensure the thermal transfer for a better cooling of high voltage components and power transformers especially, in addition to their insulating role.
Preparation and Characterization of Nanofluid Samples
The nanoparticles used in this study were supplied from Sigma-Aldrich and have a purity of 99.9%. These were conductive (Fe 3 O 4 ) and insulating (Al 2 O 3 ). Their mean sizes were 50 nm for Fe 3 O 4 , and 13 nm and 50 nm for Al 2 O 3 . Figure 1 gives the distribution of the various nanoparticles we used in mineral oil. It was measured by using a particle size analyzer (NanoPlus, Particulate Systems, USA). Scanning electron microscope (SEM) images show that these nanoparticles were near spherical in shape even if some aggregates can be observed as shown in Figure 2a. Figure 2b shows the peaks corresponding to the species present in the nanoparticles as evidenced by energy dispersive X-ray spectroscopy (EDS) analysis. The EDS analysis confirms the presence of each sample composition at the atomic percentage. The characteristic parameters of the basic mineral oil we used are shown in Table 1.
Note that because of the aged mineral oil we used, its water content is higher than that of the fresh oil, which generally does not exceed 8 ppm. This fact would affect the experimental results. However, as all nanofluid samples were prepared with the same base oil, the comparison of our results/comparison will be done on the same basis.
Mineral-oil nanofluids were prepared by dispersion of nanoparticles in a concentration ranging from 0.05 g/L to 0.4 g/L. After the magnetic stirring process for 30 min, the NFs samples were submitted to an ultra-sonication process (i.e., NFs were placed in the ultrasonic homogenizer) for 2 h to avoid agglomeration and clusters due to attractive and repulsive forces.
In order to reach a stable suspension of nanoparticles in oil, further ultrasonification was applied for only a 2 min duration using a Sonics Vibra-cell sonicator (750 W power rating, 20 kHz capability, and 0.5 inch probe) to avoid reunion of nanoparticles without the need of adding any surfactant. Then, all samples were moved into a vacuum chamber of 0.16 MPa for 24 h for drying and removal of internal micro bubbles formed during the ultrasonication process.
The stability of NFs we prepared was checked through the measurements of the zeta potential (ζ-potential), by using Malvern Zeta sizer nano ZS 90-UK, which is a key indicator of the stability of suspensions. The magnitude of the ζ-potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles in a dispersion. For particles that are small enough, a high zeta potential refers to higher stability and, consequently, the dispersion becomes more stable without agglomerations. Table 2 gives the zeta potential for four NFs and mineral oil for comparison. It was observed that the absolute value of the ζ-potential increases with the concentration of nanoparticles. Additionally, for a given concentration (0.3 g/L for instance), this value was higher for Al 2 O 3 nanoparticles than that for Fe 3 O 4 .
Energies 2018, 11, x FOR PEER REVIEW 3 of 13 USA). Scanning electron microscope (SEM) images show that these nanoparticles were near spherical in shape even if some aggregates can be observed as shown in Figure 2a. Figure 2b shows the peaks corresponding to the species present in the nanoparticles as evidenced by energy dispersive x-ray spectroscopy (EDS) analysis. The EDS analysis confirms the presence of each sample composition at the atomic percentage. The characteristic parameters of the basic mineral oil we used are shown in Table 1.
Note that because of the aged mineral oil we used, its water content is higher than that of the fresh oil, which generally does not exceed 8 ppm. This fact would affect the experimental results. However, as all nanofluid samples were prepared with the same base oil, the comparison of our results/comparison will be done on the same basis.
Mineral-oil nanofluids were prepared by dispersion of nanoparticles in a concentration ranging from 0.05 g/L to 0.4 g/L. After the magnetic stirring process for 30 min, the NFs samples were submitted to an ultra-sonication process (i.e., NFs were placed in the ultrasonic homogenizer) for 2 h to avoid agglomeration and clusters due to attractive and repulsive forces.
In order to reach a stable suspension of nanoparticles in oil, further ultrasonification was applied for only a 2 min duration using a Sonics Vibra-cell sonicator (750 W power rating, 20 kHz capability, and 0.5 inch probe) to avoid reunion of nanoparticles without the need of adding any surfactant. Then, all samples were moved into a vacuum chamber of 0.16 MPa for 24 h for drying and removal of internal micro bubbles formed during the ultrasonication process.
The stability of NFs we prepared was checked through the measurements of the zeta potential (ζ-potential), by using Malvern Zeta sizer nano ZS 90-UK, which is a key indicator of the stability of suspensions. The magnitude of the ζ-potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles in a dispersion. For particles that are small enough, a high zeta potential refers to higher stability and, consequently, the dispersion becomes more stable without agglomerations. Table 2 gives the zeta potential for four NFs and mineral oil for comparison. It was observed that the absolute value of the ζ-potential increases with the concentration of nanoparticles. Additionally, for a given concentration (0.3 g/L for instance), this value was higher for Al2O3 nanoparticles than that for Fe3O4. The measurements of electrical conductivity show that the Fe 3 O 4 based NF were higher than that of Al 2 O 3 for the same concentration of nanoparticles, as shown in Table 2. This was due to the fact that Fe 3 O 4 conducts more than Al 2 O 3 , which is known as a good insulating material. Figure 3 depicts the real and imaginary parts of the dielectric permittivity of the investigated NF versus the frequency up to 100 Hz. These measurements were performed by a HIOKI-LCR meter IM3536, Japan. We observed that the real part of the dielectric permittivity decreases abruptly in the range of 0 Hz-40 Hz and then tends to be constant. The measurements of electrical conductivity show that the Fe3O4 based NF were higher than that of Al2O3 for the same concentration of nanoparticles, as shown in Table 2. This was due to the fact that Fe3O4 conducts more than Al2O3, which is known as a good insulating material. Figure 3 depicts the real and imaginary parts of the dielectric permittivity of the investigated NF versus the frequency up to 100 Hz. These measurements were performed by a HIOKI-LCR meter IM3536, Japan. We observed that the real part of the dielectric permittivity decreases abruptly in the range of 0 Hz-40 Hz and then tends to be constant.
At 50 Hz-60 Hz, which corresponds to industrial frequencies, the real part of the dielectric permittivity of nanofluid samples was practically the same as that of the base oil (mineral oil). Similarly, this was also observed with the imaginary part of the dielectric permittivity. Table 2. Zeta potential and electrical conductivity of the tested nanofluids. At 50 Hz-60 Hz, which corresponds to industrial frequencies, the real part of the dielectric permittivity of nanofluid samples was practically the same as that of the base oil (mineral oil). Similarly, this was also observed with the imaginary part of the dielectric permittivity.
Breakdown Voltage Measurement
The breakdown voltage (BDV) measurements were achieved according to the IEC 60156 standard [27] and using a test cell of a 500 mL volume and an oil tester (Foster Oil Test 90 type). The electrode arrangement consisted of two copper hemispheres of a 12.5 mm diameter; the gap distance between the electrodes was kept at 2.50 ± 0.05 mm. Both electrodes and the test cell were also prepared according to the IEC 60156 specifications. The voltage was continuously applied at the electrodes at a uniform rise rate of 2 ± 0.2 kV/s until the breakdown occurred. The breakdown voltage was the average of 16 successive measurements and the time delay between successive measurements was 2 min. Sixteen (16) is a proper number for a Weibull statistical analysis. This enables us to have a power of two (2 4 = 16) and to deduce the slope of the Weibull graphs [28]. Table 3 gives the AC breakdown voltage at breakdown probabilities of 1%, 10%, and 50% for the three investigated NFs, which were read from the distinctive Weibull plots. Further, these tables show the incremental percentage of the mineral oil with different concentrations of nanofluids. The breakdown voltage at the 1% cumulative probability is estimated to be the minimum possible breakdown voltage, and so, refers to the reliability of oil. Generally, the BDV of nanofluids were higher than that of the pure mineral oil whatever the type and size of nanoparticles.
Experimental Results
The indications within the inserts are: (1) The shape parameter that is equal to the slope of the line in a probability plot, which affects the shape of the curve; (2) the scale parameter, which is related to the scattering of the data, and indicates the degree of failure; (3) the Anderson-Darling (AD) value that is the Anderson-Darling measure of the area between the fitted line and the empirical distribution function, which is based on the data points; (4) N is the number of breakdown voltage data points; and (5) the p-value is a probability that measures the evidence against the null hypothesis.
Breakdown Voltage Measurement
The breakdown voltage (BDV) measurements were achieved according to the IEC 60156 standard [27] and using a test cell of a 500 mL volume and an oil tester (Foster Oil Test 90 type). The electrode arrangement consisted of two copper hemispheres of a 12.5 mm diameter; the gap distance between the electrodes was kept at 2.50 ± 0.05 mm. Both electrodes and the test cell were also prepared according to the IEC 60156 specifications. The voltage was continuously applied at the electrodes at a uniform rise rate of 2 ± 0.2 kV/s until the breakdown occurred. The breakdown voltage was the average of 16 successive measurements and the time delay between successive measurements was 2 min. Sixteen (16) is a proper number for a Weibull statistical analysis. This enables us to have a power of two (2 4 = 16) and to deduce the slope of the Weibull graphs [28]. Table 3 gives the AC breakdown voltage at breakdown probabilities of 1%, 10%, and 50% for the three investigated NFs, which were read from the distinctive Weibull plots. Further, these tables show the incremental percentage of the mineral oil with different concentrations of nanofluids. The breakdown voltage at the 1% cumulative probability is estimated to be the minimum possible breakdown voltage, and so, refers to the reliability of oil. Generally, the BDV of nanofluids were higher than that of the pure mineral oil whatever the type and size of nanoparticles.
Experimental Results
The indications within the inserts are: (1) The shape parameter that is equal to the slope of the line in a probability plot, which affects the shape of the curve; (2) the scale parameter, which is related to the scattering of the data, and indicates the degree of failure; (3) the Anderson-Darling (AD) value that is the Anderson-Darling measure of the area between the fitted line and the empirical distribution function, which is based on the data points; (4) N is the number of breakdown voltage data points; and (5) the p-value is a probability that measures the evidence against the null hypothesis. It was observed that the BDV of mineral oil-based Fe 3 O 4 increased with the concentration of nanoparticles as shown in Figure 4. With a concentration of 0.4 g/L, the average BDV of NF exceeds twice that of mineral oil alone (Figure 7). This is of a great interest for the oil-filled apparatus, especially for the power transformer. While for mineral oil-based Al 2 O 3 with nanoparticles of 13 nm, the average BDV increased up to a maximum value (optimal), which was reached at a small concentration of 0.05 g/L; this increase is of 72% with respect to that of mineral oil and then decreased, but remained higher than that of mineral oil (Figure 8). While with nanoparticles of 50 nm, the optimal average BDV was reached at a higher concentration of 0.3 g/L and the increase was of 69% (Figure 9). Table 4 summarizes the median and average breakdown voltages of the tested MO/NFs samples. It was observed that the BDV of mineral oil-based Fe3O4 increased with the concentration of nanoparticles as shown in Figure 4. With a concentration of 0.4 g/L, the average BDV of NF exceeds twice that of mineral oil alone (Figure 7). This is of a great interest for the oil-filled apparatus, especially for the power transformer. While for mineral oil-based Al2O3 with nanoparticles of 13 nm, the average BDV increased up to a maximum value (optimal), which was reached at a small concentration of 0.05 g/L; this increase is of 72% with respect to that of mineral oil and then decreased, but remained higher than that of mineral oil (Figure 8). While with nanoparticles of 50 nm, the optimal average BDV was reached at a higher concentration of 0.3 g/L and the increase was of 69% (Figure 9). Table 4 summarizes the median and average breakdown voltages of the tested MO/NFs samples. It was observed that the BDV of mineral oil-based Fe3O4 increased with the concentration of nanoparticles as shown in Figure 4. With a concentration of 0.4 g/L, the average BDV of NF exceeds twice that of mineral oil alone (Figure 7). This is of a great interest for the oil-filled apparatus, especially for the power transformer. While for mineral oil-based Al2O3 with nanoparticles of 13 nm, the average BDV increased up to a maximum value (optimal), which was reached at a small concentration of 0.05 g/L; this increase is of 72% with respect to that of mineral oil and then decreased, but remained higher than that of mineral oil (Figure 8). While with nanoparticles of 50 nm, the optimal average BDV was reached at a higher concentration of 0.3 g/L and the increase was of 69% (Figure 9). Table 4 summarizes the median and average breakdown voltages of the tested MO/NFs samples.
Discussion
It appears from Table 4 that at concentrations of 0.05 g/L and 0.20 g/L, the highest average breakdown voltage was obtained with Al2O3 nanoparticles of 13 nm; the average BDV was increased by 76.6% with respect to mineral oil. While with Al2O3 nanoparticles of 50 nm, the increase was 36.3 % for the same concentration. This increase was only 28.9% with Fe3O4.
With concentrations of 0.3 g/L and 0.4 g/L, the maximum average BDV was obtained with Fe3O4 nanoparticles. The improvement was 75.3% and 108.8% with respect to mineral oil, respectively. Table 3 presents a comparison of different NFs of different volume concentrations with mineral oil. The fact that with the same kind of nanoparticles and concentration, the average BDV was higher with smaller nanoparticles is due to the large volume fraction of interfaces in the bulk of the material and the ensuing interactions between the charged nanomaterial surface and the liquid molecule.
It was observed that improvements with the same nanoparticles, such as Madawan et al. [23], are two times higher than the ones reported by these authors. Also, our findings confirm those reported by a number of authors [13,[19][20][21][22][23] and contradict others [26] regarding concerns of the influence of Al2O3 and Fe3O4 nanoparticles on the breakdown voltage of mineral oil.
The increase of the breakdown voltage of liquid (MO) when adding some amounts of nanoparticles results from their influence on the physicochemical processes evolving during the pre-breakdown phase that are mainly the conduction, the initiation, and/or propagation of streamers. The question is: How and which mechanism(s)? In the following, we analyze the possible implication of each one of these mechanisms. If the nanoparticles (NPs) act on the conduction, they likely act as charge carriers' scavengers. Thus, by reducing the number of charges by trapping and, consequently, the total space charge, the liquid becomes less conductive and its breakdown voltage increases.
Discussion
It appears from Table 4 that at concentrations of 0.05 g/L and 0.20 g/L, the highest average breakdown voltage was obtained with Al 2 O 3 nanoparticles of 13 nm; the average BDV was increased by 76.6% with respect to mineral oil. While with Al 2 O 3 nanoparticles of 50 nm, the increase was 36.3 % for the same concentration. This increase was only 28.9% with Fe 3 O 4 .
With concentrations of 0.3 g/L and 0.4 g/L, the maximum average BDV was obtained with Fe 3 O 4 nanoparticles. The improvement was 75.3% and 108.8% with respect to mineral oil, respectively. Table 3 presents a comparison of different NFs of different volume concentrations with mineral oil. The fact that with the same kind of nanoparticles and concentration, the average BDV was higher with smaller nanoparticles is due to the large volume fraction of interfaces in the bulk of the material and the ensuing interactions between the charged nanomaterial surface and the liquid molecule.
It was observed that improvements with the same nanoparticles, such as Madawan et al. [23], are two times higher than the ones reported by these authors. Also, our findings confirm those reported by a number of authors [13,[19][20][21][22][23] and contradict others [26] regarding concerns of the influence of Al 2 O 3 and Fe 3 O 4 nanoparticles on the breakdown voltage of mineral oil.
The increase of the breakdown voltage of liquid (MO) when adding some amounts of nanoparticles results from their influence on the physicochemical processes evolving during the pre-breakdown phase that are mainly the conduction, the initiation, and/or propagation of streamers. The question is: How and which mechanism(s)? In the following, we analyze the possible implication of each one of these mechanisms. If the nanoparticles (NPs) act on the conduction, they likely act as Energies 2018, 11, 3505 10 of 13 charge carriers' scavengers. Thus, by reducing the number of charges by trapping and, consequently, the total space charge, the liquid becomes less conductive and its breakdown voltage increases. Table 2 seems to contradict that hypothesis since the conductivity of liquid increases when increasing the amount of nanoparticles. Therefore, the nanoparticles would act on the streamers' phenomena. The influence of electronic scavenger additives (especially halogenated molecules) has been investigated by many researchers and it was established unanimously that such additives accelerate the streamer propagation velocity [29][30][31][32]. However, the interpretations concerning the effect of this streamer acceleration on the breakdown voltage were contradictory. Most authors deduced that the fact that the streamers are more rapid leads to the decrease of the breakdown voltage [29]. Beroual [31,33], and Beroual and Aka [34] showed that the electronic scavengers' additives increase the initiation threshold voltage of streamers, thus increasing the voltage and therefore its energy: The streamer, being more energetic, results in its velocity also being higher [31].
In our case, the nanoparticles acted at two levels: (1) At the electrodes interfaces by constituting a barrier, which reduces the injected charges into the liquid and their mobility. Therefore, the initiation threshold voltage of streamers increased and the breakdown voltage was higher; and (2) at the nanoparticles/hosting liquid interfaces by constituting double layers. The charge carriers were trapped up to saturation. The fact that there was an optimum concentration of NPs is likely due do to some saturation of NPs/hosting oil interfaces. On the other hand, the dielectric permittivity (real part) of Fe 3 O 4 being significantly higher than that of Al 2 O 3 results in a higher surface charge with the Fe 3 O 4 NPs/base oil. Fe 3 O 4 , being more conductive, could easily be charged and polarized than Al 2 O 3 . These phenomena (polarization, double layer, and trapping) explain the greater improvement with conductive nanoparticles.
According to Hwang et al. [35], nanoparticles with different conductivity or permittivity than those of the matrix oil enhance the breakdown voltage strength while the insertion of nanoparticles with a conductivity and permittivity comparable to those of the matrix oil results in the decrease of the breakdown voltage. It is difficult to accept such an interpretation because in some cases, beyond a certain concentration (optimal concentration) of nanoparticles, the breakdown voltage decreases.
The role of nanoparticles as electronic scavengers has been also advanced by Peppas et al. [36] and Makmud et al. [37] to explain the improvement of the breakdown voltage. They attributed this improvement to the effective electron scavenging by the nanostructures, which results in delaying the development of streamers and reducing their propagation velocity.
The mechanism of trapping electrons has also been proposed by some researchers [26,38,39] to explain the higher breakdown strength of conducting nanofluids compared to base oils. The conductive nanoparticles capture very rapidly fast moving electrons and convert into slow negatively charged nanoparticles, resulting in the slowing of the streamer propagation (i.e., reduction of streamer velocity) and therefore increasing the breakdown voltage.
As indicated above, such an interpretation through the slowing propagation of streamers remains a subject of discussion; the electronic scavenger additives accelerate the propagation of streamers [29][30][31][32]. Another possibility, which would explain the improvement of the breakdown voltage, is the result of electron trapping by nanoparticles: Conductive nanoparticles trap electrons by charge induction, such is the case of Fe 3 O 4 , while nonconductive nanoparticles, which are Al 2 O 3 , trap electrons due to polarization [39].
On the other hand, the fact that for the same concentration of nanoparticles, the dielectric strength is higher for smaller nanoparticles is due to the large volume fraction of interfaces (more surfaces for charges accumulation) in the bulk of the liquid and the ensuing interactions between the charge nanoparticles' surface and the molecular structure of the liquid. There are few displacements toward the opposite electrode, thus slowing the propagation of streamers.
Conclusions
In this work, the AC breakdown voltages of mineral oil-based Fe 3 O 4 and Al 2 O 3 nanofluids (NFs) were investigated. The preparation and physicochemical characterization of these nanofluids were presented.
It has been highlighted that the AC breakdown voltage of these NFs are higher than that of mineral oil. The magnitude of this improvement depends on the concentration, and the size and type of nanoparticles. For a given type of nanoparticles, the effect is more marked with the smallest nanoparticles. The conductive nanoparticles (Fe 3 O 4 ) offer a higher enhancement of the dielectric strength compared to insulating nanoparticles based nanofluids. With Fe 3 O 4 , the breakdown voltage (BDV) can exceed twice that of mineral oil and it increases by more than 76% with Al 2 O 3 . The possible mechanisms implicated in the improvement of BDV were discussed. Therefore, these nanofluids not only have very good cooling performances, but they can also be considered for use in high voltage power transformers. | 2019-04-16T13:29:04.802Z | 2018-11-06T00:00:00.000 | {
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243615283 | pes2o/s2orc | v3-fos-license | Day 3 versus day 5 embryo freezing: which is better, A comparative study
Group 2 (n =150), will underwent embryo freezing at day 5 then will undergo FET (Blastocyst) The duration of the study had been from 6 to 12 months, Results: the results revealed that there is high significant difference between the studied groups as regard Total number of survival more survival was observed in blastocyst group, while there is no significant difference between the studied groups as regard completed transfer. Conclusion: The maintenance of embryo culture until day 5 may be a more sensible approach for the correct identification of best quality embryos with the highest probability of success for both transfer and freezing.
Introduction
Freezing of embryos and gametes is considered one of the cornerstones of ART. Its application increases both IVF safety and efficiency (Rienzi et al., 2017). The percentage of frozen transfer cycles compared to fresh transfer cycles is increasing. It represents more than 50% in some countries ( Embro transfer is done at cleavage stage or blastocyst stage (Martins et al., 2017) either fresh or frozen. Day 5 transfer is supposed to allow more synchrony between females uterus and transferred embryos. This also allow transfer of viable embryos leading to a higher pregnancy rate (Glujovsky et al., 2012). On the other hand, theoretically, due to the superiority of in vivo environment to that in vitro, some embryos may be implanted if transferred at day 3 and blocked if extended in vitro (Racowsky C, 2000). Also, invitro culture after embryonic genome activation may be harmful to the embryo (Martins et al., 2017).
There are multiple studies compared day 3 and day 5 fresh transfer outcome showing that day 5 transfer is associated higher clinical pregnancy and live birth rate (Glujovsky et al., 2012) and decrease aneuploidy. However, there are few studies compared embryo freezing at day 3 and day 5. There is no evidence to suggest the superiority of any of these two options. Therefore, in our study we will compare day 3 and day 5 embryos cryopreservation as regards the outcome of frozen embryo transfer. Ovarian reserve testing (serum antimullarian hormone (AMH), basal serum follicular stimulating hormone (FSH) and basal antral follicular count (AFC) by transvaginal ultrasonography (TVUS)).
Hormone Analysis
Blood samples were collected on the day of the ovulation trigger, and serum P levels were measured using a chemilumi-nescent immunoassay for quantitative determination of the hormone (Diagnostics Biochem Canada Inc), with a sensitivity of 0.1 ng/mL. 3. Uterine cavity examination ( bytrasvaginal 3-dimentional ultrasound or office hysteroscopy) 4. Routine investigations (Complete blood count, blood grouping, liver function tests, kidney function tests, prothrombin time , prothrombin concentration , serum bloog sugar) 5. Evaluation of male factor (husbnad semen analysis). Ovarian stimulation: All the patients in the study had been undergo stimulation by gonadotrophins and had been used GnRH long agonist protocol during their ICSI cycles. Final oocytes maturation:Triggering of ovulation had been done by using Human chorionic gonadotropins (HCG) 10000 IU(ovitrelle250 microgram /0.5 ml-2 ampoules IM) 34 -36 hours prior to ovum pick-up (OPU). Transvaginal ultrasound-guided oocyte retrieval: Egg retrieval was performed by aspiration of follicular fluid by passing a hollow needle through the wall of the vagina into the ovarian follicles under sonographic guidance. The fluid aspirate was then inspected under the microscope to do egg collection. Intracytoplasmic sperm injection (ICSI): After oocytes denudation .a single sperm was injected into the cytoplasm of each mature oocyte using RI micromanibulator, oocytes had been cultured in Global Total media microdroplets under oil in co2 incubator , then fertilzation check had been done 16 hours post injection.
Eligible women are divided into two groups: 1. Group 1 (n =150), has undergone embryo freezing at day 3 then had been undergo FET 2. Group 2 (n =150), has undergone embryo freezing at day 5 then had been undergo FET Embryos had been frozen by vitrification method using Dimethyl sulphoxide (DMSO) (Global DMSO Vitrification kit,LifeGlobalGroup,Canada) as cryoprotectant. Ethical consideration: Informed consent was obtained from all participants after being informed about the aims and process of the study as well as applicable objectives. The study procedures were free from any harmful effects on the participants as well as the service provided. The principal investigators have kept individual data as private information safely. There was no extra fee to be paid by the participants and the investigators covered all the costs in this regard.
Data management and analysis:
Data entry, processing and statistical analysis was carried out using SPSS version 26.0. According to the type of data qualitative represent as number and percentage , quantitative data represent by mean ± SD , the following tests were used to test differences for significance.
Results:
Population were divided into two groups: 1. Group 1 (n =150), underwent embryofreezing at day 3 then had been undergo FET(Cleaved embryo) 2. Group 2 (n =150), had been underwent embryofreezing at day 5 then had been undergo FET(Blastocyst). Table (1)shows that there is no significant difference between the two groups as regard age, BMI, marriage duration, infertility type, infertility cause or cyce of previous treatment. Table (2) shows that there is no significant difference between the studied groups as regard E2, FSH or AMH Table (3) shows that there is no significant difference between the studied groups as regards Cycle characteristics Table (4) shows that the blastocycst group has a significantly higher rate of survival when compared to the cleaved embryo group. However, the transfer rate was non significantly different.
DISCUSSION
As many as one in six couples will experience difficulty conceiving and may seek assisted reproduction to achieve a pregnancy. One of the most important steps during an assisted reproduction cycle is the transfer of the embryo from the laboratory to the uterus. Traditionally, cleavage-stage embryos were transferred on day 3, but over the past decade there has been a move to transferring blastocysts on day 5 or 6. Transfer at this stage is considered to be a more physiologically appropriate time as it more closely mimics the time of natural implantation and may improve synchrony between the endometrium and embryo development (Maheshwari et al.,2015). An extraporation of this physiological advantage of day 5 transfer of fresh transfer is the theoretical advantage of day 5 freezing.
Direct comparisons between the two stages of embryo development appear to support the use of blastocyst transfers in clinical practice. Women who undergo fresh blastocyst transfers achieve higher live-birth rates compared with those who receive fresh cleavage-stage transfers . However, the results are not quite so conclusive when the transfers of frozen embryos are considered (NICE., 2013). This is why the study was selected to be conducted to compare cleaved embryo and blastocyst freezing to determine the optimal time for embryo cryopreservation.
This randomized clinical trial included 300 cases of infertile patients who will undergo ICSI cycles and embryo freezing at the assisted reproduction unit, Qena University hospital, South Valley University, Egypt.Population were divided into two groups: Group 1 (n =150), underwent embryo freezing at day 3 then underwent FET(Cleaved embryo). Group 2 (n =150), underwent embryo freezing at day 5 then underwent FET(Blastocyst).
There is no significant difference between the two groups as regard age, BMI, marriage duration, infertility type, infertility cause or cycle of previous treatment. This finding negates the selection bias.
Various factors affect blastocyst formation and quality, including culture conditions, number of oocytes, maternal age, and male factor infertility (Jones and Trounson,1999). In addition, the quality of early-stage embryos can substantially influence rates of blastocyst formation rates (Miller et al.,1999). However, the data do not totally support the idea that the number of eight-cell embryos on day 3 and the potential for blastocyst formation are directly correlated (Racowsky et al.,2000); in particular, they do not support the assumption that the blastocysts that form do so from the day-3 eight-cell embryos. Confirmation of this assumption is only possible if all embryos are individually cultured.
The present study shows that there is no significant difference between the studied groups as regard E2, FSH or AMH.There is no significant difference between the studied groups as regard Picked Cycle characteristics. Our results are in line with study of Thuyet al.,2018 as they reported that there were no statistically significant differences in the total FSH dose and total days of stimulation.The number of retrieved oocytes, the numbers of mature oocytes using to performance of ICSI, the number of fertilized oocytes, the number of day-5 embryos and day-5 frozen embryos were recorded and analyzed. There were no statistically significant differences between the morphokineticgroup versus morphologic group. They found that there were similarities between the two groups in terms of patient characteristics and laboratory outcomes (Thuyet al.,2018).
The rationale of blastocyst transfer is based on increasing the probability of obtaining advanced embryos with the highest chance for survival, i.e., implantation. The prolongation of embryo culture to day 5 requires a relatively high number of top quality blastocysts. Good quality cleavage-stage embryos increases the likelihood of good quality blastocyst embryos. Therefore, it would be prudent to expect no advantage if only a few good quality blastocysts exist in the culture (Zech et al.,2007).
In our study we found that there is high significant difference between the studied groups as regards the total number of survivals. More survival was observed in blastocyst group, while there is no significant difference between the studied groups as regard completed transfer.There is significant difference between the studied groups as regard Chemical pregnancy, Clinical pregnancy and FHB/embryo transferred.
Previous study by Papanikolaou et al.,2008 indicated that recruitment at the blastocyst stage yields better results than selection at day 3, which merely depends on the morphologic evaluation of embryos. These studies also claim that pregnancy rates of up to 50% can be acquired by the transfer of blastocysts when compared with embryo transfer at the cleavage stage which supports our results.
This agree with the study conducted by Glujovsky et al.,2012 comparing day 3 and day 5 fresh transfer outcome showing that day 5 transfer is associated higher clinical pregnancy and live birth rate and decrease aneuploidy (Alder et al,2013)..
Conclusion
Which embryo to be transferred has represented a great dilemma in the field of ART. Along decades of trials, embryo morphology assessment is considered to be a cornerstone in choosing embryos for higher implantation and pregnancy rates. Cleavage stage has limited value for morphology assessment. Maintenance of embryo culture until day 5 has better clarifation on embryo morphology, so embryos are best evaluated during blastocyst stagefor both transfer and freezing. | 2020-11-19T09:11:18.805Z | 2019-07-01T00:00:00.000 | {
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119636090 | pes2o/s2orc | v3-fos-license | New fractional integral unifying six existing fractional integrals
In this paper we introduce a new fractional integral that generalizes six existing fractional integrals, namely, Riemann-Liouville, Hadamard, Erd\'elyi-Kober, Katugampola, Weyl and Liouville fractional integrals in to one form. Such a generalization takes the form \[ \left({}^{\rho}\mathcal{I}^{\alpha, \beta}_{a+;\eta, \kappa}f\right)(x)=\frac{\rho^{1-\beta}x^{\kappa}}{\Gamma(\alpha)}\int_a^x \frac{\tau^{\rho \eta +\rho-1}}{(x^\rho-\tau^\rho)^{1-\alpha}}f(\tau)\text{d}\tau, \quad 0\leq a<x<b \leq \infty. \] A similar generalization is not possible with the Erd\'elyi-Kober operator though there is a close resemblance with the operator in question. We also give semigroup, boundedness, shift and integration-by-parts formulas for completeness.
Until very recently, the fractional calculus had been a purely mathematical subject without apparent applications. Nowadays, it plays a major role in modeling anomalous behavior and memory effects and appears naturally in modeling long-term behaviors, especially in the areas of viscoelastic materials and viscous fluid dynamics [25,26]. Fractional integrals alone, without its counterpart, naturally appear in certain modeling and theoretical problems, for example, probability theory [1], surface-volume reaction problems [11], anomalous diffusion [28], porous medium equations [29,30], and numerical analysis [2], among other applications. Now, consider the following generalized integral. Definition 1.1. Let f ∈ X p c (a, b) [16], α > 0, ρ, η, κ ∈ R. The left-sided generalized fractional integral is defined by, if the integral exists.
It can be seen that this integral generalizes four existing integrals. For η = 0, κ = 0, the Riemann-Liouville fractional integral is obtained when ρ = 1, while the Katugampola integral is obtained if β = α, further, in this case, when ρ → 0 + , the integral coincides with Hadamard integral, which can be easily verified using L'Hospital rule. Now, for β = 0, κ = −ρ(α + η), and any η, it gives the Erdélyi-Kober(type) operator. It should be remarked that ρ 1−β is complex when β Z and ρ < 0 and can be treated using theory of complex analysis considering appropriate branches.
Fractional integrals sometimes work in pairs, specially in variational calculus [2]. The corresponding right-sided fractional integral can be defined as, if the integral exists.
In the Definitions (1) and (2), we can also consider the cases a = −∞ and b = ∞, respectively, and are known in the literature as Weyl and Liouville type integrals, respectively. Such integrals are corresponding to infinite memory effects and have applications in financial mathematics and diffusion models [5,33].
Let us note that, using the change of variable u = (τ /x) ρ , the integral (1) can be rewritten in the Riemann-Liouville form as
Riemann-Liouville integral Hadamard integral
Riemann-Liouville type integral Further the Riemann-Liouville fractional integral is used to define both the Riemann-Liouville and the Caputo fractional derivatives [23,32]. To justify the claim about the Hadamard integral, when κ = 0 and β = α > 0, using L'Hospital rule and taking ρ → 0 + , we have which is the Hadamard fractional integral [23, p.110]. It should be pointed out that a similar result is not possible with the Erdélyi-Kober operator though there is a close resemblance with the operator in (1). Recent results about the Hadamard and Hadamard-type integrals such as Hadamard-type fractional calculus [21], composition and semigroup properties [7], Mellin transforms [6], integration by parts formulae [8], Gtransform representations [22], impulsive differential equations with Hadamard fractional derivative [36,37], and Hadamard type fractional differential systems [13] can be found in the literature, among others. The Katugampola fractional integral was first introduced in [12,15] as a generalization of n−fold integral, and then a simpler version was discussed in [16] along with the corresponding fractional derivatives. The Mellin transforms of it were given in [17]. The same reference also discusses a new class of generalized Stirling numbers of 2 nd kind and a recurrence formula for such sequences. Further applications of Katugampola fractional integrals or derivatives are in probability theory [1], variational calculus [2], inequalities [9], Langevin equations [34], Fourier and Laplace transforms [20] fractional differential equations [4] and Numerical analysis [3], among others.
Main Results
For simplicity, we give the following results without proofs. The proofs of the similar results for the Erdélyi-Kober type operators can be found in the classical books by Kiryakova [24], Yakubovich For "sufficiently good" functions f, g we have the following results. 2 f, which hold in the corresponding spaces of the functions f if α 2 > 0, Similar results are also valid when a = 0 and b = ∞ and in particular for ρ = 2 and ρ = 1.
The proof of part (a) is straightforward and for completeness we shall prove parts (b) and (c) later in the paper. Here we mean by a "sufficiently good" is that f ∈ X p c (a, b) [16], that is t c−1/p f (t) ∈ L p (a, b). We need such additional conditions to guarantee convergence. Further results on such conditions can be found, for example, in [24] and [32]. In such a space we have the following boundedness result. Theorem 2.1. Let α > 0, 1 ≤ p ≤ ∞, 0 < a < b < ∞ and let ρ ∈ R and c ∈ R be such that ρ ≥ c. Then the operator ρ I α,β a+;η,κ is bounded in X p c (a, b) and Proof. The proof is similar to the case of Katugampola integral [15, Theorem 3.1]. First consider the case 1 ≤ p ≤ ∞. Since f ∈ X p c (a, b), then t c−1/p f (t) ∈ L p (a, b) and we can apply the generalized Minkowsky inequality. We thus have thus, Theorem 2.1 is proved for 1 ≤ p < ∞. For p = ∞, by taking into account the essential supremum [16,Eq. (3.2)], we have after the substitution u = x/τ . This agrees with (9) above. This completes the proof of the theorem.
For simplicity, we have only considered the cases of 0 ≤ a <≤ b < ∞. For a = −∞ and b = ∞, we obtain a different K in (8).
Next we give the semigroup properties of the integral operator.
In particular, we have Proof. For brevity we only prove the first result. The proof of the other identity is similar. Using Fubini's theorem, for "sufficiently good" function f , and Dirichlet technique [31, p.64], we have The inner integral is evaluated by the change of variable u = (τ ρ − t ρ )/(x ρ − t ρ ) and taking κ 2 = −ρη 1 into account, according to the known formulae for the beta function [23]. Substituting (14) into (13) we obtain ρ I α1,β1 are bounded in X p c (a, b), hence the relation (11) is true for f ∈ X p c (a, b). This completes the proof of the theorem 2.2.
We have the following corollary.
Now we shall prove the fractional product-integration formulae (6) for the generalized integral. A similar result is referred by some authors as fractional integration by parts formula, but in our opinion this is not similar to the integration by parts formula due to the absence of a derivative term and we shall use the former to identify it. 4 Theorem 2.4. Let α > 0, β ∈ R, 1 ≤ p ≤ ∞, 0 ≤ a < b ≤ ∞ and let ρ ∈ R and c ∈ R be such that ρ ≥ c. Then for f, g ∈ X p c (a, b) the fractional product-integration formula hold. That is, Similar results are also valid when a = 0 and b = ∞ and in particular for ρ = 2 and ρ = 1.
Remark 2.5. Instead of Eq. (2), we can also consider a more general right-fractional integral given by The drawback is that the results in Theorem 2.4 would take a more complicated form. This explains the rationale behind the choice of the parameter(s) ρη of Eq. (2).
Remark 2.6. The generalized fractional integral introduced in this paper has a corresponding generalized fractional derivative which unifies the six fractional derivatives, namely, the Riemann-Liouville, Hadamard, Erdélyi-Kober, Katugampola, Weyl and Liouville fractional derivatives in to one form and will be discussed in another article. | 2016-12-22T16:48:32.000Z | 2016-12-22T00:00:00.000 | {
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237605424 | pes2o/s2orc | v3-fos-license | Generalisations and improvements of New Q-Newton's method Backtracking
In this paper, we propose a general framework for the algorithm New Q-Newton's method Backtracking, developed in the author's previous work. For a symmetric, square real matrix $A$, we define $minsp(A):=\min _{||e||=1} ||Ae||$. Given a $C^2$ cost function $f:\mathbb{R}^m\rightarrow \mathbb{R}$ and a real number $0<\tau $, as well as $m+1$ fixed real numbers $\delta _0,\ldots ,\delta _m$, we define for each $x\in \mathbb{R}^m$ with $\nabla f(x)\not= 0$ the following quantities: $\kappa :=\min _{i\not= j}|\delta _i-\delta _j|$; $A(x):=\nabla ^2f(x)+\delta ||\nabla f(x)||^{\tau}Id$, where $\delta$ is the first element in the sequence $\{\delta _0,\ldots ,\delta _m\}$ for which $minsp(A(x))\geq \kappa ||\nabla f(x)||^{\tau}$; $e_1(x),\ldots ,e_m(x)$ are an orthonormal basis of $\mathbb{R}^m$, chosen appropriately; $w(x)=$ the step direction, given by the formula: $$w(x)=\sum _{i=1}^m\frac{<\nabla f(x),e_i(x)>}{||A(x)e_i(x)||}e_i(x);$$ (we can also normalise by $w(x)/\max \{1,||w(x)||\}$ when needed) $\gamma (x)>0$ learning rate chosen by Backtracking line search so that Armijo's condition is satisfied: $$f(x-\gamma (x)w(x))-f(x)\leq -\frac{1}{3}\gamma (x)<\nabla f(x),w(x)>.$$ The update rule for our algorithm is $x\mapsto H(x)=x-\gamma (x)w(x)$. In New Q-Newton's method Backtracking, the choices are $\tau =1+\alpha>1$ and $e_1(x),\ldots ,e_m(x)$'s are eigenvectors of $\nabla ^2f(x)$. In this paper, we allow more flexibility and generality, for example $\tau$ can be chosen to be $<1$ or $e_1(x),\ldots ,e_m(x)$'s are not necessarily eigenvectors of $\nabla ^2f(x)$. New Q-Newton's method Backtracking (as well as Backtracking gradient descent) is a special case, and some versions have flavours of quasi-Newton's methods. Several versions allow good theoretical guarantees. An application to solving systems of polynomial equations is given.
Introduction
Let f : R m → R be a C 2 function, with gradient ∇f (x) and Hessian ∇ 2 f (x). Newton's method x n+1 = x n − (∇ 2 f (x n )) −1 ∇f (x n ) (if the Hessian is invertible) is a very popular iterative optimization method. It seems that every month there is at least one paper about this subject appears on arXiv. One attractive feature of this method is that if it converges then it usually converges very fast, with the rate of convergence being quadratic, which is generally faster than that of Gradient descent (GD) methods. We recall that if {x n } ⊂ R m converges to x ∞ , and so that ||x n+1 − x ∞ || = O(||x n − x ∞ || ǫ ), then ǫ is the rate of convergence of the given sequence. If ǫ = 1 then we have linear rate of convergence, while if ǫ = 2 then we have quadratic rate of convergence.
However, there is no guarantee that Newton's method will converge, and it is problematic near points where the Hessian is not invertible. Moreover, it cannot avoid saddle points. Recall that a saddle point is a point x * which is a non-degenerate critical point of f (that is ∇f (x * ) = 0 and ∇ 2 f (x * ) is invertible) so that the Hessian has at least one negative eigenvalue. Saddle points are problematic in large scale optimization (such as those appearing in Deep Neural Networks, for which the dimensions could easily be millions or billions), see [4,5].
There are many modifications of Newton's method. The intended readers can see an overview in [13]. This paper concerns only the versions recently developed in The author's joint paper [13] defined a new modification of Newton's method which is easy to implement, while can avoid saddle points and as fast as Newton's method. It is recalled in Algorithm 1. Here we explain notations used in the description. Let A : R m → R m be an invertible symmetric square matrix. In particular, it is diagonalisable. Let V + be the vector space generated by eigenvectors of positive eigenvalues of A, and V − the vector space generated by eigenvectors of negative eigenvalues of A. Then pr A,+ is the orthogonal projection from R m to V + , and pr A,− is the orthogonal projection from R m to V − . As usual, Id means the m × m identity matrix.
Algorithm 1: New Q-Newton's method
Result: Find a critical point of f : R m → R Given: {δ 0 , δ 1 , . . . , δ m } ⊂ R (chosen randomly) and α > 0; Experimentally, on small scale problems, New Q-Newton's method works quite competitive against the methods mentioned above, see [13]. However, while it can avoid saddle points, it does not have a descent property, and an open question in [13] is whether it has good convergence guarantees. More recently, in [7], the author defined New Q-Newton's method Backtracking, see Algorithm 2 below, which resolves this convergence guarantee issue. It incorporates Backtracking line search into New Q-Newton's method. Its use of hyperparameters is more sophisticated than that of New Q-Newton's method, and we need some notations. For a symmetric, square real matrix A, we define: sp(A) = the maximum among |λ|'s, where λ runs in the set of eigenvalues of A, this is usually called the spectral radius in the Linear Algebra literature; and minsp(A) = the minimum among |λ|'s, where λ runs in the set of eigenvalues of A, this number is non-zero precisely when A is invertible.
Algorithm 2: New Q-Newton's method Backtracking
Result: Find a critical point of f : R m → R Given: {δ 0 , δ 1 , . . . , δ m } ⊂ R (chosen randomly), 0 < α and 0 < γ 0 < 1; There are two main differences between New Q-Newton's method and New Q-Newton's method Backtracking. First, in the former we only need det(A k ) = 0, while in the latter we need the eigenvalues of A k to be "sufficiently large". Second, the former has no line search component, while the latter has. Note that the w k in Algorithm 2 satisfies || w k || ≤ 1, and if || w k || < 1 then w k = w k . On the other hand, if one wants to keep closer to New Q-Newton's method, one can apply line search directly to w k , and obtain a variant which will be named New Q-Newton's method Backtracking S, see Section 2 for its description and theoretical guarantees.
In [7], there are also some other versions of New Q-Newton's method Backtracking, both theoretically and practically. For simplicity, here we concerns only the above version. It is shown in [7] that any cluster point of a sequence {x n } constructed by New Q-Newton's method is a critical point of f . Moreover, in case f is a Morse function, then it is shown that if the initial point x 0 is random, then either lim n→∞ ||x n || = ∞ or {x n } converges to a local minimum with quadratic rate of convergence. In an updated version of [13], this is used to show theoretically that one can quickly find roots of univariate meromorphic functions, and experiments show that even New Q-Newton's method works well for this task.
This suggests that New Q-Newton's method Backtracking (or New Q-Newton's method) also has good theoretical guarantees when the cost function is real analytic, or more generally if both f and ∇f satisfy Lojasiewicz gradient inequality. Besides Morse functions, the latter type of cost functions are met in many realistic applications, for example in robotics or Deep Neural Networks.
Experimentally, this is confirmed on various examples, including non-smooth ones, see [7]. In this paper, we provide more theoretical support to this, by showing that a modification of New Q-Newton's method Backtracking indeed has this good theoretical guarantee.
We start with a generalised framework, see Algorithm 3.
. . , e m,k an appropriately chosen orthonormal basis for R m There are two main changes from New Q-Newton's method Backtracking, which bring in more generalities and flexibilities as well as more convergence guarantees. First, the choice of the exponent µ is now allowed to be ≤ 1, which allows more convergence guarantees. Second, we don't need to choose e i,k to be eigenvectors of ∇ 2 f (x k ).
We now check that the new algorithm includes both New Q-Newton's method Backtracking and Backtracking gradient descent as special cases.
Roughly speaking, near a non-degenerate critical point, then the best learning rate one can choose for Armijo's condition is about 1/||∇ 2 f (x k )|| (see e.g. [11]). The above version gives a more precise value of the learning rate to start with. Compare also [10,8,7].
We now define some new special cases of New Q-Newton's method Backtracking G. We fix e 1 , . . . , e m an orthonormal basis for R m .
New Q-Newton's method Backtracking G1: We choose 0 < τ ≤ 1, and choose e 1,k , . . . , e m,k to consist of eigenvectors of A k . This is almost the same as New Q-Newton's Backtracking, except The benefit is that this allows one to prove new theoretical guarantees for real analytic cost functions, while the associated dynamical system is still C 1 near non-degenerate critical points and hence it still can avoid saddle points.
This version is probably the less computationally expensive, and hence has some favours of quasi-Newton's methods. Both New Q-Newton's method Backtracking (G1) (where we always follow rule i) and G2 (where we always follow rule ii) are special cases of New Q-Newton's method G3. Here is one way to combine both of them: New Q-Newton's method Backtracking G4: If minsp(A k ) ≥ κ||∇f (x k )|| 1/2 , then we choose e 1,k , . . . , e m,k to be eigenvectors of ∇ 2 f (x k ). Otherwise, we choose e 1.k , . . . , e m,k to be e 1 , . . . , e k .
In the remaining of this section, we present the good theoretical guarantees of the new algorithms.
We recall that if {x n } ⊂ R m has a subsequence {x n k } converging to a point x ∞ , then x ∞ is a cluster point of {x n }. By definition, the sequence {x n } is convergent if it is bounded and has exactly one cluster point. We note that the below results generalise those known for Backtracking gradient descent and New Q-Newton's method Backtracking. Note that when 0 < τ < 1, then part 2 of Theorem 1.1 gives a stronger result.
Next, we discuss the applications to cost functions f which are real analytic or more generally both f and ∇f satisfy Lojasiewicz gradient inequality (need near critical points of f only). These types of cost functions appear in many realistic applications such as in robotics and Deep Neural Network. Recall that a function f satisfies Lojasiewicz gradient inequality at a point x * if there is a small neighbourhood U of x * , a constant 0 < µ < 1 and a constant C > 0 so that for all x, y ∈ U we have We will need the following quantity in statements of the next results: Definition (Lojasiewicz exponent): Assume that f : R m → R has the Lojasiewicz gradient inequality near its critical points. Then at each critical point x * of f , we define µ(x * ) := inf{µ : there is an open neighbourhood U of x * and a constant C > 0 so that for all We say that the gradient ∇f satisfies Lojasiewicz gradient inequality at a point x * , if the function theorem, if f is real analytic (and hence F (x, y) is also real analytic), then f and its gradient satisfy Lojasiewicz gradient inequality. Hence, the next theorem can be applied to quickly finding roots of systems of (real or complex) analytic equations. Part 3 of its in particularly generalises a result in [7], which treats the case of finding roots of univariate meromorphic functions (i.e. meromorphic functions in 1 variable). The result in part 1 is new only when µ = 1 (the remaining case is covered by part 2 of Theorem 1.1). In parts 2 and 3, we do not require that f has only countably many critical points. In part 2), it is allowed that sup ∇f (x * )=0 µ(x * ) × (1 + τ ) = 1, provided the supremum is not attained at any critical point x * . On the other hand, since in general µ(x * ) ≥ 1/2, it follows that if µ(x * )(1+ τ ) < 1 then we should have τ < 1. The condition in part 3) is proven in [13] to be satisfied for the case where u = the real part of g and v = the imaginary part of g, and g is a univariate holomorphic (more generally, meromorphic) function. The next result is inspired by the mentioned result in [7], and generalises it for the case of polynomial equations.
(Note, however, the proof in [7] The remaining of this paper is organised as follows. In Section 2 we prove the main theoretical results. In the last section we will explore the feasibility of implementation of the algorithms and of the assumptions in the theoretical results.
Acknowledgments. The author is partially supported by Young Research Talents grant 300814 from Research Council of Norway.
3) By the proof of [13,8], it suffices to show that near a non-degenerate critical point, the associated dynamical system is C 1 , and hence Stable-Central manifold theorems can be applied.
So, we let x * be a non-degenerate critical point of f , and show that the assignment First of all, as argued in [7], near a non-degenerate critical point we have γ(x) = 1. Therefore, we need to show that the assignment x → x − w(x) is C 1 near x * . This is clearly the case when τ > 1, in that case the map x → ||∇f (x)|| τ is C 1 .
Even when µ ≤ 1, we still have that H(x) is C 1 . Indeed, since x → ||∇f (x)|| τ is C 1 at noncritical points, it can be checked that H(x) is C 1 for non-critical points. Since x * is the only critical point of f in a small neighbourhood of itself, it remains to check that the gradient of H at x * exists, and gives rise to a C 1 function.
We consider first the case where e 1 (x), . . . , e m (x)'s are C 1 functions near x * . Then, The first sum on the RHS is clearly defined at x * as well, and is continuous there. Hence, it suffices to show that the same is true for the second sum. We will show that indeed To this end, we recall that by arguments in [13], near x * we have δ(x) = δ 0 . Hence A(x) = By chain rule, the norm of ∇||∇f (x)|| τ is bounded by ||∇f (x)|| τ −1 . Therefore, the norm of is bounded by ||∇f (x)|| τ , which converges to 0 as x converges to x * . From this, it is easy to complete the proof.
Now we consider the remaining case that e 1 (x), . . . , e m (x) are eigenvectors of ∇ 2 f (x) near x * .
In this case, the functions x → e j (x) may not be C 1 , and hence the above argument cannot be directly applied. However, we can use the integral representations of pr A(x),± as in [13], and then the above argument can be similarly applied.
5) This is well-known for methods having the descent property in part 0.
Proof of Theorem 1.2. We can use verbatim the proof for the corresponding property for New Q-Newton's method Backtracking in [7].
Hence, by assumption, there exists C > 0 and µ < 1, and an open neighborhood U of x * as well as ǫ > 0 so that for all x ∈ U and all ||y|| = 1: Then, by replacing C with a bigger constant, and substituting x by x n k and y by e j,k we obtain also the inequality | < ∇f (x n k ), e j,k > | µ ≤ C(||∇ 2 f (x n k ).e j,k + δ||∇f (x n k )||e j,k || + ||∇f (x n k )||).
If we choose δ appropriately from the finite set δ 0 , . . . , δ m , we obtain A(x n k ).
2) Similar to [13], we need to show that if x * is a critical point of f , there exists a constant C > 0 and θ < 1 so that if a subsequence {x n k } converges to x * , then: lim k→∞ ||w n k || = 0, The equality follows from part 2 of Theorem 1.1 (if 0 < τ < 1) and from part 1 above if τ = 1.
Now we prove the inequality. We have Then, computed as in [7], we obtain From this, the claim follows by the definition of µ(x * ): we can choose θ = µ(1 + τ ) < 1 for some choices of µ ≥ µ(x * ).
3) The proof is similar to that in [13].
Feasibility
We provide some comments in the feasibility of the assumptions in the main results, as well as the implementation.
The assumptions: The assumptions that f is Morse or more generally has at most countably many critical points is a local condition and is satisfied generically.
The assumption that f has Lojasiewicz gradient inequality is satisfied by many interesting cost functions, and relevant to those used in Deep Learning.
Experimentally, we found that choosing τ > 1 gives similar performance.
The assumption µ(x * )(1 + τ ) < 1 for all critical points x * is roughly satisfied for example when the multiplicities at the critical points are bounded from above by a finite number, and τ < 1 is small enough.
For the assumption lim inf k→∞ min i∈Λ k ||A k .e i,k || max i∈Λ k ||A k .e i,k || > 0, in part 3 of Theorem 1.3: The term on the LHS is related to the "condition number" for the matrix A k . Note that if at each step k one chooses the vectors e 1,k , . . . , e m,k randomly (or if one pertube them randomly), then each e j,k would not be completely orthogonal to the eigenvector(s) of the largest eigenvalue of A k , and hence one expect that the mentioned assumption would hold. Note that actual computations on computers have inherent errors, and hence can contribute to make the process behave as random.
Implementation:
If one wants to compute minsp(A k ), then there are two ways to go. First, one can compute the characteristic polynomial p k (t) of A k , and then uses one numerical method (e.g. New Q-Newton's method or Backtracking gradient descent) to find if p k (t) has a root in the ball {z ∈ C : |z| < κ||∇f (x k )|| τ }. Second, one can use numerical methods for the optimization problem < Ax, x > 2 for x on the Riemannian manifold {||x|| = 1}.
If one does not check the condition minsp(A k ) ≥ κ||∇f (x k )|| τ , but only checks the weaker condition det(A k ) = 0, then experimental results (on various types of problems) are similar. An other method, which is both theoretically and practically sound, is to choose δ randomly. This way, with probability 1 one has minsp(A k ) "large enough" (while not need to check it or even the weaker condition that A k is invertible). | 2021-09-24T01:15:31.262Z | 2021-09-23T00:00:00.000 | {
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13895171 | pes2o/s2orc | v3-fos-license | Presynaptic I 1 -Imidazoline Receptors Reduce GABAergic Synaptic Transmission in Striatal Medium Spiny Neurons
ImidazolinereceptorsareexpressedwidelyintheCNS.Inthepresentstudy,whole-cellpatch-clamprecordingsweremadefrommedium spinyneuronsindorsalstriatumslicesfromtheratbrain,andtherolesofI 1 -imidazoline receptors in the modulation of synaptic transmission were studied. Moxonidine, an I 1 -imidazoline receptor agonist, decreased the GABA A receptor-mediated IPSCs in a concentration-dependent manner. However, glutamate-mediated EPSCs were hardly affected. The depression of IPSCs by moxonidine was antagonized by either idazoxan or efaroxan, which are both imidazoline receptor antagonists containing an imidazoline moiety. In contrast, yohimbine and SKF86466 (6-chloro-2,3,4,5-tetrahydro-3-methyl-1 H -3-benzazepine), which are (cid:1) 2-adrenergic receptor antagonists with no affinity for imidazoline receptors, did not affect the moxonidine-induced inhibition of IPSCs. Moxonidine increased the paired-pulse ratio and reduced the frequency of miniature IPSCs without affecting their amplitude, indicating that this agent inhibits IPSCs via presynaptic mechanisms. Moreover, the sulfhydryl alkylating agent N -ethylmaleimide (NEM) significantly reduced the moxonidine-induced inhibition of IPSCs. Thus, the activation of presynaptic I 1 -imidazoline receptors decreases GABA-mediated inhibition of medium spiny neurons in the striatum, in which NEM-sensitive proteins such as G i/o -type G-proteins play an essential role. The adenylate cyclase activator forskolin partly opposed IPSC inhibition elicited by subsequently applied moxonidine. Furthermore, the protein kinase C (PKC) activator phorbol 12,13-dibutyrate attenuated and the PKC inhibitor chelerythrine potentiated the moxonidine-induced inhibition of IPSCs. These results suggest that IPSC inhibition via presynaptic I 1 -imidazoline receptors involves intracellular adenylate cyclase activity and is influenced by static PKC activity in the striatum.
Introduction
Accumulating evidence suggests that there are specific binding sites recognized by imidazolines and their structurally related compounds in the peripheral and central tissues.Such binding sites have been separated into at least three subclasses, defined as I 1 -, I 2 -, and non-I 1 /I 2 -(ϭ I 3 ) imidazoline receptors (for review, see Eglen et al., 1998).Although the ␣2-adrenergic receptor ligands containing an imidazoline moiety display high affinity for imidazoline receptors, there has been no reported correlation between the distribution pattern of the imidazoline receptors and ␣2-adrenergic receptors in many brain regions (De Vos et al., 1994;Piletz et al., 2000).
The physiological functions of imidazoline receptor subtypes are not fully determined, but it seems likely that I 1 -imidazoline receptors in the brainstem participate in the regulation of arterial blood pressure (Bousquet, 1995;Ernsberger and Haxhiu, 1997).It has been reported that I 1 -imidazoline receptors are subcellularly located on the plasma membrane (Piletz and Sletten, 1993;Ernsberger and Shen, 1997).In contrast, some populations of I 2 -imidazoline receptors are located intracellularly on the outer membrane of mitochondria and inhibit monoamine oxidase activity (Carpe ´ne ´et al., 1995;Ozaita et al., 1997;Lalies et al., 1999).I 3 -imidazoline receptors have been shown to act peripherally in the regulation of ATP-sensitive K ϩ channels coupled with insulin secretion (Chan et al., 1994;Olmos et al., 1994;Zaitsev et al., 1999) and centrally in the modulation of firing activity of locus ceruleus neurons (Ugedo et al., 1998) and spinal reflexes (Kino et al., 2005).
It is widely accepted that I 1 -and I 2 -imidazoline receptors are distributed in the CNS (Bricca et al., 1989;Ernsberger et al., 1995;Lione et al., 1998;Ruggiero et al., 1998).A previous study by De Vos et al. (1994) using autoradiography has revealed that the striatum is one of the regions of the human brain that is richest in I 1 -and I 2 -imidazoline receptors.Similarly, in rodents, a moderate density of these receptor subtypes has been demonstrated (Kamisaki et al., 1990;Ruggiero et al., 1998) (but see Vauquelin et al., 1999).Evidence that I 2 -imidazoline receptor binding is altered in the putaminal tissue taken postmortem from patients with Huntington's disease (Reynolds et al., 1996) and that the I 2imidazoline receptor ligand 2-(2-benzofuranyl)-2-imidazoline increases dopamine release in the striatum shown in vivo (Sastre-Coll et al., 2001) indicates that imidazoline receptors may influence neuronal function in the striatum.However, no study has provided direct evidence of modulatory roles for I 1 -and/or I 2 -imidazoline receptors detected electrophysiologically in the striatum.In the study presented here, we focused on I 1 -imidazoline receptors and explored their roles in the modulation of excitatory and inhibitory synaptic transmission in the dorsal striatum by using moxonidine, a mixed I 1 -imidazoline and ␣2-adrenergic receptor agonist with at least 40-fold selectivity for I 1 -imidazoline receptors over ␣2adrenergic receptors (Ferry et al., 1988;Ernsberger et al., 1993) and demonstrated that activation of I 1 -imidazoline receptors reduces GABAergic inhibitory synaptic transmission by N-ethylmaleimide (NEM)-sensitive protein-mediated presynaptic mechanisms.
Materials and Methods
All of the experimental protocols used here were approved by the Animal Care and Use Committee of Nagoya City University and were performed according to the guidelines of the National Institutes of Health and the Japanese Pharmacological Society.
Patch-clamp electrophysiology.Whole-cell voltage-clamp recordings were made from visually identified medium spiny neurons in the dorsal striatum using an upright microscope with a water-immersion objective lens (40ϫ; Zeiss) with Nomarski optics.The patch electrodes (2.5-3 m tip diameter) were pulled from borosilicate glass capillaries (Harvard Apparatus, Edenbridge, UK) and had a resistance of 3-5 M⍀ when filled with the internal solution containing the following (in mM): 140 CsCl, 10 HEPES, 1.1 EGTA, 2 MgCl 2 , 3 MgATP, and 0.3 Tris-GTP, pH 7.4 adjusted with CsOH.Synaptic currents were evoked by stimulating the neighboring area (within a 40 -100 m radius of the recorded neuron) via a glass pipette filled with 1 M NaCl.A voltage pulse 0.2 ms in duration at 0.1 Hz was applied at suprathreshold intensity via the stimulating electrode.Glutamate-mediated EPSCs and GABAergic IPSCs were recorded in the presence of (Ϫ)-bicuculline methiodide [10 M (referred to simply as bicuculline)] and 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) (10 M), respectively, at a holding potential of Ϫ60 mV.When spontaneous miniature IPSCs (mIPSCs) were recorded, tetrodotoxin (TTX) (0.5 M) was further added to the extracellular solution containing CNQX (10 M).Under these experimental conditions, EPSCs, IPSCs, and mIPSCs were detected as inward deflections (Momiyama and Koga, 2001).Whole-cell currents were amplified (EPC-7; List Medical, Darmstadt, Germany), low-pass filtered at 4 kHz, and digitized at 10 kHz for computer analysis with pClamp7 software (Molecular Devices, Union City, CA).The access resistance was monitored by measuring capacitative transients obtained in response to a hyperpolarizing voltage step (10 mV, 10 ms) from a holding potential of Ϫ60 mV.All experiments were performed at 33-34°C.
The effects of moxonidine on evoked IPSCs were evaluated by comparing averaged IPSCs taken during the peak responses to the drug (six traces for 1 min) with those before drug application (18 traces for 3 min).mIPSCs were analyzed using N software (kindly provided by Dr. S. F. Traynelis, Emory University, Atlanta, GA), and the frequency and amplitude distribution of the events for 5-10 min during the peak response to moxonidine were compared with those obtained before application of the drug.The concentration-inhibition curve of IPSCs obtained with moxonidine was fitted, and the IC 50 value was determined using nonlinear regression analysis with the embedded logistic function in Origin (Microcal Software, Northampton, MA): %I ϭ E max /{1 ϩ (IC 50 /[D]) n }, where %I is the percentage inhibition of IPSCs, [D] is the concentration of moxonidine, E max is the maximal percentage inhibition of IPSCs, and n is the Hill slope.
All data are expressed as means Ϯ SEM.Student's t test (two-tailed) was used to compare the data for two groups.Two-tailed t test with Bonferroni's correction following one-way ANOVA was used for multiple comparisons between the control and the treated groups (Wallenstein et al., 1980).The Kolmogorov-Smirnov test was used for comparison of the cumulative probability distributions of mIPSCs.Differences at p Ͻ 0.05 were considered to be significant.
Inhibition of GABA A receptor-mediated synaptic currents by moxonidine in striatal neurons
Bath application of moxonidine inhibited GABAergic IPSCs elicited in cells voltage clamped at a holding potential of Ϫ60 mV in a concentration-dependent manner.The recorded GABAergic IPSCs were mediated by GABA A receptors, because they were abolished by bicuculline (10 M; data not shown).Figure 1 A illustrates the typical time course of IPSC suppression in response to moxonidine (30 M).This concentration of moxonidine superfused for 5 min reduced IPSCs by 72.0 Ϯ 3.9% (n ϭ 5), and the effect was slowly reversed during washout.The concentrationinhibition curve, constructed at a concentration range between 1 and 100 M (n ϭ 4 -5), revealed an IC 50 of 3.4 M (Fig. 1C).In contrast, moxonidine was much less effective on glutamatergic EPSCs and reduced EPSCs by only 10.9 Ϯ 5.6% (n ϭ 6) and 23.6 Ϯ 7.6% (n ϭ 5) at concentrations of 30 and 100 M, respectively (Fig. 1 B, C).Therefore, only the effect of moxonidine on GABAergic IPSCs was studied further.
Determination of the synaptic site of action of moxonidineinduced inhibition of IPSCs
To determine the synaptic site of action of moxonidine, we first examined the effect of moxonidine on the ratio of the amplitude of the second IPSC divided by that of the first one [paired-pulse ratio (PPR)] elicited by two successive stimuli of identical strength at an interval of 50 ms.A change in the PPR is considered to be attributable to a presynaptic change in release probability (Zucker, 1989;Manabe et al., 1993).A typical example of IPSCs in Figure 4, A and B, shows a change in the PPR from depression to facilitation by application of moxonidine (30 M).In four cells tested, moxonidine (30 M) increased the PPR from 0.60 Ϯ 0.05 to 1.13 Ϯ 0.10 ( p Ͻ 0.01), suggesting that moxonidine decreases the release probability of GABA presynaptically.
To further establish the mode of presynaptic action of moxonidine in the inhibition of GABAergic inhibitory synaptic transmission, the effects of moxonidine (30 M) on action potentialindependent spontaneous mIPSCs recorded in the presence of CNQX (10 M) and TTX (0.5 M) were examined.Sample recordings before and during application of moxonidine (30 M) in Figure 5A show that application of moxonidine (30 M) reduced the frequency of mIPSCs.This was clearly evidenced by the cumulative probability distributions of mIPSC interevent intervals and amplitude (Fig. 5 B, C, respectively) constructed from the same cell before and during application of moxonidine, demonstrating that moxonidine shifted significantly the interevent interval distribution to longer intervals without altering the amplitude distribution.In five cells, moxonidine (30 M) decreased the mean frequency of mIPSCs from 1.6 Ϯ 0.1 to 0.5 Ϯ 0.1 Hz ( p Ͻ 0.01) (Fig. 5D).In contrast, the mean amplitude of mIPSCs before and during application of moxonidine were 35.9 Ϯ 6.2 and 35.0 Ϯ 5.9 pA, respectively ( p Ͼ 0.05) (Fig. 5D).Together, these results demonstrate that moxonidine reduced GABAergic synaptic transmission in the medium spiny neurons by acting on presynaptic I 1 -imidazoline receptors.
Effects of NEM on moxonidine-induced inhibition of IPSCs
Previous studies have suggested that I 1 -imidazoline binding sites are coupled with G-proteins (Molderings et al., 1993;Ernsberger and Shen, 1997;Takada et al., 1997;Greney et al., 2000).To assess whether inhibition of IPSCs mediated by presynaptic I 1imidazoline receptors involves G-proteins, the effect of moxonidine on IPSCs was examined in the presence of the sulfhydryl alkylating agent NEM, which attacks sulfhydryl groups of cys-Figure 2. I 1 -imidazoline receptors mediate the moxonidine-induced inhibition of IPSCs.A, B, Moxonidine (mox; 30 M) was applied in the presence of efaroxan (efa; 10 M) or idazoxan (ida; 10 M), which block both imidazoline receptors (I 1 for efaroxan and I 1,2 for idazoxan) and ␣2-adrenergic receptors.The inhibitory effect of moxonidine on IPSCs was primarily attenuated.C, D, No impairment of the moxonidine (30 M)-induced inhibition of IPSCs in the presence of yohimbine (yoh; 10 M) and SKF86466 (SKF; 10 M), both of which are ␣2-adrenergic receptor antagonists with no affinity for imidazoline receptors.Each point represents the mean amplitude of six consecutive IPSCs evoked at 0.1 Hz.Example traces on each graph are the averaged IPSCs in control and in moxonidine (smaller trace).Calibration: 500 pA, 20 ms.E, Summary of the effects of imidazoline and/or ␣2-adrenergic receptor antagonists on the moxonidine-induced inhibition of IPSCs.Each column represents the mean Ϯ SEM.The significance of differences between the values for the moxonidine alone (n ϭ 5) and antagonisttreated groups (n ϭ 4 -5) was determined with the two-tailed multiple t test with Bonferroni's correction following ANOVA (4 comparisons in 5 groups).**p Ͻ 0.01.teine residues of the proteins, including pertussis toxin (PTX)sensitive G i/o -type G-proteins (Shapiro et al., 1994;Momiyama and Koga, 2001).As shown in Figure 6 A, NEM (50 M) alone did not affect IPSCs.However, subsequent application of moxonidine (30 M) resulted in only slight depression of IPSCs.In five cells recorded in the presence of NEM, moxonidine (30 M) reduced IPSCs by 10.4 Ϯ 8.2%, which was significantly smaller than the inhibition elicited by moxonidine alone (67.6 Ϯ 5.4%; p Ͻ 0.01; n ϭ 5).Thus, NEM-sensitive proteins such as G i/o -type G-proteins are involved in the presynaptic I 1 -imidazoline receptor-mediated inhibition of IPSCs.Although we need additional evidence to conclude that presynaptic I 1 -imidazoline receptors are coupled with G i/o -type G-proteins, the final set of experiments reported here assessing the intracellular signaling was made partly based on the assumption that G i/o -type G-proteins are involved.
Intracellular signaling mediating and affecting the moxonidine-induced inhibition of IPSCs
Because G i/o -type G-proteins are coupled negatively to adenylate cyclase activity, we first explored the possibility that a decrease in adenylate cyclase activity was the intracellular element involved in the inhibition of IPSCs by moxonidine.Bath application of forskolin (10 M), an activator of adenylate cyclase, increased IPSCs by 170.7 Ϯ 25.8% (data not shown; n ϭ 6).Subsequent application of moxonidine (30 M) in the presence of forskolin reduced IPSCs by 53.7 Ϯ 3.5% (n ϭ 6) (Fig. 7A, left, B), which was significantly different from the control value for moxonidine alone (68.1 Ϯ 5.1%; p Ͻ 0.05; n ϭ 5).This result suggests that forskolin may have opposed the decrease in adenylate cyclase activity that was involved partly in the inhibition of IPSCs via presynaptic I 1 -imidazoline receptors.
We then examined whether an increase in protein kinase C (PKC) activity, which would also be expected in the effect mediated by G i/o -type G-proteins, was involved in the inhibition of IPSCs in response to moxonidine.However, bath application of the PKC activator PDBu (0.5 M) increased IPSCs by 217.5 Ϯ 26.3% (data not shown; n ϭ 7), suggesting that PKC activation does not function in downstream signaling leading to inhibition of IPSCs after occupation of presynaptic I 1 -imidazoline receptors.Surprisingly, application of moxonidine (30 M) in the presence of PDBu decreased IPSCs by only 29.2 Ϯ 2.9% (n ϭ 7) (Fig. 7A, middle, B), which was significantly different from the control value for moxonidine alone (70.3 Ϯ 4.5%; p Ͻ 0.01; n ϭ 5).Moreover, the PKC inhibitor chelerythrine (5 M), which alone did not significantly change IPSCs, augmented the moxonidine-induced inhibition of IPSCs (88.1 Ϯ 3.7%; n ϭ 5) (Fig. 7A, right, B), which was significantly different from the control value for moxonidine alone (65.7 Ϯ 4.1%; p Ͻ 0.01; n ϭ 6).Thus, it is likely that the presynaptic inhibition of IPSCs mediated by I 1 -imidazoline receptors is influenced by how PKC is statically activated.
Discussion
There has been a complete lack of studies on the roles of imidazoline receptors in the regulation of neuronal activities in the striatum, despite the fact that the striatum has one of the highest density of I 1 -and I 2 -imidazoline receptors in the human brain (De Vos et al., 1994) and also has a moderate density of there receptor subtypes in the rodent brain (Kamisaki et al., 1990;Ruggiero et al., 1998) (but see Vauquelin et al., 1999).In the present study focusing on I 1 -imidazoline receptors, we demonstrated for the first time the novel role of I 1 -imidazoline receptors in the modulation of GABAergic inhibitory synaptic transmission in the medium spiny neurons of the dorsal striatum.This inhibitory effect on IPSCs was mediated by presynaptic mechanisms coupled with NEM-sensitive proteins culminating partly in a decrease of intracellular adenylate cyclase activity and was influenced by static PKC activity.
I 1 -imidazoline receptors mediate moxonidine-induced inhibition of IPSCs
We used moxonidine as a selective agonist of I 1 -imidazoline receptors.Nevertheless, its weak affinity for ␣2-adrenergic receptors (Ferry et al., 1988;Ernsberger et al., 1993) may have accounted for the observed inhibition of IPSCs.Hayar and Guyenet (2000) revealed that inhibition of both EPSCs and IPSCs by moxonidine in bulbospinal neurons of the rostral ventrolateral medulla is mediated via ␣2-adrenergic receptors.Indeed, in the present study, application of noradrenaline under blockade of both ␣1and -adrenergic receptors with prazosin and propranolol, respectively, resulted in ␣2-adrenergic receptor-mediated inhibition of IPSCs, as was demonstrated by the apparent lack of inhibition by noradrenaline when combined with either yohimbine or SKF86466, which are non-imidazoline antagonists selective for ␣2-adrenergic receptors.However, this was not the case for the inhibition of IPSCs by moxonidine in the striatum.Neither yohimbine nor SKF86466 influenced the effect of moxonidine on IPSCs.Only in the presence of efaroxan or idazoxan, which block both imidazoline receptors (I 1 for efaroxan and I 1,2 for idazoxan) and ␣2-adrenergic receptors, was the inhibitory effect of moxonidine on IPSCs essentially lost.We therefore conclude that moxonidine reduces IPSCs via I 1imidazoline receptors.
Presynaptic mechanisms underlie moxonidine-induced inhibition of IPSCs
Paired-pulse protocol and analysis of mIPSCs are the most widely used approaches for assessing the synaptic sites of drug action.Moxonidine elicited an increase in the PPR concurrently with a reduction of IPSCs, which is considered to reflect a presynaptic change in release probability (Zucker, 1989;Manabe et al., 1993).More importantly, we have observed a decrease in the frequency of mIP-SCs after application of moxonidine without any change in their amplitudes.From these two lines of evidence, it is most likely that the moxonidine-induced inhibition of IPSCs is primarily presynaptic in origin and not attributable to postsynaptic reduction in the sensitivity of GABA A receptors.Our results not only support the immunocytochemical study by Ruggiero et al. (1998), who revealed the presence of imidazoline receptors in axon terminals of the rat CNS, but demonstrate electrophysiologically, for the first time, the functional existence of presynaptic imidazoline receptors that are coupled with neurotransmitter release in the CNS.
G-proteins and intracellular signaling
NEM uncouples PTX-sensitive G i/o -type G-proteins from receptors (Shapiro et al., 1994;Momiyama and Koga, 2001).The present result clearly demonstrates that PTX-sensitive G i/o -type G-proteins couple with presynaptic I 1 -imidazoline receptors.Previous studies have suggested that the coupling of imidazoline receptors with G-proteins is based on the sensitivity of imidazoline-specific binding to GTP or nonhydrolyzable analogs (Molderings et al., 1993;Ernsberger and Shen, 1997;Greney et al., 2000).Moreover, Takada et al. (1997) have demonstrated in rats that intracerebroventricular pretreatment with PTX prevented the antidysrhythmic effect of systemically injected rilmenidine, a selective I 1 -imidazoline receptor agonist, on halothane-adrenaline dysrhythmias.Our present study may provide more details about the functional linkage of I 1 -imidazoline receptors and G-proteins, especially in the modulation of GABA release at GABAergic terminals in the dorsal striatum.However, we should take a possible involvement of other proteins that contain active cysteine residues and therefore are attacked by NEM into consideration.This remains to be clarified.
Our finding that the moxonidine-induced inhibition of IPSCs was reduced by forskolin suggests that the cAMP pathway is an intracellular target of I 1 -imidazoline receptors, and this is also in line with the suggested coupling with G i/o -type G-proteins (but see Greney et al., 2000).Because the activation of PKC with PDBu did not mimic the inhibitory action of moxonidine on IPSCs, at least diacylglyceride-sensitive PKC is unlikely to play a role in this action of presynaptic I 1 -imidazoline receptors.Rather, the finding that PDBu attenuated and the PKC inhibitor chelerythrine potentiated the moxonidine-induced inhibition of IPSCs could be explained by a mechanism similar to that demonstrated in the modulation of the coupling of metabotropic glutamate receptors to G-proteins by PKC (Macek et al., 1998;Poisik et al., 2003).Additional study is needed to explore this mechanism.
As a transduction pathway for I 1 -imidazoline receptors, activation of phosphatidylcholine-specific phospholipase C, which results in downstream activation of mitogen-activated protein kinase along with PKC, has been proposed previously in PC12 cells (Edwards et al., 2001;Zhang et al., 2001) and recently in the rostral ventrolateral medulla in vivo (Zhang and Abdel-Rahman, 2005).This pathway may be linked to the imidazoline binding protein showing characteristics of I 1 -imidazoline receptors whose gene has been recently cloned (Piletz et al., 2000).However, whether or not this pathway is associated with G-proteins remains undetermined.Because I 1 -imidazoline receptors could be coupled to more than one transduction pathway, as was demonstrated by Greney et al. (2000), the involvement of this pathway as well as participation of the atypical subtype of PKC (Edwards et al., 2001) should be also assessed in the presynaptic modulation of IPSCs via I 1 -imidazoline receptors.
Physiological implications
GABA A receptor-mediated IPSCs in the dorsal striatum arise from intrinsic GABAergic inputs through either neighboring medium spiny neurons via axon collaterals or interneurons.Re-moval of GABAergic inhibition on the medium spiny neurons by presynaptic I 1 -imidazoline receptors may result in augmentation of the striatal GABAergic output signals.At present, however, we are unable to conclude whether I 1 -imidazoline receptormediated disinhibition of the striatal output neurons results in a net increase or decrease of neuronal activities in the internal globus pallidus and substantia nigra, the basal ganglia output nuclei that send inhibitory projections to the glutamatergic thalamocortical neurons (Alexander and Crutcher, 1990).Decreased activities of the thalamocortical neurons in Parkinson's disease should be clinically normalized to achieve stimulation of movement via the cortex (Gerfen, 1992;Di Chiara et al., 1994).Considering that tizanidine, an imidazoline agent in clinical use for spasticity (Wagstaff and Bryson, 1997) with a higher affinity for I 1imidazoline receptors over ␣2-adrenergic receptors (Piletz et al., 1996), reduces muscle rigidity by acting at the supraspinal level (Honda et al., 2002;Kino et al., 2005), the therapeutic potential of specific agonists of I 1 -imidazoline receptors appears to be further established for the treatment of motor dysfunction related to the basal ganglia.
In our preliminary experiment, the I 1 -imidazoline receptor antagonist efaroxan alone did not alter the amplitude of IPSCs at higher concentrations (data not shown).The postulated endogenous ligands for imidazoline receptors, including agmatine (Reis and Regunathan, 2000), harmane (Parker et al., 2004), and imidazoleacetic acid-ribotide (Prell et al., 2004), could be easily washed out of slice preparations, thereby resulting in failure to detect their tonic inhibitory effects on IPSCs.Agmatine and imidazoleacetic acid-ribotide are considered to be synthesized from L-arginine (Reis and Regunathan, 2000) and histamine (Thomas and Prell, 1995), respectively, and released synaptically in the brain (Reis and Regunathan, 2000;Prell et al., 2004).However, the striatum is not enriched with agmatine (Reis and Regunathan, 2000), and the presence of imidazoleacetic acid-ribotide has been studied only in the brainstem neurons (Prell et al., 2004).Moreover, the formation and distribution of harmane in the brain have not been fully determined.The endogenous ligands may reach both I 1 -and I 2 -imidazoline receptors in the striatum.Activation of I 2 -imidazoline receptors inhibits monoamine oxidase (Carpe ´ne ´et al., 1995;Ozaita et al., 1997;Lalies et al., 1999) and elicits dopamine release (Sastre-Coll et al., 2001), both of which can result in an increase of extracellular dopamine.Hence, it is plausible that the endogenous ligands facilitate the striatal direct output pathway via both I 1 -imidazoline receptormediated disinhibition and I 2 -imidazoline receptor-mediated increase in dopamine.Additional studies to address the role of the endogenous ligands for imidazoline receptors will provide new insights into drug research in the field of neurodegenerative diseases related to the basal ganglia.
Figure 1 .
Figure 1.Selective inhibition of GABAergic IPSCs by moxonidine in striatal neurons.Wholecell patch-clamp recordings were made from medium spiny neurons in slices of the rat brain dorsal striatum.The cell was clamped close to Ϫ60 mV, and either GABAergic IPSCs or glutamatergic EPSCs were evoked at 0.1 Hz.A, A representative time course of the inhibition of IPSCs by moxonidine (mox; 30 M) bath applied for 5 min.Each point represents individual IPSCs.Inset, Example averaged traces of six consecutive IPSCs recorded during the times indicated on the graph in A. B, A representative time course showing almost no effect of moxonidine (30 M for 5 min) on EPSCs.Each point represents the mean amplitude of six consecutive IPSCs evoked at 0.1 Hz.C, Concentration-inhibition relationship for the effect of moxonidine on IPSCs (open circles; n ϭ 4 -5) and EPSCs (filled circles; n ϭ 4 -5).Each point represents the mean Ϯ SEM.
Figure 3 .
Figure 3. Yohimbine and SKF86466 antagonize the noradrenaline-induced inhibition of IPSCs.A, Noradrenaline (NA; 30 M) applied for 5 min in the presence of propranolol (prop; 10 M) and prazosin (pra; 10 M) reduced IPSCs.B, C, Attenuation of the noradrenaline-induced inhibition by yohimbine (yoh, B; 10 M) and SKF86466 (SKF, C; 10 M).Each point represents the mean amplitude of six consecutive IPSCs evoked at 0.1 Hz.D, Summary of the effects of the ␣2-adrenergic receptor antagonists yohimbine and SKF86466 on the noradrenaline-induced inhibition of IPSCs.Each column represents the mean Ϯ SEM.The significance of differences between the values for noradrenaline in the presence of propranolol and prazosin and ␣2adrenergic receptor antagonist-treated groups (n ϭ 4 each) was determined with the twotailed multiple t test with Bonferroni's correction following ANOVA (2 comparisons in 3 groups).**p Ͻ 0.01.
Figure 4 .
Figure 4. Moxonidine increases the paired-pulse ratio.To assess the effect of moxonidine (mox; 30 M) on the PPR, two successive stimuli of identical strength at an interval of 50 ms were applied (0.1 Hz).A, A typical time course of the increase in the PPR in response to moxonidine.Each point represents the individual PPR.B, Example averaged traces of six consecutive paired IPSCs recorded during the times indicated on the graph in A.
Figure 5 .
Figure5.Moxonidine reduces the frequency but not the amplitude of mIPSCs.The mIPSCs were recorded in the presence of CNQX (10 M) and TTX (0.5 M).A, Typical example traces recorded in control and during application of moxonidine (mox; 30 M). B, C, Cumulative probability distribution of interevent intervals (B) and peak amplitudes (C) of mIPSCs in control (open circles; 395 events) and in moxonidine (filled circles; 189 events) from the same cell as that shown in A. The interevent interval was increased, and the amplitude of IPSCs was unaffected by moxonidine.Inset on graph C shows superimposed traces of the averaged mIPSCs in control (395 events) and in the presence of moxonidine (189 events).D, Summary graphs showing that moxonidine decreased the mean frequency, without altering the mean amplitude, of mIPSCs.Each column represents the mean Ϯ SEM (n ϭ 5 each).Student's t test; **p Ͻ 0.01.
Figure 6 .
Figure 6.Effects of NEM on moxonidine-induced inhibition of IPSCs.A, A representative time course of the effect of moxonidine (mox; 30 M for 5 min) on IPSCs in the presence of NEM (50 M).Each point represents individual IPSCs evoked at 0.1 Hz.B, Example averaged traces of six consecutive IPSCs recorded during the times indicated on the graph in A.
Figure 7 .
Figure 7. Involvement of the cAMP pathway and modulation by PKC in the moxonidineinduced inhibition of IPSCs.A, Typical averaged traces of six consecutive IPSCs showing that the moxonidine-induced inhibition of IPSCs was attenuated by the adenylate cyclase activator forskolin (10 M; left) and the PKC activator PDBu (0.5 M; middle) and potentiated by the PKC inhibitor chelerythrine (5 M; right).Calibration: 500 pA, 20 ms.B, Summary of the effects of forskolin, PDBu, and chelerythrine on moxonidine-induced inhibition of IPSCs.Each column represents the mean Ϯ SEM (n ϭ 5-7).Student's t test; *p Ͻ 0.05, **p Ͻ 0.01. | 2017-04-15T03:48:06.730Z | 2006-02-08T00:00:00.000 | {
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231652498 | pes2o/s2orc | v3-fos-license | How to Control Behavioral Studies for Rodents—Don’t Project Human Thoughts onto Them
Abstract In neuroscience research, we often use behavior as an easy tool and assume a straightforward relationship between memory and behavior. However, many factors are often not accounted for and need to be considered when interpreting a behavioral outcome. This opinion article will discuss factors in rodent studies such as handling and how the animal views the world, that will affect whether memory leads to a certain behavior.
Introduction
In neuroscience, we like to test for a certain behavior and then use that as direct outcome to determine underlying mechanisms. This can make the forced swim test, tail suspension test, or open-field exploration to conclude about a depressed or anxiety state of the animal or any form of memory task to test whether a memory is intact or not. Since memory needs to be present to be able to observe the learned behavior, the tendency is to use the reverse inference: if the behavior is missing, we can conclude that the memory is not intact (Fig. 1A, how we like to see it). However, in the case of behavior the directionality of evidence is a one-way street and cannot simply be reversed. It is not behavior = memory. Instead, many other factors will influence whether the presence of memory (or depressed mood) leads to a certain behavior. To name just a few of the factors, what the animal is focusing on, how the animal views the world, and the appropriateness of the behavior in the current context will influence behavior (Fig. 1B,how it is). Therefore, if the behavioral expression of memory is not present, it does not mean memory is not intact.
One should keep in mind, with a few exceptions, that most such influencing factors have not been systematically tested for. Thus, much of what is described in this opinion piece is based on personal experience or anecdotes shared among behavioral researchers.
What is the animal focusing on?
Is the animal focused on the task and doing what you think it is doing? Of course, this is closely associated with the next point: is the animal motivated to perform the behavior that you want it to perform? Recent studies have shown that, what many have categorized as "noise" in neural recordings is actually associated with the animal doing other things than the intended task, such as subtle facial movements (Stringer et al., 2019). This is especially important when we use tasks that have just one trial as a test. If the animal is distracted and not focused on the task, you may not see the expected behavioral outcome. Distraction can be both internally generated (e.g., being more interested in grooming) and externally originated (e.g., changes in housing or testing environment can distract an animal). We might think that we can control for the external distracting factors; however, we need to keep in mind that animals will sense things we cannot. For example, their sense of smell is superior to ours and since their hearing range is different, they may hear sounds we do not hear. Very high-frequency or low-frequency sounds can be generated, for example, when drilling into foundations far away. Interestingly, the more one tries to control for such distractors by exclusion, the more salient and distracting they are to an animal when encountered. In contrast, if an animal is used to a very noisy environment, it seems to deal better with such distractors and is able to ignore them.
Motivation, "personality" and the "inner state" How motivated an animal is to do something will also influence if memory leads to a certain behavior. Motivation is not only affected by known factors such as introducing food deprivation to create motivation in an animal to look for food in a maze. Also the individual personality (for lack of a better term) of an animal will affect its general motivation. Most experimenters working with behavior will report certain types of personalities in rodents, especially noticeable in rats but also present in mice: across eight animals, one usually has one to two animals who never seem very motivated to do anything; these tend to be larger, heavier animals. There will also be at least one hyperactive animal that has issues focusing on the task at hand. These differences are usually discussed as anecdotes, but the regularity of the occurrence of different types within a cage of four animals does indicate that it might be a result of hierarchies in the cage and may represent a natural way of creating diversity in a population. Since one of four animals will usually have such a noticeable personality, which will have a large influence on motivation and consequently expressed behavior, one should be careful when interpreting individual memory expression. For example, the larger animal may not express the behavior associated with a memory because it is not motivated to pay attention to the task, but that does not mean the memory is not present in that animal.
Further, manipulating motivation does not always follow a simple linear relationship (Berditchevskaia et al., 2016). One good example is food deprivation: some food deprivation will lead to increased focus when searching for food and can motivate an animal to deliberate before deciding where to search for food. However, too much food deprivation will lead to faster search strategies that tend to include less deliberation. This U curve in food deprivation, with too little or too much causing worse expression of deliberation and subsequently memory performance, might be especially seen in tasks that are cognitively more demanding. An animal that is too food deprived tends to "think" less.
The inner state of an animal will also influence how it will behave in a task. The way any type of procedure will influence the inner state of the animal can show a natural variability across animals. A great example of the diversity of possible outcomes was shown by Fogel et al. (2011). All animals underwent the same avoidance-learning paradigm (footshock in the dark part of the box). However, later on some remembered and became active avoiders (i.e., the next day they avoided the shock zone). A second group seemed to "forget" and returned to the part of the box with the shock the next day, but as soon as the shock was reinstated, they left the zone again. Interestingly, a third group showed evidence for learned helplessness: they would go to the zone the next day and, even if shocked again, remain there. Thus, the same procedure resulted in some cases in an inner state of "depressedlike mood" or "learned helplessness," which influenced the behavioral response. It also remains unclear whether those animals "remembered" the initial experience or not. Similarly, a "depressed" animal would be less likely to be motivated to run for a food reward if it is feeling anhedonia. Thus, the inner state would influence performance in reward-related tasks independently whether an animal remembered learning about the reward or not.
Another example for intrinsic factors influencing results can be seen in the object exploration task. Generally, we use the discrimination index as an outcome in this task, for which we calculate the difference in exploration between the known and the new object. This index is based on the preference for novelty-neophilia-with animals on average exploring the novel object more than the known one. However, the difference in genetics, disease, or Figure 1. A, Many researchers think of behavior as a direct readout from, for example, memory and believe that since the memory has to be present for a certain behavior, the reverse logic is also true: if the behavior is not present, the memory must not be present as well. B, But actually, one can only draw the logical conclusion that memory may lead to a certain behavior if the behavior expressed is influenced by many other factors as well.
Opinion other factors that one plans to test in an experiment can potentially change the preference from general neophilia to neophobia. Subsequently, this would lead to a change from a positive discrimination index to a negative one, if the animal remembers the original event. Moreover, a mix of neophilia and neophobia will result in a discrimination index of zero even if the memory is present. This is important to consider, since the natural tendency of rats in the wild is neophobia. Only with domesticated rats do we see a natural tendency of neophilia.
Appropriateness of behaviors in the current context
Another, often neglected, factor is the appropriateness of a behavior in a certain context. In tasks involving fearconditioned responses, this is of special importance. Many memory researchers use freezing to detect the presence of memory. However, if freezing is the appropriate response to fear, it is very much dependent on the context. First, if freezing versus escaping is the correct response to fear, it is dependent on the possibility of escape (e.g., size of the box) as well as on the distance to the fear-inducing cue (Fanselow, 2018). Second, an animal may remember the fear-inducing event, but "realize" it was a one-time event and will not happen again. As a result, the animal should not show the freeze response despite the fact that the memory is intact. Or at the other extreme, if an animal is never handled by an experimenter and the only event it ever experienced was the fear-conditioning paradigm, it would be more likely to generalize the fear to any handling procedure by the experimenter and the fear response will be less specific. In sum, how an animal will respond to a fearful paradigm is mainly influenced by habituation procedures occurring before and after the memory-inducing event (Wang et al., 2009). This also means that the presence of freezing, specific or generalized, is very hard to directly associate with the presence or absence of a memory. As a point in case: the same principle of fear generalization from the original conditioning box to another box is used by memory researchers to discuss systems consolidation from the hippocampus to the cortex and by disease researchers to discuss the development of post-traumatic stress disorder after a trauma event.
The expression of appetitive behaviors is similar: an animal will not eat in an unfamiliar or changed environment. It needs to feel safe and to ensure the absence of danger before it will go to another type of behavior such as food consumption. Consequently, how familiar an animal is with the test environment will influence food search behavior and consumption.
Other influencing factors
So far, a few of the factors that can influence behavior have been mentioned, but, of course, there are many more. For example, the time of day at which training and testing is performed can have a large influence on both memory and behavior. Circadian oscillations can be seen in gene expression and kinase activity (Eckel-Mahan et al., 2008), and diseases can additionally affect these circadian oscillations; consequently, the time of day can differentially affect results in wild-type and disease models (Debski et al., 2020). Also, how animals are housedindividually or in groups-can affect disease expression (Bernard, 2019) as well as behavior. For example, in group-housed animals fear responses can be socially transmitted to nonshocked cage mates (Carew et al., 2018). Furthermore, animal-handling techniques such as cupping versus tail pickups can have a large effect on behavior (Hurst and West, 2010;Gouveia and Hurst, 2017), as can the sex of the experimenter (Sorge et al., 2014). It has been shown that male experimenters create an initial stress response in rodents that can affect experimental outcomes.
Rodents do not see the world as we do
One larger issue in rodent behavior, especially rodent learning tasks, is our preference to set up tasks using our human way of thinking and sensory input. Tasks tend to be based on visual input and cues, despite the fact that rodents are not as visually oriented as we are. The active time of rodents in nature is dusk and dawn, when not much light is present. Further, their natural home environments are complex underground burrow systems, which will not have a light source. This is why they rely more on olfactory cues as well as their sense of touch via whiskers. Rats also can use their anal glands to mark food areas as well as danger zones with specific smell cues.
Many researchers do not consider that rats perceive red as black (since they do not have the red receptors), and while they can see color they need to be trained to perceive and process colors. Further, they are short sighted and, in the case of albino rats, would even in human terms count as legally blind (http://www.ratbehavior.org/RatVision.htm). For examples of rat view, see Figure 2 and http://www. ratbehavior.org/RatCam.htm. Thus, what we humans may perceive as good cues and experimental setups will be viewed very differently from the rodent perspective.
The most extreme example of using human perception to design rodent experiments is the extensive use of virtual reality that is only based on visual input. The dissociation between any potential olfactory or tactile cues on the ball or treadmill used will make it even more difficult for the rodent. Most likely many virtual reality tasks are not measuring what they think they are. For example, Aghajan et al. (2015) showed that virtual reality setups that do not consider olfaction or tactile stimuli are more likely measuring distance estimation of the animal than spatial perception.
Which strategy is the rodent using?
One consequence of relying too much on our perception of what we think the animal should be doing is the rodent using a different strategy to solve a task than the one intended by the experimenter. A classic example would be finding a hidden platform in the water maze. While we like to think that an animal will use an allocentric strategy to learn the place of the platform within the environment, placement of cues, or even the sex of the animal, can result in the animal using a different strategy that relies on other brain areas. For example, having the platform close to a large dominant cue (e.g., the bunched up curtain hanging next to the maze) could lead to the animal using a beaconing strategy in which the animal just homes in on that one large beacon instead of using an integrated spatial representation of the environment. This strategy would be independent of the hippocampus in contrast to the allocentric spatial strategy. Another common strategy used by some animals is, instead of learning the location of the platform, learning the distance to the wall. If the animal always swims in a circle around the maze with a certain distance to the wall, it will find the platform more reliably than by using a spatial strategy.
Rat and mouse behavior
One key concept to keep in mind is that a rat is not a little human. Each species is specialized for certain behavior and will express it in their own way. We humans are very good at rule learning, but this is not the most natural form of learning for rodents. This is why it can take them quite a long time to learn tasks based on rules such as seemingly simple alternation or win-stay tasks, but can learn to navigate flexibly to a goal location, planning direct paths in a 9 Â 5 m maze within 20 min of first exposure (unpublished data from the HexMaze in the Genzel laboratory; https://www.genzellab.com/#/hexmaze2/). Also, a mouse is not just a little rat. Rats and mice differ in their baseline behavior. In a large maze environment, mice show much less goal-oriented behavior than rats (unpublished data from the HexMaze in the Genzel laboratory; https://www.genzellab.com/#/hexmaze2/). Further, Jones et al. (2017) could show that rats and mice differed in levels of baseline activity measured as shuttle rate during intertrial intervals; mice shuttled two to three times as frequently as rats. Species differences in behavioral ecology may underlie this difference, as mice need to move rapidly when outside of burrows to minimize predation risk. They tend to use bursts of speed to run from a more sheltered position to the next. Thus, when designing a behavioral experiment, we should not aim to test rats and mice in cognitive skills derived from human abilities; instead, we should test them on behaviors that fit their ecological niche.
Recently, Gomez-Marin and Ghazanfar (2019) extensively and beautifully discussed that it is impossible to treat animals as passive stimulus-response machines. Instead, we must consider their behavior in the context of their species-specific bodies and natural environment. A rat is not a little human, a mouse is not a little rat, and flies are not cheap mice. In addition, the history of an individual will influence their behavior. Variability is built into biological systems to increase possible adaptability to changes, and thus even inbred mice raised under identical conditions will show substantial variability.
Further, we tend to think of behavior as output, which is intended as our input. Instead, for the organism, what it does is much less important than the consequence of what it does. Thus, the behavioral output of an organism is aimed at influencing the input the organism receives, and we should move to this organism-centric view. For a more detailed discussion of this and how the three biological principles of materiality, agency, and historicity, as well as the idea of "Umwelts" should be considered in the study of animal behavior, please see the studies by Gomez-Marin (2019) and Gomez-Marin and Ghazanfar (2019).
A separate issue is reading human cognition into certain animal behaviors, such as "regret" when an animal is just looking to a past location. One should stick to describing the observed behavior (e.g., looking back) without reading any human emotional value into it, even if it may be tempting to do so to achieve higher impact and press mentions. Buzsáki (2020) nicely summarized these issues recently in another opinion article. In this article, I sometimes used such terms as personality, mainly for lack of better terms.
Differences can cause variability across laboratories
The International Brain Laboratory has mounted an impressive effort to create standardized behavioral tests Opinion and datasets across different laboratories. Their recent preprint highlights how variable rodent behavioral performance can be across laboratories. Mice were trained in a decision-making task in seven different laboratories, and, once training was complete, the mice performed very similarly across the different laboratories (Aguillon-Rodriguez et al., 2020). However, how long it took to complete training was very variable and ranged from 3 to 59 d. This huge variability is likely to be the result of exactly those factors described above, which causes the variability between mice within each laboratory (e.g., personality) as well as group variability between laboratories (e.g., how animals are routinely handled in each laboratory). In their impressive efforts to standardize, the International Brain Laboratory describes habituation practices to the apparatus but not how the animals are handled both before habituation as well as during training. Perhaps including standardization in those practices as well as considering the sex (Sorge et al., 2014) and consistency of the experimenter (is it always the same person or do different people train the animals?) could help reduce the variability during initial training.
What should we do?
How should we deal with these influencing factors? We want to use behavior as an outcome to be able to deduct what happened to the memory. The most important consideration is to be aware of the potential influencing factors and confounds. Only then can we even try to control for them.
For example, it is important to look at the distribution of behavior across the individuals. In the neophobia/neophilia case during object exploration, looking at clusters of animals across the positive and negative discrimination index can be helpful. Is there a normal distribution or do subclusters seem to be present?
Habituation procedures and everything that happens to the animal outside of the experimental task is also critical. These will influence what the appropriate behavior is in the context as well as the inner state of the animal. For example, how well an animal is handled and its intrinsic fear and apprehension toward the experimenter as well as general procedures will influence the outcome of any task (Hurst and West, 2010;Gouveia and Hurst, 2017). Currently, it is not common to describe general handling procedures that occur before training beyond the very short description of "animals were handled for 10 min a day before the experiment." A potentially better way to describe and therefore to standardize such procedures could be the use of videos (see https://www.genzellab. com/#/animal-handling/). It is important to include details of the habituation in the experimental description. Were animals habituated to handling with active techniques that simulate rough tumble and play? Or did the experimenter place the hand in the cage and let the animals approach? We have tested both types of handling in the laboratory. Interestingly, while active tumble and play led to less apprehensive animals that voluntarily approached the experimenter, the other approach (only putting the hand into the cage) led to more apprehensive animals and to biting the hand of the experimenter. Further, how was the animal taken from the cage? By cupping or tubing techniques or with tail pickups? How often were the animals handled outside of the experimental context? How often were they brought to the experimental room before training or testing was performed? More handling and habituation as well as nonaversive pickups will lead to less apprehensive animals and, better, to more consistent behavioral results.
Handling should also be considered before numbering/ labeling of animals and group allocation are performed. The natural personality (see above) of an animal will lead to an animal being more likely to be in the front of the cage when the experimenters first approach. As a result, if the numbering of the animals within a cage is done before any handling, the same types of animals will consistently have the same animal number. Instead, one should do the numbering of animals at a random, later time point during initial handling. For example, 10 min into the third handling day when all animals are used to the experimenter.
Experimenters need to be aware of general procedures in the animal unit as well as in the building to reduce distractors. Especially on critical days, such as a test day, one should avoid any changes or other novel experiences for the animal. This could be a new home cage because of cage cleaning or an unfamiliar person entering the housing room for maintenance or renovation two floors away in the same building. All of these factors can lead to more generally alert, less focused animals.
We also need to describe more details of the experimenter. Was it one constant experimenter throughout the whole period or were different experimenters involved? How well trained was the experimenter? Was it a new intern or an experienced animal trainer? What was the sex of the experimenter? Since male experimenters will cause a stress response in rodents independent of how familiar the animals are with the person, these details are critical. In our experiments, we ensure that the animals are brought to the experimental room and left for at least 20 min after transport before any behavior is performed. Like this, the animals can calm down after the transport experience as well as accommodate to the training room and current experimenter. Further, while multiple experimenters will train the same animals and they may be less experienced, each experimenter has "met" and handled each animal at least once before in the housing room outside the experimental context.
We should aim to use behavioral tests that fit the ecological niche of the animal. When designing an experiment, one should consider what the behavior could correspond to in the wild. Further, tasks involving fear should only be used when the research question pertains to that system. Fear memories are not a good model for "simple," declarative memory.
One approach that can help when determining the daily experimental schedule is trying to see things from the point of view of the animal, especially when it concerns the sequence of experiences the animal will undergo. As an example, we can take an appetitive task with a food cue association, during which each trial starts with a food cue that should then lead to a correct choice. If after two wrong choices the animal is "punished" by stopping the current trial and then immediately starting the next one, the animal may not perceive it as punishment since each trial starts with eating the cue pellet. Instead, it will learn that if it makes two random choices, it will be picked up and receive a reward. From the human viewpoint, it seems like a punishment; from the rodent viewpoint, random behavior is the easier way to receive a treat.
One should also consider the animal point of view when choosing the visual cues in spatial tasks. One helpful trick is filming the task environment at the height of the rodents and implementing potential rodent movement patterns such as looking up or rearing. These videos (or pictures) can then be rendered to rodent view, using techniques mentioned in http://www.ratbehavior.org/RatVision.htm. However, even without this rendering, simply viewing the layout and selection of cues from the perspective of the animal can be very enlightening for the experimenter. We generally recommend using high-contrast cues that are arranged asymmetrically (e.g., when implementing in a small box environment). Further, 3D cues are much more useful to the animal than 2D cues, especially when two 3D cues are arranged to have unique overlap patterns when viewed from different places in the testing area. To compensate for the short-sightedness of the animal, there is a simple rule of thumb: when viewing the cue with one eye closed from the height of the animal, it has to be wider than two fingers of the experimenter's hand (with the arm stretched out). Then it is large enough for the animal to see.
Finally, we should value the expertise of behavioralists. An experimenter with experience in behavior can determine, just by watching an animal, whether it is too hungry, not focused, or distracted by an external stimulus. We readily accept that it takes experience to be able to run sophisticated techniques such as optogenetics, electrophysiology, or calcium imaging, but expertise in behavior is often less valued. Many laboratories believe that they can take any behavioral paradigm and apply it without consulting experts. This is not the case.
Conclusion
In sum, behavior is not a simple tool or measurement such as measuring the activity of individual neurons or a brain area. Instead, we should always keep in mind that behavior is the outcome of many different factors. Thus, for one, we should be aware of these factors, keep track of them, and, ideally, control for them as best as we can. Currently, most such influencing factors have not been systematically tested for, with a few exceptions (Hurst and West, 2010;Sorge et al., 2014;Gouveia and Hurst, 2017). Therefore, much of what is described in this opinion piece is based on personal experience or anecdotes shared among behavioral researchers, highlighting that more systematic research on this topic is needed.
All of the additional measures we take to control for these issues, such as habituation to the experimenter and time of day of the experiment, need to be described in more detail in scientific articles than is currently common practice. The current practice of leaving out such details is most likely one of the reasons that many behavior protocols work in one laboratory but not necessarily in other laboratories. Further, we should consider moving to paradigms that are more natural and significant for each species. Finally, when coming to conclusions on the results of studies including animal behavior, we need to keep in mind that the absence of a certain behavior is not conclusive evidence on the absence of, for example, a memory trace. | 2021-01-21T06:16:25.145Z | 2021-01-01T00:00:00.000 | {
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27702914 | pes2o/s2orc | v3-fos-license | Heat diffusion in a two-dimensional thermal fuse model
We present numerical studies of electrical breakdown in disordered materials using a two-dimensional thermal fuse model with heat diffusion. A conducting fuse is heated locally by a Joule heating term. Heat diffuses to neighbouring fuses by a diffusion term. When the temperature reaches a given threshold, the fuse breaks and turns into an insulator. The time dynamics is governed by the time scales related to the two terms, in the presence of quenched disorder in the conductances of the fuses. For the two limiting domains, when one time scale is much smaller than the other, we find that the global breakdown time $t_r$ follows $t_r\sim I^2$ and $t_r\sim L^2$, where $I$ is the applied current, and $L$ is the system size. However, such power law does not apply in the intermediate domain where the competition between the two terms produces a subtle behaviour.
I. INTRODUCTION
Motivated by important aspects such as failure prediction and material improvement, resistor network models [1][2][3][4] have been intensively used for numerical studies of mechanical and electrical breakdown phenomena in disordered media. The quasi-static fuse model [1], although very simple, reproduces the basic damage regimes in breakdown phenomena [5,6]. In the infinite disorder limit [7] the percolation regime is present. The fracture is totally disorder driven, and fuses burns out randomly until global breakdown is reached. If the disorder is small, there is little precursor damage until a single fracture is developing from one end of the system to the other. This is the nucleation regime.
However, the quasi-static fuse model contains no real dynamics, and does not capture time dependency in correlations caused by the local currents. Dynamical effects has been included in the fuse model to study the elasticity problem [8]. Sornette and Vanneste [9,10] developed a model for electrical breakdown and plastic deformation, which they referred to as the dynamic thermal fuse model. The temperature of a fuse was governed by a general Joule heating term, i b /g, and a heat loss term, −aT , which can be considered as a simplification of a full spatial diffusion description. This model has been experimentally realized by Lamaignère et al. [11], and later Mukherjee et al. [12,13] by studying electrical breakdown of carbon-polymer composites. This shows that the thermal fuse model is able to capture some of the phenomenology of breakdown in disordered media, like critical behaviour in the breakdown time. However, the model does not take into account the correlations due to heat diffusion between fuses. Thermal interaction with * glenn.tora@ntnu.no † Alex.Hansen@ntnu.no neighbour fuses has previously been studied by Pennetta et al. [4,14] in a biased percolation model. But this model implies instant thermalization of the fuses, and thereby neglects time dependent effects.
With the thermal fuse model as a base we introduce spatial heat diffusion in the system. We study how the interplay of quenched disorder, current enhancement effects and heat diffusion give rise to time dependent effects which may seem counterintuitive with respect to the quasi-static fuse model and the biased percolation model.
II. MODEL
Our simulations are based on the thermal fuse model proposed by Sornette et al. [9,10]. The model consists of a square lattice oriented at 45 • with respect to the two boundaries opposite of each other, which act as busbars (see Fig. 1). Each bond in the lattice is an electric fuse which behaves like an ohmic resistor when intact. A voltage is applied over the busbars which induces a total current I in the lattice. To each fuse j we assign a conductance g j from a power-law distribution p(g) ∝ g −1+β . The conductances are generated from g j = x B j , where x j is a uniformly distributed random number in the interval (0.5,1.5), and B = β −1 . We call B the disorder parameter. The values of the conductances are set at the beginning and never changed, corresponding to a quenched disorder.
Our model differs from the thermal fuse model by Sornette et al. in the sense that the term −aT is replaced by a spatial diffusion term. We also use a fixed b = 2, which corresponds to the Joule heating effect. There is no loss of heat at the boundaries, so the system will always reach global breakdown for I > 0. We then arrive at the following heat equation for our model in two dimensions in the continuum limit.
with boundary condition ∂T /∂n = 0 on the top and bottom boundary. Periodic boundary conditions are applied in the horizontal direction. The heat capacity is C = 1 for every fuse, and D is the thermal diffusivity. The evolution in time is given by explicit Euler integration, and adaptive timestep is used. When a fuse reaches its temperature threshold T r , chosen equal for all fuses, it breaks and irreversibly turns into an insulator. This is the way the temperature field reacts back on the current field. All the fuses start at equal temperature. When a fuse burns out, the current redistributes itself instantaneously in the network. The network will then heat up until another fuse burns out. The total current is kept constant during the fracture process, with I = 1 for the results herein, if not specified otherwise. The fuses interact with the 6 nearest neighbours through heat diffusion (see Fig. 1). Note that if more than one fuse reach the threshold T r within the same time step, those fuses are broken before the temperature field is updated. This ensures that the currents are instantaneously redistributed. The current distribution is calculated by the conjugate gradient method.
III. TWO COMPETING TIME SCALES
A series of final fracture patterns for different values of D and B are shown in Fig. 2. The fracture which disconnects the network in two pieces is outlined, and the temperature field is indicated by colors. B = 1 yields a uniform distribution, while B > 1 gives a broader distribution with a tail towards large conductances. Note the lower cutoff at 0.5 B , and upper cutoff at 1.5 B in the distribution.
The time dependence is governed by the two time scales τ 0 = gCT r /i 2 and τ 1 = ξ 2 /D. The competition between these time scales generates different domains of behaviour. For τ 0 << τ 1 the domain is referred to as the large-current domain. If we consider D = 0, the large-current domain is present for all values of B. By comparing the fracture patterns for B = 1 and B = 5 we see that for B = 5 a few large cracks appear, while for B = 1 the pattern is more diffuse, with single broken fuses. Due to the broader distribution for B = 5, the small conductances will heat up much faster than the large conductances, and the cracks will grow along pathways of small conductances. This is contrary to what the quasi-static fuse model gives, where increased disorder results in more single broken fuses, and a diffuse breaking pattern. This difference can be attributed to where the disorder is applied in the two models. In our model the disorder is in the conductances, while in the quasi-static fuse model the disorder is in the thresholds of the fuses.
As D is increased, an intermediate domain appears. Since the diffusion smoothens temperature differences, the disorder in the temperature field decreases, and the effect of the redistribution of the local currents becomes more apparent. This is visible in form of a less diffuse damage pattern, and a more localized crack (see Fig. 2 for D = 0.1 and D = 0.5).
Sufficiently large values of D yields τ 0 >> τ 1 , and the small-current domain appears. This is dominated by the redistribution of the local currents, and is characterized by a single crack disconnecting the system, with little precursor damage. Similar behaviour appears for low disorder in the quasi-static fuse model.
The three domains are visible when we plot the breakdown time t r as a function of I on a log-log scale. This is shown in Fig. 3. For both the small-and large-current domain we find a good fit with the power law To recover this, it is sufficient to show that the order of local breakdown events is independent of the value of I. For the large-current domain we can neglect the diffusion term in Eq. 1. Then we realize that the first fuse to break will not change when I is changed, and that the local currents are proportional to the applied current, i ∝ I, between each breaking event. It follows that rest of the breaking sequence will follow in the same order when I is changed. A more complete derivation of Eq. 2 is given in Ref. [10].
In the small-current domain the temperature differences are small, and the value of the diffusion term in Eq. 1 will only be significant in a short period of time after each breaking event. Hence, it is reasonable to assume i ∝ I between each breaking event, and we obtain Eq. 2 for this domain as well.
The presence of the three domains for different values of I is in agreement with the thermal fuse model by Sornette et al. [9,10], and experimental results. Lamaignère et al. [11] fitted the time-to-failure data with Eq. t r ∼ (I 2 /I 2 c − 1) −1 . By letting I c → 0, which is the case in our model due to no heat loss, these fitting laws are consistent with Eq. 2. In the intermediate domain there is a crossover from the small-current to the large-current domain. In this domain the order of local breakdown events depends strongly on the value of I, and the time dependence is more subtle. Fig. 4 shows the breakdown time versus D. We find that larger values of D result in longer t r , since high temperatures are more effectively smoothened out. This result is qualitatively in agreement with the biased percolation model [4]. However, a probabilistic approach to the disorder was used, while our model uses quenched disorder in the conductances of the fuses. This difference manifests itself in the impact D has on the percolation threshold p c . This is shown in the inset of Fig. 4. Diffusion causes less disorder in the temperature field, hence we find that p c decreases as D increases. This is contrary to what the biased percolation model gives, which approaches ordinary percolation (p c = 0.5) for vanishing disorder in the temperature field. We also find that the difference in t r between the two limiting domains for D = 0 and D >> 0 in Fig. 4 is smaller for B = 1 than for B = 5. This difference reflects the time scale, τ 1 , at which temperature differences are smoothened out, and τ 1 decreases with decreasing disorder.
IV. SCALING ANALYSIS
Based on analogy to percolation theory [15], we assume the following finite-size scaling relation Fig. 5 shows a log-log plot of t r as a function of L. The total electric resistance R is independent of L in a homogeneous two-dimensional system. We assume this for our model also, and we get i ∝ 1/L. Since i ∝ I in both the small-and large-current domains, it follows that t r ∼ L 2 . Hence, for small values of L, the large-current domain appears (τ 0 << τ 1 ). A crossover to the smallcurrent domain (τ 0 >> τ 1 ) is observed for sufficiently large values of L with D > 0.
However, in the intermediate domain i ∝ I is not a valid assumption, and I can not be replaced by 1/L in the function for t r .
V. ROUGHNESS OF THE FRACTURE
We define the width of the fracture surface as the height-height fluctuations w = ( z 2 − z 2 ) 1/2 , where z is the height from the bottom of the network to the fuses that belong to the fracture surface, i.e the crack which disconnects the network. Studies of the quasi-static fuse model in two dimensions establish that the width scales as w ∼ L ζ , with ζ = 0.7 within 10% accuracy for different threshold distributions [16,17].
We generated between 1000 and 100 samples of sizes from L = 10 to L = 100, for various values of B and D. A log-log plot of w as a function of L is shown in Fig. 6. The slopes of the linear fits give the exponents ζ listed in Tab. I. The roughness exponent ζ seems to be independent of which time scale is dominating for B = 5, i.e large disorder. We obtain ζ = 0.75 ± 0.1, which is in the range of the global roughness exponent observed in the quasi-static fuse model. For B = 1 the roughness exponent is highly dependent on D, and is approaching unity for increasing D. It means that the fracture is guided by the structure of the network, and this is a trivial regime of behaviour.
VI. DIVERGENCE OF THE RESISTANCE
In the study by Lamaignère et al. [11] they found that the total electrical resistance R follows the power law in a critical region close to t r . A value of α 3D ≈ 0.65 was obtained. A log-log plot of R versus (t r − t)/t r for our model is shown in Fig. 7. The obtained value is α = 0.28 ± 0.05. This is in agreement with the reported value for the thermal fuse model [10], and seems to be independent of the thermal diffusivity and disorder parameter. However, the critical region for power law scaling moves closer to t r as the small-current region is approached, and as the disorder in the conductances is decreased [18].
VII. CONCLUSION
We have investigated a dynamic thermal fuse model with heat diffusion. The time dependence is governed by the two competing time scales τ 0 = gT r C/i 2 and τ 1 = ξ 2 /D. The breakdown time follows t r ∼ I −2 in both the small-current domain (τ 0 >> τ 1 ) and largecurrent domain (τ 0 << τ 1 ). This is in agreement with experiments on electrical breakdown of carbon-polymer composites [11,13]. In the intermediate domain, competition between the two time scales produces a more complex behaviour. A characteristic feature of this domain is that increasing the thermal diffusivity lengthens the lifetime t r of the system.
Heat diffusion introduces new subtleties to the thermal fuse model, which still remain to be investigated. However, the power law behaviour in the divergence of the resistance and in the roughness of the fracture has proven to be robust, and seems independent of the thermal diffusivity and the disorder in the conductances. | 2011-01-02T18:09:15.000Z | 2009-11-17T00:00:00.000 | {
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266457088 | pes2o/s2orc | v3-fos-license | Adaptive Streaming Transmission Optimization Method Based on Three-Dimensional Caching Architecture and Environment Awareness in High-Speed Rail
: In high-mobility scenarios, a user’s media experience is severely constrained by the difficulty of network channel prediction, the instability of network quality, and other problems caused by the user’s fast movement, frequent base station handovers, the Doppler effect, etc. To this end, this paper proposes a video adaptive transmission architecture based on three-dimensional caching. In the temporal dimension, video data are cached to different base stations, and in the spatial dimension video data are cached to base stations, high-speed trains, and clients, thus constructing a multilevel caching architecture based on spatio-temporal attributes. Then, this paper mathematically models the media stream transmission process and summarizes the optimization problems that need to be solved. To solve the optimization problem, this paper proposes three optimization algorithms, namely, the placement algorithm based on three-dimensional caching, the video content selection algorithm for caching, and the bitrate selection algorithm. Finally, this paper builds a simulation system, which shows that the scheme proposed in this paper is more suitable for high-speed mobile networks, with better and more stable performance.
Introduction
With the development of network technology, the demand for streaming media is increasing.Video traffic accounts for a gradually increasing proportion of the total network traffic [1].Nowadays, high-speed rail has become one of the most popular modes of intercity travel.In the high-speed rail network environment, users move fast, and frequent base station handovers cause frequent network channel quality jitter, frequent data retransmissions, increased media data transmission delay, and buffer overflow, which seriously reduce the user's media experience.
Adaptive transmission technology can reduce the negative impact of network quality jitter on the user's media experience [2].Its method is to divide the meida data into different bitrates, store them on the media server, and adaptively select the appropriate bitrate data for the user according to the changes in the network bandwidth.Common transmission protocols include Dynamic Adaptive Streaming over HTTP (DASH) and HTTP Live Streaming (HLS).Many scholars have studied the transmission of variable-bitrate videos.Feng et al. [3] studied the impact of video segment length on user experience, and proposed an adaptive variable-length video segment scheme, which makes dynamic decisions based on real-time network data and user information, and achieves a balance between the accuracy and overhead of variable-length segmentation.The machine learning prediction of the client segment request bitrate implemented at the network edge was studied in [4], and the client segment request prediction problem was formulated as a supervised learning problem, i.e., to predict the bitrate of the next segment request of the client, so as to be prefetch it at the mobile edge and improve the user's experience.The above research results reduce the impact of channel quality jitter on the user's media experience, but are based on low-speed networks; and more constraints need to be considered in high-speed networks.
Cache placement can reduce video transmission delay and the negative impact of network bandwidth jitter.Cache technology is also one of the techniques commonly used by scholars to reduce video transmission delay.Xu et al. [5] proposed a user-assisted base station (BS) storage and cooperative prefetching scheme for high-speed railway (HSR) communication, which improves the system throughput.However, it does not consider the environmental factors of users watching videos.A mobile edge network streaming media optimization framework based on privacy-preserving learning was proposed in [6], which included a user data privacy scheme and dynamic video cache algorithm.However, the strategy proposed by the literature is in the scenario of low mobility speed, and does not consider some characteristics of the high-speed railway mobile scenario.In high-speed networks, users move very fast, so the calculation of cache placement location and time is also a very critical challenge.
This paper considers the characteristics of the high-speed railway network scenario, such as high-speed mobility and severe network channel quality jitter.Under the above constraints, this paper proposes an adaptive streaming media transmission method based on three-dimensional cache and environment awareness (TDCEA).The three dimensions referred to in this paper are the time dimension, the space dimension, and multilevel caching from server to user.In the time dimension, the multimedia data need to be placed on different base station severs according to time.The space dimension refers to caches located at different locations, such as base station, high-speed rail, and client.Multilevel caching refers to the multitier cache architecture formed by different base stations, highspeed rails, and clients based on the spatio-temporal attributes.
According to the user's channel quality and the environmental parameters of the user watching the video, the transmission of streaming media based on the three-dimensional cache architecture is mathematically modeled, the optimization goal of this paper is summarized, and the corresponding solution algorithm is proposed, aiming to reduce the transmission delay and improve the user's QoE.The solution proposed in this paper needs to solve the following problems: The first is which data to store.On the high-speed rail, a large number of users watch videos, and it is a challenge to choose which media data to store.The second is how to choose the cache location (at the base station or on the high-speed rail).The third is when to store the data.The user moves at a high speed and the cache has a short validity period, so the cache placement time is also very critical.The fourth is how to choose the media bitrate so as to improve the user's media experience.
The rest of the paper is organized as follows.Section 2 introduces the related research status of video cache technology and adaptive bit rate technology.Section 3 proposes the system architecture and system model.In Section 4, some algorithms to solve the problem are proposed.The simulation is given in Section 5. Section 6 analyzes the results of the experiments, and discussion in Section 7. Section 8 provides a conclusion.
Related Works
Scholars have carried out a great deal of research on cache technology in order to improve users' media experience.In [7], Mamduhi et al. proposed a cross-layer controller for handling file transfer at different levels and applied it to wireless network transmission, thus better managing network resources and improving the network transmission efficiency. in [8], the evolution of 360-degree video view requests was predicted by a long short-term memory (LSTM) network, and the predicted content is prefetched to the temporary storage.To further improve the video quality of transmission, users located in the overlapping areas of multiple small base stations are allowed to receive data from any of these small base stations, thereby enhancing the user's video experience.With the development of edge computing technology, scholars have proposed streaming media transmission optimization strategies based on edge computing.In [9], the user's QoE was used as the key indicator to drive the edge device offloading, and limited computing resources in were used to provide better service for the user.Nguyen et al. [10] dynamically allocated wireless resources to optimize the user's QoE in LTE wireless cellular networks, used the cross-layer information of the network.
Setting memory in the streaming media transmission process is one of the effective method to reduce video transmission delay and improving user QoE.Many researchers have carried out a large amount of research on placement problems.The technical challenges and development directions of mobile edge cache were analyzed in [11].In [12], Pham et al. stored media data on edge servers to reduce data transmission delay and proposed an algorithm to increase the data hit rate.Wang et al. [13] analyzed the current state of research on caching in edge computing and proposed a collaborative strategy for streaming media content in cloud-edge collaborative environment.For the cache data selection problem, researchers generally believe that storing popular content can minimize the energy consumption of data delivery.The caching data selection algorithm was studied by [14], by using small base stations to store the most popular files.In [15], Li et al. studied the relationship between multiple cache points and user service, and found that, in some cases, randomly caching files results in better performance than caching the most popular data.
Li et al. [16] studied the system modeling, large-scale optimization, and framework design of hierarchical edge caching in device-to-device assisted mobile networks.A layerbased non-orthogonal transmission scalable media cache technology was proposed in [17], which utilized the layer features in scalable video.In [18], Zhang et al. studied the challenges brought by the different levels of content popularity and different viewing demands of scalable video, and proposed different types of content corresponding transmission schemes.In [19], Tran et al. proposed a multirate cache (MRC) scheme for video offloading in dense device-to-device (D2D) networks.A scalable video coding collaborative cache architecture for drones and clients was proposed in [20], which provided high transmission rate content distribution and personalized viewing quality in hotspot areas.A reactive content cache strategy based on deep reinforcement learning was proposed in [21], which made decisions for new requested content.The main goal was to obtain higher cache gain with lower space cost.Xu et al. [22] proposed a new heterogeneous network video content secure edge cache scheme.In order to motivate the participation of edge devices, the Nash bargaining game was used to model the negotiation between content providers and edge storage, and the optimal request cache space of content providers and the optimal price of each edge device were analyzed together.In [23], Kim et al. studied the caching of part of the content, rather than the entire content, to enrich users' QoE.And They proposed a cache strategy for data blocks of different quality.A 360°video cache solution based on viewpoint reconstruction was proposed in [24].
At present, many scholars have conducted in-depth research on the adaptive technology of streaming media.A novel AQM algorithm was proposed in [25], which was based on the hotspot technology of deep learning, and used a long short-term memory (LSTM) neural network to enhance the users' perception of video quality.Meanwhile, the optimization of the buffer was also presented in [26].Santos et al. designed a new rate-adaptive algorithm, which aimed to minimize the amount of rebuffering and maximize the video quality.Gupta et al. [27] proposed a data cache video adaptive bit-rate switching algorithm.The algorithm dynamically constructed a smooth factor based on the throughput and cache state, and performed exponential weighted averaging on the bandwidth to maintain the buffer balance and ensure the video quality.
The above literature studied the cache technology and variable-bitrate algorithm for streaming media, and proposed many valuable methods.However, the above research is based on low-speed mobile networks.In high-speed mobile networks, cache is not only related to space, but also to time, and frequent base station handovers cause severe jitter in user channel quality; these factors all cause the performance of the above methods to decline.Therefore, this paper proposes an adaptive streaming media transmission optimization method based on three-dimensional cache and environment awareness.
The main contributions of this paper are as follows: 1.This paper proposes an adaptive streaming media transmission architecture based on three-dimensional cache and environment awareness, which considers the utility of the cache in time and space dimensions.
2.
This paper digitizes the adaptive streaming media transmission scheme based on the three-dimensional cache and environment awareness, and summarizes the problems to be optimized.
3.
This paper proposes an algorithm to solve the problems, which are the selection of video data, cache location, and time and bitrate selection, and verifies the performance of the proposed method via simulation.
Architecture and Model
The adaptive streaming media transmission architecture based on a three-dimensional cache and environment awareness proposed in this paper is shown in Figure 1: First, the base station collects the user's media requests and analyzes the popularity of the videos according to the request files of a large number of users, and then stores these data in the sever of the base station or the high-speed rail.Then, the user selects the appropriate bitrate video according to the environmental parameters of watching the video, and first searches in the high-speed rail cache.If the required content is found, the current quality of the wireless channel does not need to be considered.If it is not found, the data request is reapplied according to the quality of the current network channel quality and is searched in the base station cache.If it is found, it is directly sent to the user.If it is not found, the data is resent from the server or transcode (if there are other bitrate data).
User's QoE Model
Assume that the current time is t, n base stations are passed along the way, and the video is evenly divided into m segments of length T s .In this paper, in the high-speed mobile scenario, the distance between the user equipment (UE) and the base station (BS), and between the rail speed and other viewing environment, as well as the objective quality of the video, have a great impact on the user's QoE, which is defined by Equation (1): where the function σ represents the objective quality.The objective quality σ can be expressed by the quality evaluation parameters PSNR and SSIM [27].The function ϕ represents the impact of cache miss and retranscoding on the user's QoE, and T r represents the impact of data retransmission delay after cache miss on the user's QoE.The parameter RJ s represents the jitter of video quality, and the parameter N rt represents the number of rebuffering instances.The parameters α, β, Θ, ι, and ρ represent the impact coefficients of each factor.
where RJ s denotes a monotonically increasing function of S x , where S x is the number of bitrate switches that user x has experienced in the previous segments.J ′ is the current bitrate of user.J is the video bitrate index.Normalize the value of QoE:
Video Data Cache Model
Content popularity: Content popularity is used to describe the popularity of streaming media, reflecting the probability of customers requesting streaming media files at a certain time.In this paper, the content popularity of streaming media files can be calculated by Equation ( 4): Assume a streaming media file where m i represents the i-th video segment in M. Assign m i with a popularity level number, and the probability of accessing m i is p i , for all 1 ≤ i ≤ j ≤ N, p i ≥ p j .Therefore, p i can be called the popularity of m i in the set M , and the popularity distribution in M is: Starting from each time segment, the base station collects requests from users, and the cache content is divided into two types: (i) New content, i.e., video segments requests that have not been cached yet.(ii) New version, i.e., the new version of the current video segment requested.Content popularity is modeled as a Zipf distribution.In this distribution, content of a higher popularity may have a higher probability of being requested.According to [28], p i can be obtained as follows: where s ≥ 0 reflects the skewness of the popularity distribution.
where s c = 1 means storing the video data in the base station cache, and s c = 0 means storing the video data in the high-speed rail cache.The parameters ψ and λ represent the popularity thresholds, which can be defined by the user.When s c = 1, the base station and the storage time of the cache data need to be calculated.Assume that the user connects to the base station BS t at time t, the current speed of the user is V t , the distance to BS t is dis t , and the distance to BS t+1 is dis t+1 .Therefore, the cache base station selection function is Assuming that the base station cache is selected at BS t+1 , the cache validity can be calculated by Equation ( 9)
Transmission Model
The objective quality of QoS can be calculated by Equation (10): where f is the calculation function of video objective quality, and V Bitrate represents the bitrate selected.
When the user needs to obtain data from the base station, the video bitrate chosen by the user should be less than the current network bandwidth bandwidth t .The current network bandwidth is related to the user's channel quality.
bandwidth t = r(SI NR). ( Due to multipath effects, the signal from the base station reaches the user through M different paths; therefore, the average signal-to-interference-plus-noise (SINR) ratio of a single user at a given point is expressed as [29]: with where M is the number of multipaths, τ i (m) is the propagation delay with base station i (for base station 0, τ 0 (m) = 0), δ j is the additional delay that is added by path j, q − i(m) = d i a 10 ε i 10 is the path loss from base station i, α is a constant and ξ i a lognormal variable due to shadowing.P p is the average power that is associated with the j-th path, T cp is the length of the cyclic prefix (CP), and T u is the length of the useful signal frame, and N 0 is the noise power.
The value of SI NR is related to the base station that the user connects to.
where γ is a function, and C BS is the base station to which that the user connects.The base station C BS is related to the current viewing environment of the user, such as the user's moving speed, the distance between the user and different base stations, the size of video data transmission.
where w is a function, V t is the user's moving speed at time t, dis t is the distance between the user and the base station BS t , dis t+1 is the distance between the user and the base station BS t+1 , and V Bitrate is the bitrate.When the user needs to obtain data from the high-speed rail cache, the bitrate is independent of the current network condition of the user.
where V Bitrate ′ represents the data bitrate in the high-speed rail cache.When the video data requested by the user is not found in the base station, there are two choices: request the video data with the corresponding bitrate from the server, or transcode the media data based on the computing module of the base station.If transcoding is chosen, there is an additional cost, which has a negative impact on the user's QoE.
where t s represents the playback time of a video segment, and δ represents the processor frequency of the edge service.The higher the frequency, the lower the cost.If the user chooses to retransmit the data, the data retransmission will cause delay T r .
Algorithm 4.1. Three-Dimensional Cache Placement
In this paper, the base station collects requests from user devices in periods and processes the corresponding data requests in parallel.According to the popularity of the media, some data will be cached in the base station.When the user sends a request for mi, it will first search the high-speed rail B uk .If B uk has the requested content, it will directly call mi from B uk .If it does not, the BS will search for mi in B K .If found, it will directly call mi from B K .If none of the above is found, the content will be obtained from the video server.Algorithm 1 shows the three-dimensional cache access strategy.For BK and B uk , when receiving the content mi, if there is content mi in the old version data, it will replace the old version with the new content.If the user has extra storage space, the content mi will be cached directly.If both storage spaces are full, the content in B u k is deleted, and then the content in B K is deleted.Construct m i (t) which obey Zipf.BS collects m i (t).end for 25: end for
Cache Content Selection
Prestore content selection can be regarded as a multiple knapsack problem.The cache is regarded as a knapsack, and b bytes of space are allocated to UEk in Bk (the total capacity allocated to N users is less than or equal to the total capacity), so the capacity of is bB.Similarly, the capacity of hight-speed rail is Buk.There are n mi that need to be downloaded to the cache, each mi occupies space, and the popularity pi of mi is the value of mi.The specific content selection strategy is shown in Algorithm 2. The algorithm can achieve the local optimal solution of the cache strategy, and compared with other algorithms, it can significantly reduce the delay caused by the user not finding the target file.
for j = 0 to bandwidth do 4: for j = 0 to bandwidth do 15:
Bit Rate Adaptive Selection Algorithm
This paper adopts edge computing and cross-layer information for bit rate adaptive algorithm.It is assumed that the video segment has multiple fixed bitrates: The value of Bc is calculated according to Equation ( 21) where V h represents the bitrate of the highest quality level, and Vm is initially set to V 1 .
Then, the user's QoE is calculated respectively, at the bitrates of V h and V m .If the bitrate of V h is chosen, the user's QoE is denoted as QoE
Simulation
In this section, we conducted a simulation experiment to verify the effectiveness of the proposed algorithm.First, we compared the cache performance, such as the average lifetime and the average hit probability of the cache data.Then, we compared the user's QoE under the HLS, BSBC and BBA algorithms to verify the performance of the whole algorithm proposed in this paper.
Cache Performance Comparison Algorithm
In this paper, the average lifetime and the average hit probability of the cache data are defined as follows: The average lifetime of the cache data: This is used to describe the timeliness of the data collected by the system.The peak age of the content represents the maximum value of the content in the system; the age of the content is defined as follows: where t represents the current time, and g(t) represents the generation time (also known as the generation time stamp) of the latest data packet received by the receiver.Therefore, the average information age is the sum of all instantaneous information ages divided by the amount of information: where N represents the total number of streaming media files.Average hit probability: The efficiency of cache is usually measured by the hit rate.In this paper, the hit rate refers to the probability that the file requested by the user is in the corresponding cache.The hit rate is defined as the ratio of the number of successful accesses to the total number of accesses.Specifically, it is the probability that a given access to a memory location will be satisfied by the cache.The hit rate can be expressed as follows: where, N h represents the number of times that the requested file matches, and N s represents the total number of user requests.The average hit probability is defined as follows: This paper uses Python to build a simulation environment, and the simulation parameters are shown in Table 1.The bandwidth data used in the simulation experiment are real bandwidth data collected in a real high-speed rail environment, and Figure 2 shows the network bandwidth in the high-speed rail environment used in this experiment.In order to compare the user's QoE under different cache algorithms, the parameters in the QoE model are set as follows: α = 3.5, β = 0.15, γ = 1.5, ψ = 0.2, ρ = 0.2.The transmission cost factor δ = 1.35.In this paper, eight videos named Boat, FoodMarket, RitualDance, Crosswalk, TunnelFlag, Tango, Dancers, and DinnerScene are used as data for the streaming media experiment [30].In the simulation experiment, each video is divided into 10 s segments, and the video is played in a loop, using the Joint Scaling video model (JSVM, version 9.19) [31] to encode the data with different quality levels.The following are the cache algorithms compared in this paper: Content-Oriented Strategy: This strategy focuses on optimizing the content distribution in the streaming media system.The core idea of this strategy is to cache the requested content as close as possible to the user's location in order to reduce latency and improve user experience.
Version-Oriented Strategy: Version-Oriented Strategy focuses on optimizing version management in the streaming media system.The core idea of this strategy is to cache different versions of the same content on different nodes to meet the needs of different users.
The following are the bitrate selection algorithms compared in this paper: BSBC [32]: BSBC is the abbreviation of buffer-based video bitrate adaptation, which aims to solve the problem of low average bitrate in the DASH standard.The algorithm analyzes the data in the video cache, calculates the average bitrate of the current frame, and dynamically adjusts the bitrate to improve the user experience.Specifically, the BSBC algorithm divides the video into multiple blocks, with each block containing multiple frames.For each block, the algorithm first calculates the total bitrate and total cache of the block, and then calculates the average bitrate of each frame according to these values.Finally, the bitrate is dynamically adjusted according to the average bitrate to improve the user experience.
HLS [33]: HTTP Live Streaming algorithm is a protocol and method for implementing the online streaming of audio and video content.This technology, developed by Apple, aims to use the HTTP protocol to transmit audio and video content efficiently and stably on the Internet, while automatically adjusting the bitrate to meet the needs of different network environments.
BBA [34]: The buffer-based adaptive (BBA) algorithm is a method of selecting bitrate based on buffer filling state.When the buffer is full, it chooses a higher bitrate; when the buffer is empty, it chooses a lower bitrate to avoid buffer overflow or playback interruption.
Results
Figure 3 shows the average lifetime of the cache data of the three algorithms as the number of files requested by UEk increases.As can be seen, as the total number of files increases, the average lifetime of the content decreases.This means that the files become increasingly fresh.The lower the freshness of the files, the higher the stability of the files obtained by the user, so our proposed algorithm has higher robustness.As shown in Figure 4, as the number of file requests increases, the cache hit rate is higher because the cache content increases, so the overall QoE rises.However, the performance of the above two strategies is unstable, because their content requests are more random, and when the file is not found in the cache, the performance of the above two strategies will be decreased.Therefore, the strategy proposed in this paper has higher applicability in dealing with streaming media transmission in high-speed rail.Figure 5 shows the average hit probability of the three algorithms as the number of files requested by UEk increases.As the number of requested files increases, the files become increasingly difficult to hit.Our strategy ensures that the hit rate of the files is higher when the number of files is large.As shown in Figure 6, as the number of users increases, the user's QoE decreases because the cache space occupied by each user decreases.At this time, when the user requests a file, the probability of finding the requested file in the sever decreases, so the QoE decreases.However, the performance of the strategy proposed in this paper is still better than those of the above two strategies.The strategy proposed in this paper is more suitable for video transmission in high-speed mobile networks.
In Figure 7, the content movement of the video Boat is slow, so the encoded data are small, and the required bandwidth is small, resulting in the highest bitrate video playing smoothly for the user.The algorithm proposed in this paper can quickly find the local optimal solution, thus obtaining high QoE performance.In addition, in Figure 8, the QoE of the HLS algorithm is lower than that of other algorithms, especially at the beginning of the transmission, because the algorithm randomly selects the video bitrate at the beginning, making it more unstable.However, the algorithm proposed in this paper performs well in the initial bitrate selection.In Figure 9, watching the video RitualDance, because there are many elements in the video and the scene changes faster, the encoded data are larger, and thus users cannot watch the high bitrate video smoothly, resulting in lower QoE.However, the algorithm we proposed still maintains good performance, indicating that it can still perform well under high load.In general, the algorithm we proposed is relatively superior in high-speed environments.Figure 10 shows the QoE of high-speed rail users when each user randomly selects one of eight videos to watch under different algorithms.The results show that the TDCEA method proposed in this paper achieves the best performance.Compared to high-speed rail users watching a single video individually, the average user QoE obtained in this experiment is lower than that of users watching the same video.This is because the increase in the number of videos available occupies more cache space, thus reducing the cache hit rate and the average user QoE.The results in Figure 11 are obtained from simulations under the scenario where highspeed rail users randomly select one of eight videos to watch.In this paper, both the base station cache and the high-speed rail onboard cache vary with different levels.This shows the impact of different levels on the average user QoE.As illustrated, the proposed TDCEA method exhibits a significant increase in average user QoE with larger cache sizes.This suggests that expanding the onboard cache capacity in high-speed rail enables more video data to be cached, thereby reducing the demand for wireless resources and improving user video experience.The QoE for other methods also increases with larger cache levels, but to a lesser extent.This is because in high-speed rail scenarios, the bottleneck for video transmission lies between the base station and client side.Under the special networking environments of high-speed rails, it is difficult to store more data on the client side.
Discussion
In the high-speed rail network environment, the frequent switching of users causes the congestion algorithm to intervene frequently, resulting in a decline in the stability of the network bandwidth estimation accuracy.This paper proposes a new transmission method called TDCEA, which constructs a three-dimensional cache architecture to prestore media data at suitable locations and times.The simulation results demonstrate that our proposed TDCEA achieves the best performance.The main reason for this is that TD-CEA has been designed specifically for high-mobility scenarios and is thus more suitable for such environments.As shown in Figure 11, TDCEA performs better with increased cache capacity because prefetching data in the high-speed rail environment improves the communication quality between the rail and clients.Moreover, the channel between the stationary high-speed rail environment and users is more stable.Therefore, clients can receive video data from the rail cache more smoothly.In summary, our proposed TDCEA delivers superior performance and robustness in highly mobile environments.
The method proposed in this paper requires cache servers to be deployed onboard high-speed rails.Also, due to the high speed of these rail systems, the timeliness of cached content is reduced, resulting in additional hardware costs.However, the trade-off between increased hardware expenses and improved user quality of experience has not been extensively discussed in this paper.This will be the focus of our future research by incorporating caching costs into our model to enable a more comprehensive cost-benefit analysis.
Conclusions
This paper proposes a new transmission method called TDCEA to address video delivery challenges in high-mobility environments.It solves the following three problems: one, when to prestore data; two, where to prestore data; and three, which data bitrate to select.Simulation experiments demonstrate that our proposed TDCEA delivers superior performance and robustness compared to existing methods under high-speed mobility scenarios.
Figure 1 .
Figure 1.The architecture interactive model proposed in this article.
11 :
Download M from VS to B k according to dp[i][j].12: for the user requestM∥b u = max(b u ) do 13:for i = 1 to |M| do 14: d w i d t h ( m b p s ) t i m e ( s ) B a n d w i d t h
Figure 2 .
Figure 2. Bandwidth data in high-speed rail 5G environment.
Figure 3 .
Figure 3. Average age of content with increasing number of files.
Figure 4 .
Figure 4. Average hitting probability with increasing number of files.
Figure 5 .
Figure 5. QoE with increasing number of files.
Figure 10 .
Figure 10.The users' QoE when watching the eight videos.
Figure 11 .
Figure 11.The users' QoE with different cache levels.
Author Contributions:
Conceptualization, J.G. and J.Z.; methodology, J.G.; software, Y.Z.; validation, Y.Z. and F.S.; formal analysis, Y.Z.; investigation, H.G.; resources, J.G.; data curation, Y.Z.; writingoriginal draft preparation, J.G.; writing-review and editing, J.G.; visualization, Y.Z.; supervision, Y.T.; project administration, Y.T.; funding acquisition, J.G., Y.Z., J.Z., F.S., H.G., and Y.T.All authors have read and agreed to the published version of the manuscript.Funding: This work was substantially supported by the National Natural Science Foundation of China under Grant No. 62002263 and No. 62002025, in part by Tianjin Municipal Education Commission Research Program Project under 2022KJ012 and Open Foundation of State key Laboratory of Networking and in part by Switching Technology (Beijing University of Posts and Telecommunications) (SKLNST-2021-1-20).
Algorithm 2 :
Cache Content Selection Download Algorithm (CCSDA) 1 , and if the bit rate of B m is chosen, the user's QoE is denoted as QoE 2 .Compare the values of QoE 1 and QoE 2: if QoE 1 >= QoE 2 , then V c = V m ; otherwise, set V c = V h .Repeat the above steps until h = m.Then, the user's QoE 3 when choosing the bitrate is compared with the user's QoE 4 when choosing the video data stored in the high-speed rail.If QoE 3 > QoE 4 , the data with the bit rate V c are chosen, and if QoE 3 < QoE 4 , the video data are requested from the high-speed rail cache.The pseudocode of the algorithm is shown in Algorithm 3. | 2023-12-22T16:24:58.315Z | 2023-12-20T00:00:00.000 | {
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36311015 | pes2o/s2orc | v3-fos-license | The Massive Goldstone (Higgs) mode in two-dimensional ultracold atomic lattice systems
We discuss how to reveal the massive Goldstone mode, often referred to as the Higgs amplitude mode, near the Superfluid-to-Insulator quantum critical point (QCP) in a system of two-dimensional ultracold bosonic atoms in optical lattices. The spectral function of the amplitude response is obtained by analytic continuation of the kinetic energy correlation function calculated by Monte Carlo methods. Our results enable a direct comparison with the recent experiment [M. Endres, T. Fukuhara, D. Pekker, M. Cheneau, P. Schau{\ss}, C. Gross, E. Demler, S. Kuhr, and I. Bloch, Nature 487, 454-458 (2012)], and demonstrate a good agreement for temperature shifts induced by lattice modulation. Based on our numerical analysis, we formulate the necessary conditions in terms of homogeneity, detuning from the QCP and temperature in order to reveal the massive Goldstone resonance peak in spectral functions experimentally. We also propose to apply a local modulation at the trap center to overcome the inhomogeneous broadening caused by the parabolic trap confinement.
I. INTRODUCTION
Collective modes are important for understanding dynamic susceptibilities, which include experimentally observed spectral functions and transport properties. The situation becomes particularly intriguing in strongly coupled systems, where a simple description in terms of weakly interacting excitations is unreliable and perturbation theories fail even qualitatively. Under these conditions, the role of underlying collective modes on dynamic susceptibilities becomes increasingly more important but is hard to calculate 1 . This is exactly what happens in the vicinity of the two-dimensional (2D) Superfluid-to-Mott insulator quantum critical point (SF-MI QCP) 2 . Though it is considered to be one of the best studied strongly coupled systems, its quantum critical dynamics is still poorly understood, both theoretically and experimentally.
In superfluids near SF-MI quantum criticality, the ef-fective field theory in terms of a complex scalar order parameter Ψ features an emergent particle-hole symmetry and Lorentz invariance, and is expected to have two types of collective modes. 3 The first one originates from fluctuations of the phase of Ψ and describes a massless Bogoliubov-Nambu-Goldstone mode. The second one describes amplitude fluctuations and is associated with a massive Goldstone (MG) mode 3 , often referred to as a Higgs amplitude mode. The fate of the MG-mode in 2D is an intriguing and controversial issue because its decay into lower-energy gapless modes is found to be strong. The mode was argued to become either completely overdamped (i.e., without any resonance type feature in spectral functions 4 ) or be detectable as a well-defined resonance peak in certain spectral functions on the superfluid side away from (but not on approach to) the critical point [5][6][7] .
Recent progress on ultracold atom in optical lattice experiment 19 , as well as Monte Carlo simulations 10-13 , 1/N corrections to higher order 14 , and non-perturbative renormalization group methods 15,16 have presented solid evidence in favor of yet another scenario: A critical resonance in the universal scaling regime. Unlike MG-modes in three (and higher) dimensions which become sharper when approaching the QCP, the ratio of the width of the resonance peak over its mass remains constant in (2+1) dimensions.
In order to detect the MG-mode experimentally, the scalar spectral function A(ω) (i.e., the correlation function of |Ψ 2 |) is considered to be the best probe 7 . On the SF side of the transition point, in the scaling limit, A(ω) takes the form 1 where ∆ is the Mott insulator gap for the same amount of detuning from the QCP, and ν = 0.6717 is the correlation length exponent for the U (1) ≡ O(2) universality in (2 + 1) dimensions 8,9 . The universal function Φ SF (x) starts as Φ SF (x → 0) ∝ x 3 and saturates to a quasi-plateau with weak ω dependence Φ SF (x 1) ∝ x 3−2/ν ≈ x 0.0225 . The intermediate regime between the two limits can be constructed numerically, where a welldefined resonance peak associated with the critical MGmode is observed at x = 3.3(8) 12 . Monte Carlo data also suggest that a similar universal resonance, though less pronounced, may equally well be seen on the other side of the transition, as well as in the quantum critical normal liquid 12,13,15 . These conclusions are yet to be confirmed or refuted experimentally. The bottleneck of the numerical analysis is analytical continuation of data for correlation functions from imaginary to real frequencies, A(iω n ) → A(ω), where iω n = 2πnT with n = 0, ±1, ±2 . . . are Matsubara frequencies. This procedure is a notorious, ill-posed problem that requires application of certain regularization schemes 17,18 , and thus independent experimental studies are required for final understanding.
The ultracold atom experiment of Ref. 19 aimed at detecting the MG-mode in A SF (ω) and confirmed the expected softening of the quantum critical spectrum implied by (1) but remained inconclusive with regards to the existence of a well-defined MG resonance. To obtain A(ω), a 2D Bose Hubbard system was gently "shaken" by modulating the lattice laser intensity (lattice depth) and probed by in situ single-site-and single-atom-resolved measurements. The observed signal (through temperature increase) featured a broad maximum whose onset softened on approach to QCP, in line with the scaling law (1). The onset correlates remarkably well with the energy of the MG-mode, while the ratio of the onset width to its frequency was measured to be approximately constant when approaching the critical point. However, a resonance-type peak with diminishing width was not detected, which can be interpreted either as evidence for the MG-mode being overdamped in the critical state or as broadening caused by finite temperature and system inhomogeneity (tight confinement) effects 10 . Thus a direct comparison between numerical calculations and experimental measurements with a common setup is crucial to settle the controversy.
In this paper, we employ an ab initio numerical procedure based on quantum Monte Carlo simulations and numerical analytic continuation 18 to calculate spectral functions for ultracold atoms in optical lattices. The final result for the temperature increase as a function of modulation frequency successfully reproduces the main data of Ref. 19 for the experimental setup "as is"; i.e, in the spirit of the quantum simulation paradigm 20 . The consistency between numerical results and experimental measurements establishes the reliability of both approaches, and, in particular, validates the analytic continuation procedure. Moreover, simulations performed for various system parameters indicate several improvements/requirements with regards to the experimental setup that will help revealing the resonance peak in the spectral function. They include (i) the system should be effectively homogeneous to avoid inhomogeneous broadening, which can be achieved through confining the lattice depth modulation locally at the parabolic trap center; (ii) the detuning from the QCP should be small, j/j c ≤ 1.05, where j = J/U is the dimensionless coupling parameter for the Bose Hubbard model introduced below [see Eq.(2)], and j c is its critical value; and (iii) the system's temperature should be at least as low as the Berezinskii-Kosterlitz-Thouless transition point T c . Our results suggest that a direct observation of a well-defined resonance peak and understanding the fate of the MGmode experimentally is challenging but not impossible.
The rest of the paper is organized as follows. We introduce the model in Sec.II and describe the numerical procedure in Sec. III. The comparison between the temperature response from simulations and experimental measurements in a specific setup from Ref. 19 is presented in Sec.IV. We discuss requirements and possible experimental improvements to reveal the MG resonance in Sec.V.
II. THE MODEL
Ultracold bosons in optical lattices offer unique possibilities to study the SF-MI quantum phase transition in 2D 21,22 . At low enough temperatures the physics of the system is restricted to the lowest Bloch band, and can be described by the Bose-Hubbard (BH) model 2,23 , which is parametrized by a hopping amplitude J and an on-site interaction energy U , where b † i (b i ) creates (annihilates) a particle on the site i, and i, j denotes the sum over nearest neighbors on the square lattice. In the BH model, the dimensionless coupling parameter j = J/U is easily tunable via the lattice depth, and the dimensionality of the system can be reduced to 2D by suppressing the hopping in the third direction. The total particle number N is controlled by the global chemical potential µ. Finally, an ultracold atomic gas is trapped by a confining potential V i , which is usually harmonic, V i = 1 2 mω 2 d 2 R 2 i (with m the mass of atom, d the lattice spacing, and R i the distance of site i from the trap center measured in units of d). Within the local density approximation picture (LDA), µ − V i plays the role of a local chemical potential.
Ideally, without the V i term in Eq. (2), the system is a homogeneous 2D Bose Hubbard model and its phase diagram is known with high accuracy 24-26 at both zero and finite temperature. In the ground state, the system undergoes a second order phase transition from the SF to the MI when decreasing the ratio j = J/U at fixed integer filling factor. At filling factor n = 1, the transition occurs at j −1 c = 16.7424(1) and µ c /J = 6.21 (2) and features a QCP with emergent particle-hole symmetry, which enlarges the Galilean invariance to Lorentz invariance (the system is actually conformally invariant). The SF phase is supposed to have the critical MG-mode according to the discussion in the previous section.
When the V i term is presented as in current experimental implementations 19 , due to the inhomogeneous local chemical potential, the particle density decreases to zero when moving away from the center of the trap. Any conclusion regarding the existence of the GM-resonance in the homogeneous case cannot be naively applied to the realistic experiment, even if the center of the atomic system is fine-tuned to be in the vicinity of QCP. A careful bottom-up calculation of the scalar spectral function is required in order to understand the experimental signal.
III. SPECTRAL FUNCTION MEASUREMENT: THEORY
In this section, we revisit the generic mathematical framework for the measurement of the scalar spectral function in ultracold atoms.
In the BH model, the total kinetic energyK = is the simplest operator with nontrivial dynamics leading to strong scalar response. Thus, we may consider adding an external perturbation term δf (t)K to the Hamiltonian (2). Within standard linear response theory, the total kinetic energy response is proportional to the external field, and the ratio defines the response function χ(ω, T ) ≡ δ K (ω) T /δf (ω) where ... T denotes the thermal average at temperature T . The spectral function is defined as the dissipative part of the response function, A(ω, T ) ≡ 2Imχ(ω, T ), so that A(ω, T ) is proportional to the energy absorbed by the system, which, in turn, determines the temperature change of the system. To learn about the spectral function, one can measure either the total kinetic energy response or the temperature change. Though being rather indirect, the latter one is the quantity that is measurable in the ultracold atom experiment 19 .
Experimentally, a small uniform modulation δV 0 cos(ωt) of the optical lattice depth V 0 is applied in the 2D plane to generate the external perturbation term 19,27 . In the parameter regime where the BH model is a valid approximation, the lattice depth in units of the recoil energy E r = π 2 /2md 2 is much larger than unity and controls both parameters J and U : J where the effective dimension D = 2. Substituting V 0 with V 0 + δV 0 cos(ωt) in J and U , and keeping terms to first order in δV 0 , the perturbed BH Hamiltonian readŝ Here, the generalized forces δf (t) = δf 0 cos(ωt) with V0 are linear in δV 0 . Note that the second term in Eq.(3) commutes with H 0 and yields no contribution to the spectral function. Furthermore, we argue that the effect from the confining potential term (third term) is also negligible compared to the kinetic energy term (fourth term) if one of the following conditions is satisfied, i) |δg 0 /δf 0 | = 2/|(4 V 0 /E r − 1)| 1; ii) the trap is large enough and LDA is valid; namely, it is possible to decompose the system into independent mesoscopic regions whose sizes are larger than the correlation length but are still small enough to be regarded as homogeneous systems with the local chemical potential µ − V i . This implies, on the one hand, that the total response of the system can be approximated by a sum over independent contributions from mesoscopic regions, while, on the other hand, the confining potential term in each mesoscopic region is proportional to the particle number in this region and thus commutes with the local H 0 (i.e., it is not dynamic under LDA). The combined effect is that the confining potential does not contribute to the linear response. The validity of LDA for critical systems has been addressed before in Ref. 28 For ultracold atoms whose dynamics is dominated by the kinetic energy term, the energy dissipation rate is proportional to the kinetic energy spectral function A(ω), Here P (ω, T ) is the heating power from other mechanisms (ultracold atoms are always coupled to the photon subsystem and are subject to collisions with the background gas). In the leading approximation, P does not depend on the small lattice-depth modulation, and we expect P (ω, T ) P (T ). Assuming that the system is quasistatic, i.e., the relaxation time is small enough, τ 1/ω, the thermodynamics can applied at all times and the final temperature shift can be deduced from the following self-consistent equation, where t is the period of the modulation and C(T ) is the heat capacity. We do not assume here that under the linear response conditions one is allowed to neglect time dependence of temperature on the r.h.s. of Eq. (5). This is because over long modulation times the temperature change might be substantial. In the experiment 19 , the initial temperature is chosen to be frequency independent, T (ω, 0) = T ini , and the modulation time to be a certain fixed number of modulation cycles t = t(ω) = 2πM/ω. Then the final temperature dependence on frequency, T fin (ω) = T (ω, t(ω)), is directly related to A(ω), and any sharp resonance structure in T fin (ω) can be traced back to the spectral function; i.e., the temperature response provides a practical probe to detect the MG-mode as demonstrated in Ref. 19.
IV. MEASURING THE SPECTRAL FUNCTION IN SIMULATIONS
In this section, the experimental setup from Ref. 19 is used as a benchmark system for calculation of the temperature response from first principles. The parameters closest to the QCP include the lattice depth V 0 = 10E r , which gives a dimensionless coupling parameter j/j c = 1.2 (or U = 14J), and the particle number N = 190(36). Combined with the unity filling requirement at the trap center, this corresponds to the harmonic confinement V i /J = 0.0915(x 2 i +y 2 i ). 10 The small dimensionless modulation of the lattice depth is δV 0 /V 0 0.03, which corresponds to the generalized forces |δf 0 | = 0.087 and |δg 0 | = 0.015. Those parameters define the perturbed BH model (3).
We argue that the external potential term in Eq. (3) is negligible, since both conditions discussed in the previous section are fulfilled, i) |δg 0 /δf 0 | = 0.17 1 ; ii) the correlation length near the trap center at a typical experimental temperature is about one lattice spacing 10 , so that the LDA also holds in the vicinity of the trap center, which dominates the total response.
In the experiment, the modulation protocol consists of two stages. First, ultracold atoms are modulated for M = 20 oscillation cycles. Second, the system is held to thermalize for some time such that the sum of modulation and hold time is constant at 200 milliseconds for all modulation frequencies. During the first stage, both the modulation and the heating power P (T ) contribute to the system's energy dissipation, while during the second stage, only the heating power P (T ) contributes to the energy increase. The integral over time in Eq. (5) must hence be divided into two segments in order to correctly account for both modulation and holding stages. The advantage of keeping the two-stage time at the same value is that the contribution of P (T ) is essentially constant for all modulation frequencies. Finally, the temperature T fin (ω) is determined by slowly ramping the system to the atomic limit and measuring the atomic parity in-situ with single-site resolution.
We rely on path-integral quantum Monte Carlo (MC) simulations with worm-type updates 29 to calculate the scalar spectral functions and the specific heat. Since the particle loss in the experiment is negligible, we simulate the system in the canonical ensemble.
In MC simulations, it is straightforward to measure the imaginary time correlation function for the kinetic energy, T , which is related to A(ω) via the spectral integral with the finite-temperature kernel, N (τ, ω; T ) = 2(e −ωτ + e −ω(1/kBT −τ ) )/(1 − e −ω/kBT ). We employ the same protocol for collecting and analyzing data as in Ref. 10 and 12. More precisely, we collect statistics for the correlation function at Matsubara frequencies ω n and recover χ(τ ) by a Fourier transform. In the pathintegral representation, χ(iω n ) has a direct unbiased estimator, | k e iωnτ k | 2 , where the sum runs over all hopping transitions in a given configuration. Once χ(τ ) is obtained from χ(iω n ) with an accuracy up to 10 −4 the analytic continuation method described in Ref. 18 is applied to extract the spectral function A(ω). We present the analytically continued results at different temperatures in Fig.1, where we see that the curves look qualitatively similar at all temperatures in the range between 0.5J/k B and 3.33J/k B ; values of A(ω; T ) at any temperature in this range can be estimated using linear interpolation. All spectral functions vanishing at zero frequency reflects the absence of dissipation in response to a static external field. Another way to understand this result is through the dissipation-fluctuation theorem, A(ω; T ) = (1 − e −ω/kBT )S(ω; T ) where S(ω; T ) is the dynamic correlation function of kinetic energy. Zero value of A(ω = 0; T ) is a natural outcome of a finite S(ω = 0; T ), see Fig. 6 in Ref. 10. We also would like to point out that the analytical continuation result becomes unreliable at very low frequency ω 1/β when the spectral weight is relatively small.
The heat capacity for a canonical-ensemble system has also been calculated and is shown in Fig. 2. It is seen that the heat capacity becomes much smaller in the superfluid phase than in the normal phase, which may lead to more rapid heating.
To solve Eq.(5) self-consistently, the initial temperature T ini and the heating power P (T ) are also required. However, both quantities were not addressed by the previous experiment nor is P (T ) computable by Monte Carlo simulations. Thus, we are forced to consider both quantities as fitting parameters. In Fig. 3, we show two possible temperature responses to modulation obtained by solving Eq. (5), which both fit the experimental data well despite having rather different (but realistic) sets of (T ini , P ). Excellent agreement between the numerical and experimental results not only ensures that the analytical continuation procedure (as routinely applied on the kinetic energy correlation function [10][11][12][13]15,16 ) is reliable, but also validates various assumptions made in the first-principle calculation, such as quasi-static thermodynamics. We also would like to point out that there are no fundamental difficulties in experiment to measure T ini and P (T ), and thus an even more stringent test avoiding any fitting can be envisioned in the future. Heat capacity C(T /J) per atom as a function of temperature T /J. For the homogeneous system, the Berezinskii-Kosterlitz-Thouless transition temperature is at kBTc 1.04J. 25 Notice that below Tc, the heating of the system gets boosted due to the smallness of C(T /J) . Temperature response to the lattice-depth modulation that reveals the spectral function for the ultracold atomic system. The vertical axis represents final temperature. Filled circles connected by a black dashed line are final temperatures measured experimentally 19 . The solid lines (red and blue) are two predictions based on numerical calculations, with parameters (kBTini = 0.56J, P = 0.27J/sec) and (kBTini = 1.12J, P = 0.45J/sec) respectively. In the calculations, we assume that the heating power P is independent of temperature.
V. ON THE EXPERIMENTAL OBSERVATION OF THE MASSIVE GOLDSTONE PEAK
Comparing the temperature response in Fig. 3 with the spectral function for a homogeneous system with j/j c = 1.2 (or U = 14J) (see Fig. 2 of Ref. 10), we find that while the steep onset of the spectral weight correlates remarkably well with the GM-mode energy, the resonance structure is lost in the experimental system. As mentioned previously, this may occur for two unrelated reasons: either because the MG-mode is overdamped, or the resonant signal is broadened by finite temperature and system inhomogeneity (tight confinement) effects 10 .
Our simulations indicate that the second scenario is far more likely. Previous work on the homogeneous case established that a detuning smaller than j/j c = 1.05 (or U = 16) is required to clearly see the MG-resonance on top of the high-frequency continuum. Let us therefore take a system with j/j c = 1.05, particle number N = 800 and unity filling factor at the trap center and perform a numerical thought experiment: In order to reduce inhomogeneity effects, we limit the lattice-depth modulation to a mesoscopic area around the trap center, where the confining potential is nearly flat. For simplicity, we choose a square area with side length R. In Fig.4, we show spectral functions for different values R. Resolving the resonance structure hiding in the inhomogeneous signal is dramatically improved when the modulation area is reduced. Though no resonance structure is seen when the entire system is modulated (R/d = ∞), it emerges when the modulation region is reduced to R/d = 16 or R/d = 8 at low enough temperature T ∼ T c (where T c 0.45J/k B is the BKT temperature for a homogeneous model with j/j c = 1.05). Converting spectral density to temperature response does not change this observation qualitatively, see Fig. 4, even though the contrast for observing the resonance feature is diminishing. This thought experiment demonstrates that by taking care of response homogeneity, detuning from the QCP, and temperature, the MG-peak can be seen in the kinetic energy spectral function using existing technology. Spectral functions for different modulation areas (a square of size R × R whose center coincides with the trap center) and temperatures for a system with with N = 800 atoms and j/jc = 1.05. The MG-resonance emerges at temperatures T ∼ J/kB when the modulation is limited to a mesoscopic area of linear size R = 16d, where d is the lattice spacing.
Combining the numerical results for the homogeneous and inhomogeneous model from Ref. 10 and 12 and from this work, we deduce that the following three conditions are to be met in order to reveal the MG-peak: First, the modulation area should be restricted to the region with nearly constant chemical potential to ensure that the temperature response is measured for a homogeneous system. To achieve it, a straightforward approach would be to replace the harmonic confinement with the flat-bottom plus sharp walls potential. This approach, however, may lead to problems with controlling system's density and entropy, and , thus, detuning from the QCP. An alternative approach is to restrict modulation to a We assume the system's heating power to be P = 0.2J/sec and the initial temperature kBT /J = 0.5. To optimize the contrast, the number of modulation cycles is set to be three and the sum of modulation and hold time is kept constant at 19 milliseconds. The best modulation strength are found to be δV0/V0 = 0.02, 0.03, 0.06 for R/d = 8, 16, ∞ respectively. mesoscopic area around the trap center, as shown is done in Fig. 5. One promising experimental implementation would be to apply a localized modulation of the scattering length 30 using a laser beam induced Feshbach resonance 31 . The technique has recently been shown in ultracold atom without optical lattice 32,33 . The size of the modulation area can be engineered by tuning the size of the laser beam (e.g. using a mask). Such a modulation would result in a time-dependent on-site interaction U in the modulation area. As pointed out in Sec. III, by subtracting a term proportional to H 0 , the perturbation in potential energy can be replaced with the perturbation in kinetic energy, meaning that the MG-mode can be studied using the same temperature-response protocol as in the current experiment. Second, the system has to be close enough to the QCP so that a Lorentz invariant action provides an adequate description of physics. Our simulations indicate that a detuning j/j c 1.05 is sufficient to reveal the MGresonance, while a smaller detuning j/j c 1.02 is required to recover the universal spectral function Eq. (1) including the critical pseudo-plateau at large frequencies 12 . Third, the system temperature has to be low enough. For a homogeneous system, simulations suggest that the resonance peak survives at temperatures as high as the BKT transition temperature T c , but gradually goes away at T > 2T c 10 . Thus, having initial temperatures below T c is recommended. For example, in the test system with j/j c = 1.05, N = 800, and localized modulation size R = 8d, which heats from T c up to 2T c ( T c 0.45J/k B ), the resonance peak will remain visible in temperature response according to Fig. 4(C).
To conclude, we would like to point out that the quantum critical dynamics in the MI and normal liquid phases is also of great interest. Numerical simulations indicate the presence of a universal resonance structure in the spectral function not only in the SF phase but also in phases with un-broken U (1) symmetry, and at temperatures T T c (normal quantum critical liquid) 12 . The existence of such universal resonances is unexpected within the current weak-coupling theory and their nature requires further study. Verification of this prediction from ultracold atom experiments would be crucial to solve this puzzle. | 2015-11-27T06:24:35.000Z | 2015-09-23T00:00:00.000 | {
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260407931 | pes2o/s2orc | v3-fos-license | Impact of COVID-19 Pandemic on Well-Being, Social Relationships and Academic Performance in a Sample of University Freshmen: A Propensity Score Match Evaluation Pre- and Post-Pandemic
The COVID-19 pandemic has impacted freshmen, compromising their mental health, lifestyles, and academic performance. There are few studies that have investigated changes in the health status and lifestyles of freshmen before and after the pandemic. The aims of this study were: (1) to carry out a pre–post-COVID-19 pandemic comparison between two freshmen samples, in order to detect differences in their socio-demographic characteristics and in some clinical variables; (2) to assess the effect of the COVID-19 pandemic on the social and academic lives of the second sample of freshmen. The samples recruited in 2019 and 2022, matched by propensity score procedure (N = 553), were mostly female (57.3% vs. 55.3%); the mean age was 22.9 and 20.9 years, respectively. The freshmen recruited after the pandemic had less psychological distress and substance use than freshmen recruited before the pandemic. Seventy-eight percent of the freshmen stated that the pandemic had an impact on their social relationships. This effect was greater for females and Italian students. Forty-seven percent reported that the pandemic has worsened their academic performance, while 60% stated that pandemic has improved their grades. The results of this study can provide valuable insights into the impact of the pandemic on freshmen, in order to implement interventions to mitigate the consequences of the pandemic in some subgroups of this target population.
Introduction
The COVID-19 pandemic has brought about significant concerns regarding the mental health of university students. Beyond the physical health risks, the pandemic has emerged as a major stressor that has potentially compromised the mental health of students, leading to lifestyle changes and potential consequences for their overall well-being, levels of anxiety, academic performance, and self-efficacy [1]. Students who showed a higher level of anxiety expressed more negative emotions and perceived themselves with less academic selfefficacy. The pandemic, together with illness and the death of a relative/friend due to COVID-19, increased anxiety and jeopardized the perception of academic self-efficacy [2].
Previous research has highlighted the first year of college as a critical period characterized by heightened vulnerability to psychological distress and its subsequent impact on academic performance [3,4]. There are several factors that make this population vulnerable: freshmen can struggle to orient themselves in college, which requires different time and study management than high school; they must learn to self-regulate their knowledge, using different cognitive strategies; they often live off-site, away from their family, and it can take time to make friends, so they can live for a long time without social support; finally, students can enroll in university without a real intrinsic motivation, but following pressure from others (e.g., parents, etc.). All these factors, together with the changes in social roles and in the re-definition of identity that occurs in this stage of life [5], can represent important stressors. In fact, the transition from high school to university occurs during emerging adulthood. This coming together of multiple challenging development tasks at the same time may be stressful for some students. The COVID-19 pandemic, and above all the health measures implemented to deal with it, may have been an additional stress factor contributing to the difficult adaptation of freshmen to academic life. The Global Survey Report of the International Association Universities (IAU) on the "Impact of COVID-19 on higher education around the world" showed the important degree of stress and constraint experienced by higher education institutions during the pandemic [6]. Almost all institutions are affected by the COVID-19 pandemic and the pandemic has affected all institutional activities. With the emergence of the COVID-19 pandemic, university students have faced several challenges during their transition to higher education in the midst of a global health crisis. The sudden closure of universities and campuses, coupled with the abrupt shift to remote learning formats, has had a profound impact on the social and academic lives of university freshmen. The biggest change was the distance learning, which influenced the academic performance and the emotional factors. Universities had to quickly adapt to this new teaching methodology. Many studies on this topic have appeared in the literature, evaluating students' satisfaction with remote teaching and showing several degrees of appreciation [7]. The IAU Report showed that remote activities were possible to some extent, but could have had negative effects on the quality of activities, also increasing the inequality of learning opportunities [6]. Only in the future will we be able to evaluate the real effects of e-learning [8]. With respect to the emotional aspects, the loss of face-to-face interactions with peers, faculty, and support services has contributed to heightened feelings of loneliness and social isolation [9,10]. Not attending face-to-face lessons made the students worried, nervous, and anxious [11].
These disruptions may have exacerbated the challenges already faced by university freshmen as they adapt to a new academic environment and establish new social networks. Furthermore, the uncertainty surrounding the duration and long-term consequences of the pandemic added an additional layer of stress for university freshmen. Concerns about the impact of COVID-19 on academic progress, future career prospects, and financial stability contributed to heightened anxiety and decreased psychological well-being [12,13]. Recognizing the specific challenges faced by freshmen is of utmost importance due to their heightened susceptibility to psychological distress, which has been associated with lower academic performance and an increased risk of dropout [14,15].
Despite the pandemic being a major stressor that has compromised the mental health of university freshmen and changed their lifestyles, leading to significant consequences for their well-being and academic performance, there have been few studies investigating changes in the health status and lifestyles of university freshmen before and after the COVID-19 pandemic. This is primarily due to the difficulty in gathering large prospective data samples from anonymous surveys. To overcome this limitation, we have adopted the propensity score matching technique, applied to samples of freshmen recruited through two different web surveys conducted before (2019) and after (2022) the COVID-19 pandemic. Although the World Health Organization (WHO) officially declared the end of the COVID-19 pandemic on 5 May 2023, in many countries, the emergency ended in 2022. In Italy, the closure of the state of health emergency declared on 31 January 2020 to counter the spread of the COVID-19 pandemic took place on 31 March 2022 [16]. For this reason, we considered it possible to talk about a post-pandemic timeframe in our research, considering a sample recruited from May to June 2022, when the state of emergency in Italy was over.
With this study, we wanted to fill the gap existing in the current international literature on the impact that COVID-19 has had on freshmen. Even with regard to Italian studies, this is still an unanswered topic. The published studies are almost always on university students and not on freshmen and have always been conducted during the health emergency, before the declaration of the end of the state of emergency issued by the Italian government [7,17]. To our knowledge, there is only one Italian study on freshmen that evaluated the role played by emotional processing and differentiation of self for psychological well-being during the pandemic, but this study was also conducted during the health emergency and therefore did not assess the impact of the COVID-19 pandemic on freshmen [18].
The aims of this study were: (a) to carry out a pre-post-COVID-19 comparison between two samples of freshmen, in order to detect possible differences in their socio-demographic characteristics and in some clinical variables such as well-being, mental distress, substance use, and suicide risk; (b) to assess the effect of the COVID-19 pandemic on the social and academic lives of the second sample of freshmen.
Study Design
The present study took place in a University in Northern Italy, composed of about 15,000 students. The courses of study that the university offers are grouped into 4 areas: medicine, engineering, economics, and law. The study involved two samples of freshmen: the first was recruited from May to June 2019 and the second from May to June 2022. The study was aimed at all freshmen attending university, and the participation was voluntary. For this reason, there were no inclusion or exclusion criteria. The freshmen received a first email asking them to participate in a web-survey, together with the link to access the survey and a complete description of the study. Through the web-link, freshmen were asked to confirm their informed consent to participate. The web-survey was created with LimeSurvey (www.limesurvey.org, accessed on 1 May 2019 and on 1 May 2022), an online survey tool that allows for completely anonymous data collection. The survey was implemented following the guidelines proposed by Pealer and Weiler [19]. To increase response rate, we used some of the strategies proposed by Edwards et al. [20] such as using user-friendly questions, choosing close-ended options for answers, and sending reminders.
Instruments
The tools listed below were administered to the two samples. Table S1 shows the detailed description of the tools.
The General Health Questionnaire (GHQ-12) is composed of 12 items that assess psychophysical well-being [21]. Each item scores from 0 (much less than usual) to 3 (more than usual). A score of 0 was assigned to the first two low-stress alternatives and a score of 1 was given to the two high-stress alternatives. The maximum score was 12, with a cut-off point > 3 indicating psychological distress. The GHQ-12 proved to be a reliable instrument, as indicated by a Cronbach's alpha of 0.81 [22].
The University Stress Scale (USS) is composed of 21 items that capture the cognitive appraisal of demands across the range of environmental stressors experienced by students [23]. Students are asked to rate on a 4-point Likert scale, ranging from 0 (not at all) to 3 (constantly). The total score ranges from 0 to 63: the higher the score, the higher the perceived stress level (cut-off point ≥ 13 is predictive of mental distress). The USS proved to be a reliable instrument as indicated by a Cronbach's alpha of 0.83 [24].
The University Connectedness Scale (UCS) is composed of 18 items that assess the degree of support and membership perceived by students with respect to their university [25]. Students are asked to rate on a 7-point Likert scale, ranging from 1 (not at all) to 7 (all the time). The total score ranges from 18 to 126, where the higher the score, the higher the student's perception of belonging and support within their own university. The UCS has a strong internal consistency with a Cronbach's alpha of 0.88 [26].
A modified version of World Health Organization-ASSIST v3.0, which is a questionnaire, based on the self-report adaptation of Barreto et al. [27], to detect harmful and hazardous drug use [28]. It contains 8 questions about 10 substance categories: tobacco, alcohol, marijuana, cocaine, methamphetamine/amphetamine type stimulants, inhalants, sedatives, hallucinogens, opioids, and other drugs. Each response corresponds to a score, ranging from 0 to 6, with the total score summation ranging from 0 to 39 for each substance. Total scores between 0 and 3 (0-10 for alcohol) are considered "low risk" (occasional or non-harmful use), 4 and 26 (11-26 for alcohol) indicate "moderate risk" (more regular use or harmful/hazardous use), and scores higher than 26 indicate "high risk" (frequent high-risk use or suggestive of dependence). The ability of the ASSIST to classify patients based on degree of drug use has been extensively validated [29,30]. Cronbach's alpha values range from 0.71 to 0.90 [27].
The Brief COPE Inventory [31] is composed of 28 items grouped into 14 sub-scales that represent modalities for facing stress conditions: self-distraction, active coping, denial, substance use, use of emotional support, use of instrumental support, behavioral disengagement, venting, positive reframing, planning, humor, acceptance, religion, and self-blame. Each item scores from 1 (I haven't been doing this at all) to 4 (I've been doing this a lot) and each scale ranges from 2 to 8: a high score is related to a greater ability to cope. The Brief COPE proved a reliability for each scale ranging between 0.50 and 0.90 [31].
The P4 Screener is a brief tool to assess potential suicide risk, which includes a prescreening question about thoughts of self-harming [32]. If a positive answer is given to this pre-screening question, there are subsequent questions on the "4 Ps": Past suicide attempts, Plan, Probability of completing suicide, and Preventive factors. Potential suicide risk is classified as minimal, lower, or higher. There is considered to be a 'minimal' risk when there is no past history, no suicide plan, and a "not at all likely" probability of an attempt. 'Lower' risk refers to respondents who indicated a plan and/or past history but responded "not at all likely" to the question regarding probability and noted there were factors preventing them from taking action. 'Higher' risk respondents are those who reported the probability of a suicide attempt as being either "somewhat likely" or "very likely" and/or reported there were no factors preventing them from taking action. If respondents give a negative answer to the pre-screening question, they are classed in the "did not trigger" category of risk.
The freshmen recruited in the second survey also completed the following instruments. The Academic Motivation Scale (AMS) is a questionnaire developed on the basis of the Self-Determination Theory [33]. For this study, we used the adapted form developed by Biasi et al. [34] to answer the question: Why are you attending the degree program you are enrolled in? The AMS is composed of 20 items rated on an 11-point Likert scale ranging from 0 (not at all true) to 10 (completely true). The items are grouped into 5 sub-scales: lack of motivation, external regulation, introjected regulation, identified regulation, and intrinsic regulation. The total score ranges from 0 to 40, where a higher score corresponds to a greater adherence to the motivational construct of the single sub-scale. The AMS showed good psychometric properties with Cronbach's alpha values ranging from 0.73 to 0.91 [35].
The Perceived School Self-Efficacy Scale (PSSES) is composed of 9 items and structured on a 5-point Likert scale ranging from 1 (not capable at all) to 5 (fully capable). For this study, we used the reduced and adapted form developed by Biasi et al. [34]. The focus of this scale is the perception that students have about their ability to regulate and focus on the studying process. The total score ranges from 9 to 45, where a higher score corresponds to a higher level of self-efficacy perceived by the student in terms of study and academic skills. The PSSES proved to be a reliable instrument with Cronbach's alpha values ranging from 0.83 to 0.87.
The Self-Regulated Knowledge Scale-University (SRKS-U) is a questionnaire developed on the basis of Pintrich's theory of self-regulated knowledge that assesses the frequency with which students implement different cognitive strategies [36]. The SRKS-U consists of 15 items rated on a 5-point Likert scale ranging from 1 (never) to 5 (always or nearly always). The SRKS-U is composed of 5 sub-scales evaluating the use of predefined cognitive processes: knowledge extraction, knowledge networking, knowledge practice, knowledge critique, and knowledge monitoring. The score of each subscale ranges from 3 to 15, where a higher score corresponds to a greater use of that cognitive strategy. The SRKS-U proved to be a reliable instrument with Cronbach's alpha values ranging from 0.70 to 0.80 [37].
The Shortened Achievement Emotion Questionnaire (AEQ-S) [38] is a condensed version of the original Achievement Emotions Questionnaire, designed to assess college students' achievement emotions [39]. In this study, we used the sub-scale "Learningrelated Emotions" that measures students' enjoyment, hope, pride, anger, anxiety, shame, hopelessness, and boredom in situations of studying. This sub-scale consists of 32 items rated on a 5-point Likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). The sub-scale is computed by summing the items and taking the mean (no cut-off). Cronbach's alpha values range from 0.75 to 0.93.
The South Oaks Gambling Screen (SOGS) is a screening tool to assess the presence of a gambling addiction [40]. It consists of 20 elements and includes 3 previous items that do not count towards the total score and are used to evaluate the type of game or bet, the maximum amount staked, and whether they are close to other people with gambling issues. The response options for items are dichotomous ("yes" or "no"). Scores on the SOGS are determined by scoring one point for each question that shows the "at risk" response indicated and adding the total points. The maximum score is 20 and a cut-off score of ≥5 indicates that the respondent is a Probable Pathological Gambler (PPG). The SOGS has a strong internal consistency with a Cronbach's alpha of 0.97.
Furthermore, all freshmen were requested to fill out an assessment form, which provided information regarding their socio-demographic and academic characteristics.
For the evaluation of the effect of pandemic on social relationship and on academic life, ad hoc questions were set for the second (post-COVID-19 pandemic) survey. In detail, freshmen were asked to reply to the following three questions (one regarding social life and two regarding academic life): (i) Did the pandemic impact your social relationships? (ii) Did the pandemic improve your academic grade? (iii) Did the pandemic worsen your academic performance? Grades and academic performance were assessed at the end of the first semester. The responses were set on a 5-point Likert scale (strongly disagree; disagree; moderately agree; agree; strongly agree) and were then dichotomized as 'no vs. yes' responses by merging 'strongly disagree + disagree' vs. 'moderately agree + agree + strongly agree').
Statistical Analysis
Categorical data are presented as absolute (n) and percentage values (%). Differences between the two survey groups were tested using the Pearson's Chi-squared test (or Fisher's exact test when appropriate). Continuous variable distributions were described by mean, median, and standard deviation (SD). Differences between continuous variables were analyzed using a t-test or corresponding non-parametric Mann-Whitney U-test for comparing two groups of Gaussian or non-Gaussian distributed variables respectively.
Propensity score was calculated using a logistic regression model. Potential confounding variables/predictors were chosen in agreement with all available socio-demographic variables in common between the two surveys. Those . We set the matching tolerance to 0.2. Matching was performed in a 1:1 ratio of nearest neighbor without replacements. We excluded freshmen with incomplete information on predictors.
To assess the association of the socio-demographic and clinical variables with the outcomes regarding the effect of the pandemic on personal life (one outcome assessing the perception of the pandemic's impact on social relationships) and on academic life (two outcomes assessing the perception of the pandemic's impact on examination grades and on the overall academic performance, respectively), univariate and multivariable logistic regression models were applied. For the choice of the best predictors in the multivariable models, the stepwise method was applied.
Data analysis was performed using the R software v.4.2.2. For the propensity score, the MatchIt package was used, while for the stepwise method, the step AIC function of package MASS was used.
Comparison between Two Freshmen Samples
The pre-COVID-19 sample collected in 2019 was composed of 553 freshmen while the post-COVID-19 sample collected in 2022 counted 721 freshmen. Considering the original survey collected data (i.e., before the propensity score matching), the differences between the two samples concerned age, marital status, place of residence, working student status, GHQ-12 total score, some scales of the Brief COPE, and the use of some substances categories included in the ASSIST (Table S2). Table 1 shows the differences between the two samples after the propensity score matching for socio-demographic variables (age, gender, nationality, marital status, university status, living status). Matching for age failed due to a severe imbalance between the two surveys for this variable; for this reason, all the subsequent analyses were then adjusted for age. The two samples differed by the GHQ-12 total score: freshmen recruited after the COVID-19 pandemic had lower scores than freshmen recruited before the pandemic, even though the score was indicative of psychological distress in both samples (cut-off > 3). With respect to coping strategies, freshmen recruited in 2022 showed lower scores, compared to freshmen recruited before the pandemic, in: planning, i.e., the attitude to identify the most suitable strategies to resolve the situation; acceptance, that is, the ability to live with difficulties; and self-blame, that is, the attitude to attribute the occurrence of events to oneself. Freshmen recruited after the pandemic showed instead higher scores, compared to pre-pandemic freshmen, in: active coping, indicative of a greater attitude to focus on the situation and to develop strategies to improve it; and substance use (two Brief COPE questions on using alcohol and drugs to cope with stress and feel better). In contrast to the latter data, the post-COVID sample reported lower scores, indicative of lower use, in some substances evaluated through the ASSIST: tobacco, alcohol, marijuana, and cocaine. As regards the other substances, there were no differences between the two groups.
Effect of the COVID-19 Pandemic on Social Relationships
Considering the post-pandemic survey, the majority of freshmen (78%) declared that the pandemic has had an impact on their social relationships (Table S3). The association between the socio-demographic and clinical variables and the perception of the pandemic's impact on social relationships in the sample of freshmen recruited after the pandemic was evaluated by univariate logistic models. The significant variables were: gender (p = 0.021), nationality (p = 0.004), USS total score (p = 0.002), emotional support (p = 0.005), instrumental support (p = 0.003), venting (p = 0.030), lack of motivation (p = 0.042), external regulation (p = 0.020), pride (p = 0.022), shame (p = 0.014), and hopelessness (p = 0.005) ( Table 2). By applying a multiple (multivariable) logistic model with all these significant factors as independent variables, and by applying the step AIC variable selection procedure for finding the best fitting, the best obtained model is shown in Table 2. The results showed that being female, compared to being male, increased the probability by 69% (OR = 1.69, 95%CI: 1.09, 2.62) of saying that the pandemic has had an effect on social relationships compared to saying that it has had no effect. The probability of saying that the pandemic has had an effect on social relationships, compared to saying that it has had no effect, was 76% lower among non-Italian freshmen than among Italian ones. This means that Italian students have suffered more negative effects on social relationships due to the COVID-19 pandemic. As the USS increased by 10 points, the probability of saying that the pandemic has had an effect on social life increased by 40% (OR = 1.04, 95%CI: 1.01, 1.07). Having an external regulation increased the probability of saying that the pandemic had an effect on social relationships compared to saying that it had no effect (OR = 1.035, 95%CI: 1.002, 1.06). Similarly, using instrumental support increased the probability of saying that the pandemic had an effect on social relationships (OR = 1.17, 95%CI: 1.03, 1.34).
Effect of the COVID-19 Pandemic on Academic Performance
The effect of the pandemic on academic life was evaluated by considering both the effects on the examination grades and on the overall perceived academic performance. Regarding the examination grade, 60% of freshmen declared that the pandemic contributed to improving their examination grade (Table S3). The socio-demographic and clinical variables significantly associated with the perceived impact of the pandemic on examination grade were: active coping (p = 0.043), humor (p = 0.049), venting (p = 0.011), sedatives (p = 0.007), knowledge monitoring (p = 0.020), intrinsic regulation (p = 0.028), IAUQ total score (p = 0.018), and SOGS total score (p = 0.032) ( Table 3). Applying the multivariable logistic model and selecting the most predictive variables using the stepwise procedure, the best estimated model included venting, sedatives, intrinsic regulation, and SOGS total score ( Table 3). The results showed that a unit increase in venting and in sedatives score increased the probability by 15% (OR = 1.15, 95%CI: 1.03, 1.29; 95%CI: 1.05, 1.31, respectively) of saying that the pandemic has improved the examination grade compared to saying that it has had no effect. Similarly, a unit increase in SOGT total score increased the probability by 24% of saying that the pandemic has improved the examination grade. Differently, there was an inverse association between intrinsic regulation and perception of improvement in examination grades: a higher score in intrinsic regulation was associated with a reduction of the perception of the improvement in examination grade of 2% (OR = 0.98, 95%CI: 0.96, 0.99). Finally, when freshmen were asked about their perception of the pandemic's impact on their overall academic performance, their response was quite balanced between the perception of worsened (47%) and not worsened (53%) performance (Table S3). Interestingly, using the univariate logistic regression models, we found almost two-thirds of the investigated socio-demographic and clinical variables were associated with the dichotomous outcome variable 'worsened academic performance [yes vs. no]'. When applying the multivariable logistic regression model, the most important features directly associated with the perception of a negative impact of the pandemic on academic performance were: UCS total score, USS total score, introjected regulation, and boredom. An increased score in such variables was associated with a higher probability of perceiving a negative impact of the pandemic on overall academic performance (OR = 1.01, 1.05, 1.02, 1.37, respectively, see Table 4). Differently, a higher age and a higher score on pride were associated with a reduced probability of 7% and 32% (OR = 0.93 and OR = 0.68, respectively) of perceiving a negative impact of the pandemic on academic performance.
Discussion
This study aimed to evaluate the impact of the COVID-19 pandemic on the social relationships and academic lives of a sample of Italian university freshmen. To do this, the survey data collected during the year 2022 (post-pandemic) were compared to data collected in the pre-pandemic year, after the application of the propensity score matching. This procedure allowed us to highlight the psychological and clinical differences pre-postpandemic aside from the potential socio-demographic differences and, in turn, to deduct that the differences may have been induced by the COVID-19 pandemic. The comparison made in this study found a lower, albeit still significant, total score on the GHQ-12 in the post-COVID-19 sample. This indicates that, even after the onset of the pandemic, freshmen continue to experience notable levels of mental distress. While the decrease in the total score might suggest a slight improvement, it is crucial to recognize that the overall value still falls within the range indicating significant mental distress. These findings corroborate the existing literature, which consistently highlights that freshmen are a particularly vulnerable population susceptible to elevated levels of psychological challenges and emotional strain during their transition to university life [4,41].
Results show that in the post-COVID-19 sample there was a decrease in substance use, such as alcohol, marijuana, and tobacco. Several factors may have contributed to this. Firstly, the COVID-19 pandemic and associated restrictions (i.e., social distancing measures, limitations on social gatherings) may have limited access to substances and reduced opportunities for peer influence [42]. The disruption in social contexts and changes in social dynamics during the pandemic may have led to a decrease in substance use among college students. Additionally, the increased focus on health and well-being during the pandemic may have influenced attitudes and behaviors related to a healthier lifestyle [43,44]. Heightened awareness of the potential health risks associated with substance use, the importance of maintaining a strong immune system, and the promotion of healthier lifestyles may have contributed to a decrease in substance use among university freshmen. At the same time, with the reduction in substance use assessed by the ASSIST, there was in the post-COVID-19 sample an increase in substance use as a coping strategy (Brief COPE), which can be explained as students needing to vent and feel less worried/fearful/angry/stressed or lonely [45].
Concerning the coping strategies, the results show that there has been an increase in the use of active coping among post-COVID-19 freshmen. This result can be attributed to several factors. The pandemic has brought about significant disruptions and challenges, such as remote learning, social isolation, and health concerns [46,47]. These unprecedented circumstances may have prompted students to adopt more active and problem-focused strategies to cope with the stressors associated with the pandemic [48]. Furthermore, the unpredictability of the pandemic may have increased students' recognition of the importance of proactive coping [49]. Active coping strategies empower individuals to take control and directly address challenges, which may have become even more crucial in light of the uncertainties surrounding the pandemic and its impact on several aspects of life.
Another result concerns the decrease in the use of the planning coping strategy among post-COVID-19 freshmen. The pandemic has caused significant disruptions and uncertainties in various aspects of life [46,47]. The unpredictable nature of the pandemic and the need for immediate adaptation may have shifted students' focus towards more immediate coping strategies rather than long-term planning and problem-solving. The pandemicrelated restrictions, such as social distancing measures and remote learning, could have restricted students' opportunities for future-oriented planning. The lack of certainty and control over the future, combined with the daily challenges posed by the pandemic, may have prompted freshmen to prioritize coping strategies that address immediate stressors rather than engaging in extensive planning. The psychological impact of the pandemic might have influenced students' cognitive processes and prevented their ability to engage in effective planning [50]. The emotional burden during the pandemic may have redirected students' coping efforts toward emotion-focused strategies rather than future-oriented problem-solving.
Post-COVID-19 freshmen also show a decrease in the acceptance coping strategy. During the pandemic, students may have experienced heightened difficulty in accepting and adapting to the uncertainties and changes imposed by the pandemic, which could have influenced their coping strategies [51]. Moreover, the social and physical distancing measures implemented during the pandemic may have limited freshmen social support networks and reduced opportunities for face-to-face interactions [52]. Social support is known to be an important resource for accepting and coping with difficult situations [53]. The reduction in available social support may have hindered the utilization of acceptancebased coping strategies among post-COVID-19 university freshmen.
Several factors may contribute to explaining the observed decrease in the use of the self-blame coping strategy. For instance, the pandemic and its associated challenges, such as academic disruptions, social isolation, and increased stressors, may have needed a shift towards more adaptive and effective coping strategies. Students may have recognized the limitations and negative consequences of self-blame as a coping strategy and sought alternative approaches. The increased awareness and availability of mental health support during the pandemic in universities [54] may have facilitated the adoption of more constructive coping strategies among university freshmen. The provision of online counseling services, mental health resources, and peer support networks could have promoted the exploration and utilization of healthier coping mechanisms, leading to a decrease in selfblame. Furthermore, the emphasis on resilience-building and psychological well-being in educational institutions during the pandemic may have influenced university freshmen to adopt coping strategies that promote self-compassion and self-care [55,56]. The recognition of the importance of self-acceptance and seeking external support may have contributed to the reduction in the use of self-blame as a coping mechanism.
The findings of this study suggest a noteworthy relationship between the perception of high levels of stress, as measured by the USS, and increased perceptions of the pandemic's impact on social relationships among freshmen. The transition to university life is inherently stressful, and the added stressors brought on by the pandemic, such as remote learning, reduced social interactions, and uncertainty, may have compounded these stress levels [52,57]. Consequently, freshmen who have a higher perception of stress may be more inclined to perceive the pandemic as having a detrimental effect on their social relationships.
The results show a significant gender disparity in freshmen's perception of the pandemic's impact on social relationships: females reported a greater impact than males. This result confirms what was found in another Italian study, that females had a worse psychological condition than males [17]. This may be due to gender differences in coping strategies and social support utilization [58]. Females tend to rely more heavily on interpersonal relationships for emotional support and social connectedness [59]. The disruptions caused by the pandemic, such as physical distancing measures and increased isolation, may have had a more pronounced effect on females' social relationships, leading to a heightened perception of the pandemic's impact. Additionally, societal gender norms and expectations surrounding females' roles as caregivers and nurturers may have influenced their heightened perception of the pandemic's effect on their social relationships [60].
The results show that Italian freshmen reported a greater impact on their social relationships than foreign freshmen. Italians, being immersed in the local culture and societal expectations, may have internalized a stronger sense of collective responsibility, which, in turn, could have contributed to heightened disruptions in their social relationships [61,62]. Conversely, non-Italian freshmen may have maintained connections within their own cultural communities, experienced different cultural norms surrounding socialization, or relied on alternative means of social interaction, leading to a diminished perception of the pandemic's influence.
Moreover, the more freshmen used instrumental support as a coping strategy, the greater the impact of the pandemic on their social relationships. This may be because instrumental support tends to be more strongly associated with problem-focused rather than emotion-focused coping. So, it is probable that those who have not been able to manage the strong emotions related to the pandemic have also suffered more in terms of the detriment of social relationships. Similarly, the more freshmen had external regulation, the greater the impact of the pandemic on their social life. People with this type of regulation are in fact motivated by external elements: control comes from the outside and not from individuals [63]. It is therefore probable that, for them, the pandemic had a greater impact on social relationships, linked to an absence of internal control over the events of their life.
Concerning the effect of the pandemic on academic performance, the sample was equally divided between freshmen who said that the pandemic has improved academic performance and freshmen who said it has worsened it. On the other hand, 60% of the freshmen said they had an improvement in their grades. This result confirms other research in which the academic performance of college students unexpectedly improved during the online learning period [64]. In contrast, an Italian study showed that only 30% of the students declared an improvement in their grade point average [7]. In particular, freshmen who were older and expressed more pride as a learning-related emotion reported a reduced likelihood of perceiving a negative impact of the pandemic on their grades. This result suggests that emotional experiences are important and could have made students more resilient toward COVID-19-related challenges and helped them learn more effectively online. On the contrary, those who reported more distress and the emotion of boredom linked to studying said that the pandemic had had a negative impact on their performance. This study has a few limitations, primarily concerning the generalizability of the results, as the data were collected from a sample consisting of freshmen from a single university. Additionally, the use of web-based surveys may exclude students who are not digitally connected, potentially leading to a lack of representation for certain social groups with distinct socio-economic characteristics and lifestyles. Consequently, conducting an exclusively online survey may introduce significant bias and under-represent certain segments of the population.
Conclusions
This is one of the few studies that have investigated changes in the personal, social, and academic lives of freshmen before and after the COVID-19 pandemic. The results show that freshmen recruited after the pandemic had less psychological distress and substance use, indicative of better psychological well-being. Moreover, these freshmen used coping strategies that addressed immediate stressors rather than engaging in extensive planning.
It is possible that the psychological effect of the pandemic has led freshmen's coping efforts toward emotion-focused strategies rather than future-oriented problem-solving. This result highlights the importance of implementing interventions to mitigate the emotional impact of the pandemic to help freshmen regain perspective for the future. The pandemic seems to have impacted academic performance less and social relationships more, especially for females and Italian freshmen. Specific supportive interventions should therefore be developed for these target students. Further studies are needed to understand the longterm effects of the COVID-19 pandemic on university students, in order to identify the most effective strategies to support their well-being over time. | 2023-08-03T15:28:51.826Z | 2023-07-31T00:00:00.000 | {
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247173475 | pes2o/s2orc | v3-fos-license | Mangrove Utilization as Sources of Ruminant Feed in Belawan Secanang Subdistrict, Medan Belawan District
Mangroves hold several benefits, one of which is barriers from marine abrasion, food sources, aquatic habitats, carbon sinks and storages, places for education and training, ecotourism sites, and sources of ruminant feed. This study aimed to determine the potential of mangroves as sources of ruminant feed and its carrying capacity for sustainable mangrove utilization. The research was conducted in Belawan Sicanang Subdistrict by using purposive sampling for vegetation analysis and questionnaire method. Avicennia marina, Bruguiera sexangula, B. gymnorrhiza, Nypa fruticans, Rhizophora apiculata, Sonneratia alba, and Thespesia populnea were among the mangrove species used by the farmer. The total capacity of mangrove species as ruminant feed in the animal unit (AU) was obtained as dry matter (835.48 AU), crude protein (481.24 AU), and total digestible nutrient (873.77 AU). The carrying capacity of mangroves as represented in the form of dry matter (13.74), crude protein (7.91), and total digestible nutrient (14.36), were categorized as safe. In addition, the potential additional populations based on the safe level of carrying capacity (2.5) was 273 AU, based on crude protein for 131 AU, and based on total digestible nutrients for 288 AU. Keyword: Animal Unit, Carrying Capacity, Mangrove, North Sumatra, Ruminant Feed Received 29 April 2021 | Revised 29 June 2021 | Accepted 1 July 2021
tolerate tidal waves, storms, and tides at any time, thereby reducing coastal abrasion.
Ecologically, mangroves have a function as a source of germplasm, spawning grounds, and nesting sites for marine organisms. Mangroves are also considered highly productive ecosystems because they serve as nutrient cycle reservoirs and a source of carbon and nitrogen for aquatic species. From a socioeconomic aspect, mangroves can be utilized as intercropping areas by protecting economically valuable brackish fish species, also known as silvofishery, and as tourism destinations [2]. Feed is an important element of the livestock industry; in fact, feed management is crucial to the success of any livestock business. The leaves of the api-api (Avicennia spp.) are used as animal feed by the local community in coastal areas of Indonesia. A study on the analysis of Avicennia marina leaves revealed the nutritional content of vitamin B (2.64 mg/100 g), vitamin C (15.32 mg/100 g), fiber (8.7%), and carbohydrates (13%) with high mineral contents promoting its utilization as a source of forage in animal feed [3]. The abundant mangroves in Belawan Sicanang Subdistrict could be studied being used by the local community as a potential source of ruminant feed. The research was carried out to determine mangrove species that could be used as a source of ruminant feed and the carrying capacity of mangroves in the area.
Field Sampling
The research was conducted in Belawan Sicanang Subdistrict from October to December 2020.
Belawan Sicanang Subdistrict is one of the six subdistricts located within Medan Belawan District, Medan City that develops into fishery sites, trading services, settlements, and others.
The subdistrict holds an area of 1,786.91 ha with a mangrove area of 895.242 ha (50.1%).
Sampling sites for vegetation analysis used purposive sampling by placing plots (5×5 m) to determine the mangrove species in the area for a total of 20 observation plots by considering the density of vegetation and geographical location ( Figure 1). The observation sites were determined purposively by considering the geographical conditions and vegetation density that represent the actual biodiversity in the area. The additional method was employed by questionnaire to collect information from 22 goat breeders, 4 sheep breeders, 1 cattle breeder, and 1 buffalo breeder. The measuring tools consisted of GPS (Geographics Position System) to measure geographical coordinates, measuring tape to determine the area of observation plots, ropes to mark the observation plots, calipers to measure the tree diameters, field guide, and identification books to identify mangrove species and questionnaires to collect information from farmers using a list of questions previously prepared. The type of data obtained in this study was presented in Table 1.
Data Analysis
Data analysis in this study based on the ecological parameters of vegetation analysis which
Determination of Carrying Capacity
Potency of mangrove species to be utilized as ruminant feed in Belawan Sicanang Subdistrict was converted into dry matter with desired water content (<74%) dan volume in the field. The The minimum feed requirement of ruminants for one livestock unit or animal unit (AU) is calculated with the assumption that 1 AU of ruminants requires an average of 6.25 kg of dry matter/day or 2,282.25 kg/year [7], crude protein of 0.06 kg/day or 240.9 kg/year and total digestible nutrient of 4.3 kg/day or 1,569.5 kg/year [8]. The average nutrient content of mangrove leaves as a source of ruminant feed is presented in Table 2. In general, the requirement for forage (fresh) by ruminants is 10 percent of body weight.
Nutrient requirements are fulfilled in an appropriate and balanced state if the capacity of feed consumed by the livestock can support their productivity [9]- [10]. The feed carrying capacity index (CCI) is an index that indicates the standard criteria for the safety level of supply of forage [11]- [12]. The feed CCI was obtained from the multiplication of the number of livestock population (AU) and nutritional requirement (kg/AU) divided by the total production (kg). The criterion of an ideal condition based on feed CCI is presented in Table 3.
Vegetation Analysis of Mangrove Species in Belawan Sicanang Subdistrict
The study focused on the community structure formed by sapling species with girth characteristics are <10 cm and height >1.5 m. The species composition and its ecological parameters are presented in Table 4.
Livestock Population in Belawan Sicanang Subdistrict
Based on the survey of breeders namely 22 goat breeders, 4 sheep breeders, 1 cattle breeder, and 1 buffalo breeder, the livestock population in Belawan Sicanang Subdistrict is presented in Table 5 and Table 6.
Mangrove Productivity as Ruminat Feed
Based on the calculation and conversion of raw production and utilization of mangrove species in Belawan Sicanang Subdistrict, the mangrove productivity can be estimated (
Mangrove Carrying Capacity as Ruminant Feed in Belawan Sicanang Subdistrict
The mangrove carrying capacity as depicted from the feed CCI is presented in
Estimation of Additional Livestock Population in Belawan Sicanang Subdistrict
Based on the sustainable level of carrying capacity (CCI = 2.5) of mangrove in Belawan Sicanang Subdistrict, additional livestock population may still be adjusted with the dry matter, crude protein, and TDN of 273, 131, and 288 AU respectively.
Conclussion
Our study reported that the most utilized mangrove species by farmers were Avicennia marina, Bruguiera sexangula, B. gymnnorrhiza, Nypa fruticans, Rhizophora apiculata, Sonneratia alba, and Thespesia populnea. The total capacity of mangrove species as ruminant feed in the animal unit (AU) was obtained as dry matter (835.48 AU), crude protein (481.24 AU), and total digestible nutrient (873.77 AU). The carrying capacity of mangroves as represented in the form of dry matter (13.74), crude protein (7.91), and total digestible nutrient (14.36), were categorized as safe. In addition, the potential additional livestock populations based on the safe level of carrying capacity (2.5) was 273 AU, based on crude protein for 131 AU, and based on total digestible nutrients for 288 AU. | 2022-03-02T16:27:23.166Z | 2022-02-28T00:00:00.000 | {
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8329393 | pes2o/s2orc | v3-fos-license | Src family kinases participate in the regulation of encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages
Src family kinases (SFKs) are non-receptor tyrosine kinases that have been implicated as regulators of the inflammatory response. In this study, the role of SFK activation in the inflammatory response of macrophages to encephalomyocarditis virus (EMCV) infection was examined. Virus infection of macrophages stimulates the expression of cyclooxygenase-2 (COX-2), interleukin (IL)-1β and inducible nitric oxide synthase (iNOS). Inhibition of SFK attenuates EMCV-induced COX-2 expression and prostaglandin E2 production, iNOS expression and subsequent nitric oxide production, and IL-1β expression. EMCV-induced COX-2 expression requires the activation of nuclear factor-κB and the mitogen-activated protein kinase p38. Consistent with these previous findings, inhibition of SFKs attenuated the phosphorylation of p38 in response to EMCV infection, suggesting that SFKs may act upstream of p38. These findings provide evidence that SFK activation plays an active role in the regulation of inflammatory gene expression by virus-infected macrophages.
INTRODUCTION
Macrophages play a crucial role in the inflammatory response to pathogens by producing inflammatory mediators, which recruit and activate a variety of cell types. The immune response of macrophages to pathogens is initiated by the recognition of pathogen-associated molecular patterns by pattern-recognition receptors (PRRs) (Medzhitov, 2007). Activation of these immune receptors initiates signalling pathways, resulting in the expression of inflammatory genes.
Src family kinases (SFKs) have been implicated in the regulation of the macrophage inflammatory response to various stimuli recognized by PRRs (Boulet et al., 1992;English et al., 1997;Leu et al., 2006;Orlicek et al., 1999;Stovall et al., 2004;Ziegler et al., 1988). SFKs are non-receptor tyrosine kinases important in the regulation of a variety of cellular processes including cell proliferation, survival and the inflammatory response (Lowell, 2004;Okutani et al., 2006;Parsons & Parsons, 2004). Pharmacological inhibition of SFKs attenuates cyclooxygenase-2 (COX-2) expression and nitrite production by rat peritoneal macrophages in response to lipopolysaccharide, a ligand for the PRR Toll-like receptor 4 (TLR4) (Leu et al., 2006). Furthermore, SFK inhibition attenuates tumour necrosis factor (TNF) production and inducible nitric oxide synthase (iNOS) expression by RAW264.7 murine macrophages in response to treatment with bacterial DNA mimetic CpG, a ligand for TLR9 (Stovall et al., 2004). Whilst SFK activation appears to participate in the inflammatory gene response to a number of TLRs, little is known concerning the role of SFKs in regulating virusinduced inflammatory responses of infected macrophages.
Virus infection of macrophages stimulates an inflammatory response that is characterized by the expression of COX-2, iNOS and interleukin (IL)-1b. COX-2 catalyses the oxidation of arachidonic acid to prostaglandin H 2 , which is subsequently isomerized to various prostanoids, including prostaglandin E 2 (PGE 2 ) (Smith et al., 2000). PGE 2 participates in the inflammatory response by enhancing the replication of many viruses, whilst inhibiting replication of other viruses (Steer & Corbett, 2003). The expression of iNOS and subsequent production of nitric oxide attenuates virus replication, in part, by nitrosylation and inactivation of viral proteins required for replication (Karupiah et al., 1993;Saura et al., 1999). IL-1b participates in the inflammatory response through recruitment of immune cells and has been implicated in the pathophysiology of many inflammatory diseases (Arend et al., 2008).
In support of a regulatory role of SFKs in the inflammatory macrophage response to virus infection, recent studies have shown that SFK inhibition in dendritic cells results in attenuation of antiviral signalling in response to dsRNA, a viral replication intermediate (Johnsen et al., 2006). Additionally, SFKs have been implicated in the regulation of TNF-a production in response to human immunodeficiency virus type 1 envelope protein in human macrophages (Tomkowicz et al., 2006). In response to encephalomyocarditis virus (EMCV), Yoon and co-workers showed that intraperitoneal administration of the SFK inhibitor PP2 attenuated the accumulation of inflammatory cytokine mRNA in peritoneal macrophages (Choi et al., 2001). These studies suggest a potential role for SFKs in antiviral signalling in macrophages.
In this study, we examined the role of SFKs in the regulation of COX-2 production by macrophages in response to EMCV, a picornavirus that has been used as a prototype virus to study antiviral responses (Iordanov et al., 2000). Inhibition of SFKs attenuates EMCV-induced COX-2 expression and PGE 2 production, iNOS expression and subsequent nitric oxide production, and IL-1b expression. We have shown previously that p38 activation is required for poly(I : C)-and EMCV-stimulated COX-2 expression, and we now provide evidence that SFKs may function upstream of mitogen-activated protein kinase (MAPK) p38 in the regulation of COX-2 expression. Our findings suggest a novel role of SFKs in the regulation of the inflammatory response of macrophages to virus infection.
SFK inhibition attenuates EMCV-induced COX-2 expression and PGE 2 production by macrophages
To examine whether SFKs participate in EMCV-induced COX-2 protein expression (Steer et al., 2003), RAW264.7 cells were treated with EMCV for 24 h in the absence or presence of the SFK inhibitor PP2. As shown in Fig. 1(a), pre-treatment with 10 mM PP2 resulted in~90 % inhibition of EMCV-induced COX-2 protein expression (determined by densitometry). PP3, an inactive analogue of PP2, did not modify EMCV-stimulated COX-2 expression by RAW 264.7 cells (Fig. 1a). SU6656 is a structurally distinct SFK inhibitor that also attenuated EMCV-induced COX-2 expression (Fig. 1b), providing evidence that two structurally different SFK inhibitors attenuate the stimulatory actions of EMCV on COX-2 expression. PP2 treatment also attenuated EMCV-induced COX-2 protein expression in primary mouse peritoneal macrophages (~80 % inhibition; Fig. 1c). The inhibitory actions of PP2 on COX-2 expression appeared to be mediated by attenuation of mRNA accumulation. Similar to previous studies, EMCV stimulated the accumulation of COX-2 mRNA expression following a 6 h treatment (Steer et al., 2006). Both PP2 and SU6656 reduced EMCV-stimulated COX-2 mRNA accumulation in RAW264.7 cells in a concentration-dependent manner (Fig. 1d). COX-2 catalyses the oxidation of arachidonic acid to prostaglandin H 2 , which is subsequently isomerized to various prostanoids, including PGE 2 (Smith et al., 2000). Consistent with the inhibition of COX-2 mRNA and protein expression, PP2 also attenuated EMCV-stimulated PGE 2 production by RAW264.7 cells. Following a 30 min pre-treatment, PP2 reduced EMCV-induced PGE 2 production by~70 % (Fig. 1e). These findings support a role for SFKs in regulating the expression of COX-2 in virus-infected macrophages.
To confirm the pharmacological approaches described in Fig. 1, molecular approaches were used to examine the role of SFKs in the regulation of COX-2 expression by macrophages. RAW264.7 cells were transiently transfected with a control vector or a vector encoding an inactive Src mutant that contains a K296R point mutation in the kinase domain and a Y528F mutation at the phosphorylation site that provides negative regulation of the kinase. There was attenuation in the stimulatory effects of EMCV on COX-2 expression in RAW 264.7 cells expressing this dominantnegative Src mutant compared with cells expressing the empty vector or control macrophages. EMCV-induced COX-2 protein accumulation was reduced by~70 % in RAW264.7 cells expressing dominant-negative Src plasmid (Fig. 2). These findings, which are consistent with the inhibitory actions of PP2 on EMCV-stimulated COX-2 expression, provide molecular evidence to support a role for SFK activation in the expression of COX-2 in EMCVinfected macrophages.
SFK inhibition attenuates EMCV-induced iNOS and IL-1b expression by macrophages
In addition to COX-2 expression, the macrophage response to virus infection also includes the expression of IL-1b and iNOS , and each of these inflammatory genes appears to be regulated by signalling pathways that are common to each gene product and also by pathways that are selective for the target gene of interest. Nuclear factor (NF)-kB is the common signalling pathway that regulates each of these inflammatory genes, whilst the calcium-independent phospholipase A 2 and cAMPresponse element-binding protein appear to be selective for iNOS (Maggi et al., 2002), extracellular signal-regulated kinase (ERK) is selective for IL-1b (Maggi et al., 2003), and c-Jun N-terminal kinase (JNK) and p38 are selective for COX-2 expression (Steer et al., 2006). To determine whether SFKs control the expression of inflammatory genes in addition to COX-2, the effects of EMCV infection on iNOS expression, nitric oxide production and IL-1b expression were examined. EMCV-induced iNOS expression was attenuated by PP2 in a concentration-dependent manner with a reduction of~90 % at 10 mM (Fig. 3a). Consistent with iNOS expression, PP2 inhibited EMCVinduced nitrite production in a concentration-dependent manner (Fig. 3a). Similar results for iNOS expression and nitrite production were observed using a second Src inhibitor, SU6656 (not shown). In addition to iNOS, PP2 also attenuated EMCV-induced pro-IL-1b expression in peritoneal macrophages (~60 % reduction with 10 mM PP2; Fig. 3b). Consistent with the inhibitor studies, transfection with the dominant-negative Src plasmid attenuated EMCV-induced pro-IL-1b protein expression by RAW264.7 cells (not shown). These findings suggested that SFKs participate in the regulation of other EMCVinduced inflammatory genes, in addition to COX-2.
EMCV rapidly induces Src phosphorylation in macrophages
To confirm that EMCV activates SFKs in macrophages, EMCV-induced Src phosphorylation was examined at Y416, the autophosphorylation site that is indicative of activation (Lowell, 2004). As shown in Fig. 4, EMCV stimulated a rapid fourfold increase in Y416 phosphorylation of Src at 15 min post-infection, and the PP2 inhibitor prevented this EMCV-induced phosphorylation of Src. This rapid activation of Src by EMCV is consistent with the Primary macrophages were isolated by peritoneal lavage, adjusted to a final concentration of 4¾10 5 in 400 ml complete CMRL-1066 and pre-treated for 30 min at 37 6C with 10 mM PP2 as indicated. Macrophages were subsequently infected with EMCV (m.o.i. 1) for 24 h, and COX-2 protein expression was determined by Western blot analysis of whole-cell lysates. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control in (a)-(c). (d) RAW264.7 cells (2¾10 5 in 400 ml DMEM) were pre-treated with PP2 or SU6656 for 30 min and infected with EMCV (m.o.i. 1) for 6 h, and total RNA was isolated. The accumulation of COX-2 mRNA was determined by real-time PCR and normalized to GAPDH mRNA levels. (e) RAW264.7 cells (2¾10 5 in 400 ml DMEM) were pre-treated with PP2 (10 mM) for 30 min at 37 6C and then cultured for an additional 24 h in the presence of EMCV (m.o.i. 1). Cell culture supernatants were harvested and PGE 2 accumulation was determined by ELISA. The results are representative of two or three independent experiments (a-c) or the mean±SEM of three independent experiments (d, e). *, P,0.05 compared with the EMCV-treated condition. rapid activation of NF-kB and MAPK signalling pathways in macrophages in response to EMCV (Moran et al., 2005). However, in contrast to the transient activation of MAPK by EMCV (Moran et al., 2005), Src phosphorylation remained at an increased level 1 h after EMCV treatment (not shown). The prolonged increase in Src phosphorylation for greater than 1 h has been observed previously for other ligands, including dsRNA (Johnsen et al., 2006) and granulocyte-macrophage colony-stimulating factor (Suh et al., 2005).
SFKs are not required for EMCV-induced IkB degradation
Activation of NF-kB is required for expression of the inflammatory genes COX-2, IL-1b and iNOS by macrophages in response to EMCV infection (Moran et al., 2005;Steer et al., 2003). As SFKs appear to participate in the regulation of these inflammatory genes, the potential role of SFKs in the activation of NF-kB was examined. Using the degradation of IkBa as an index for NF-kB activation, we showed that inhibition of SFKs using PP2 did not attenuate EMCV-induced IkBa degradation (Fig. 5a). We examined IkBa expression at multiple time points after EMCV infection, and found no effect of PP2 on the kinetics of IkB degradation (not shown). These findings suggest that SFKs do not regulate the inflammatory response by acting upstream of NF-kB activation.
DISCUSSION
In this study, the role of SFKs in the inflammatory response of macrophages to EMCV was examined. We showed that chemical and molecular inhibition of SFKs attenuated EMCV-induced inflammatory gene expression by macrophages. Inhibition of SFKs attenuated EMCV-induced COX-2, IL-1b and iNOS expression, as well as PGE 2 and nitrite accumulation by macrophages. SFKs have been implicated in the regulation of the macrophage inflammatory response to various PRR ligands. Our current study extends the role for SFKs in the macrophage inflammatory response to include a role in the response to virus infection.
Recent studies have identified a number of pathways involved in the regulation of inflammatory gene expression in virus-treated macrophages. NF-kB plays a primary role in regulating macrophage expression of iNOS, COX-2 and IL-1b in response to EMCV Maggi et al., 2003;Steer et al., 2003). SFKs have been shown to participate in cytokine-induced COX-2 expression in human epithelial cells by activating IkB kinase, resulting in IkBa degradation and NF-kB activation (Huang et al., 2003). Although NF-kB signalling is required for EMCVinduced COX-2, IL-1b and iNOS expression by macrophages Steer et al., 2003), we showed that SFK inhibition does not appear to prevent EMCV-stimulated IkBa degradation. These findings suggest that SFKs do not act upstream of NF-kB. In addition to NF-kB, the activation of a secondary signalling pathway that is selective for the target gene of interest is also required for EMCV-induced inflammatory gene expression by macrophages. These secondary signalling pathways include JNK and p38 activation for COX-2 (Steer et al., 2006), ERK activation for IL-1b (Maggi et al., 2003) and calcium-independent phospholipase A 2 for iNOS (Maggi et al., 2002). The ability of PP2 to attenuate EMCVstimulated p38 activation suggests that SFKs participate in EMCV-induced COX-2 expression through the activation of p38. SFK inhibition did not modify EMCV-stimulated JNK and ERK phosphorylation, suggesting that activation of these pathways by virus infection occurs by SFKindependent processes. In accordance with these findings, SFKs have been shown to act upstream of p38 in the activation of neutrophils (Mocsai et al., 2000). At present, it is unclear how SFKs participate in the regulation of macrophage expression of iNOS and IL-1b in response to EMCV. Whilst we have previously identified a primary role for ERK in the regulation of IL-1b expression, and for NF-kB in the regulation of IL-1b and iNOS expression in response to EMCV, inhibition of SFKs does not modify activation of these pathways in response to EMCV infection, suggesting the participation of pathways in addition to NF-kB, ERK and SFKs. The proposed mechanism by which SFKs regulate EMCV-induced COX-2 expression is depicted in Fig. 6.
Infection of macrophages with EMCV results in the rapid phosphorylation of Src; however, the mechanisms by which SFKs are activated have yet to be determined. Whilst the dsRNA-dependent protein kinase R (PKR) initiates various antiviral responses in infected cells, a number of studies have shown that EMCV-stimulated MAPK activation and inflammatory gene expression do not require the presence of PKR (Moran et al., 2005;Steer et al., 2003). Recently, SFKs were shown to participate in antiviral signalling (interferon regulatory factor-3 activation and STAT1 phosphorylation) in dendritic cells by associating with TLR3 (Johnsen et al., 2006). However, the inflammatory response of macrophages to EMCV, including MAPK activation and COX-2 expression, occurs in the absence of the dsRNA receptor TLR3 (Steer et al., 2006). Furthermore, the intracellular dsRNA receptor melanoma differentiation-associated gene 5 (mda5) (Gitlin et al., 2006) does not appear to mediate the inflammatory response to EMCV (J. Corbett & B. Christmann, unpublished data). Thus, EMCV activates inflammatory pathways independent of PKR, TLR3 and mda5. The signalling receptor activated by EMCV is currently unknown. The intracellular dsRNA receptor retinoic acid inducible gene I (RIG-I) may mediate the EMCV-induced inflammatory response of macrophages (Yoneyama et al., 2004). However, we believe that RIG-I is not a likely candidate, because EMCV capsid void of virus RNA is capable of activating MAPK and NF-kB pathways and iNOS expression, suggesting that the signalling pathways regulating inflammatory gene expression do not require viral RNA accumulation or viral protein expression (Moran et al., 2005). These findings suggest that EMCV capsid protein may activate signalling pathways by interacting with a cell-surface receptor. Consistent with this possibility, MAPK and NF-kB signalling pathways, as well as SFKs, are rapidly activated after a 15 min EMCV infection. SFKs are known to be activated in response to stimulation of various cell-surface receptors, including G-protein-coupled receptors (GPCRs) (Thomas & Brugge, 1997). SFKs can be activated through the G-protein subunits Ga s and Ga i (Ma et al., 2000), as well as independently of G proteins (McGarrigle & Huang, 2007;Sun et al., 2007). We are currently examining the potential role of GPCRs as regulators of inflammatory gene expression by virus-infected macrophages.
METHODS
Materials and animals. RAW264.7 cells and L929 cells were obtained from the Washington University Tissue Culture Support Center (St Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM; containing 10 % heat-inactivated fetal calf serum and 16 Lglutamine), CMRL-1066 tissue culture medium, L-glutamine, penicillin and streptomycin were from Gibco-BRL. C57BL/6J mice were purchased from Harlan. PP2, PP3 and SU6656 were purchased from Calbiochem. Rabbit anti-COX-2 and anti-iNOS antiserum were obtained from Cayman Chemicals. 3ZD monoclonal anti-pro-IL-1b was obtained from the Biological Resources Branch at the NCI (National Institutes of Health, Bethesda, MD, USA), rabbit antiphospho-ERK, anti-phospho-p38 and anti-phospho-JNK from Promega, rabbit anti-IkBa and rabbit anti-STAT1 antiserum from Santa Cruz Biotechnology, mouse monoclonal anti-Src (clone GD11) from Upstate, rabbit anti-phospho-Src (Y416) from Cell Signaling Technology and mouse anti-GAPDH antiserum from Ambion. Horseradish peroxidase-conjugated donkey anti-rabbit and donkey anti-mouse antibodies were obtained from Jackson ImmunoResearch. The PCR primers for COX-2 and GAPDH were purchased from Integrated DNA Technologies. The dominant-negative Src in the pUSEamp expression vector was from Upstate.
Peritoneal macrophage isolation and cell culture. Primary macrophages were obtained from wild-type C57BL/6J mice by peritoneal lavage as described previously (Beckerman et al., 1993). Briefly, the peritoneal cavity was injected with 10 ml ice-cold Hanks' balanced salt solution, the lavage was extracted and peritoneal exudate cells were collected by centrifugation, washed and plated at a density of 4610 5 cells in 400 ml complete CMRL-1066. Peritoneal cells were washed with CMLR-1066 to remove non-adherent cells.
RAW264.7 cells were removed from growth flasks by treatment with 0.05 % trypsin/0.02 % EDTA at 37 uC, washed with DMEM and plated at the indicated concentrations. Macrophages were cultured for a minimum of 2 h under an atmosphere of 95 % air, 5 % CO 2 prior to initiation of the experiments.
Virus propagation and infection. The B variant of EMCV was a generous gift from Dr Ji-Won Yoon (University of Calgary, Alberta, Canada) and has been described elsewhere (Bae et al., 1989). EMCV was propagated in L929 cells, supernatants were clarified by centrifugation and titres were determined by plaque assay. Cell monolayers were infected at an m.o.i. of 1 by the addition of EMCV to the culture medium at 37 uC for the times indicated.
Real-time PCR. Total RNA was isolated from macrophages using an RNeasy kit according to the manufacturer's instructions (Qiagen). First-strand cDNA synthesis was performed using oligo(dT) and a reverse transcriptase Superscript pre-amplification system (Invitrogen) following the manufacturer's recommendations. Realtime PCR was performed using iQ SYBR Green Supermix (Bio-Rad) and a Research DNA Engine Opticon II thermocycler with continuous fluorescence detection (MJ Research). The mRNA levels of COX-2 were normalized to those of GAPDH. The PCR primer sequences for COX-2 were: 59-TTTGTTGAGTCATTCACCAGACA-GAT-39 (forward) and 59-CAGTATTGAGGAGAACAGATGGGATT- Src regulation of virus-stimulated COX-2 expression 39 (reverse). The PCR primers for GAPDH have been described elsewhere (Arnush et al., 1998).
Transfection. RAW264.7 cells were transiently transfected using an Amaxa Nucleofector electroporator (Amaxa Biosystems). The cells were removed from growth flasks by treatment with 0.05 % trypsin/ 0.02 % EDTA at 37 uC, washed with DMEM and incubated for 2 h at 37 uC. The cells (2610 6 ) were resuspended in electroporation buffer and electroporated with 2 mg plasmid using program D-032. A total of 4610 5 cells in 400 ml DMEM were plated per condition. Six hours after transfection, the medium was replaced and the cells were cultured overnight at 37 uC. The cells were washed twice with PBS before initiation of experiments. We obtained a transfection efficiency of~50 %, as determined by electroporation with a plasmid expressing enhanced green fluorescent protein.
Determination of nitrite and PGE 2 . Nitrite production was determined by the addition of 50 ml Greiss reagent to 50 ml macrophage cell culture supernatant (Green et al., 1982). Absorbance at 540 nm was measured and nitrite concentrations were quantified by comparison to a sodium nitrite standard curve. PGE 2 release in supernatants was determined using a PGE 2 enzyme immunosorbent assay according to the manufacturer's specifications (Cayman Chemicals).
Statistics. Statistical comparisons between groups were made using one-way analysis of variance. Significant differences between groups (P,0.05) were determined by Newman-Keuls post-hoc analysis. Western blots were quantified by densitometry using ImageQuant software (GE Healthcare) and values were normalized to loading controls. | 2016-05-12T22:15:10.714Z | 2010-09-01T00:00:00.000 | {
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234873077 | pes2o/s2orc | v3-fos-license | Lipids Abnormality and Type 2 Diabetes Mellitus: Causes and Consequences
Dyslipidemia and diabetes both are important risk factors for cardiovascular disease. Emerging evidence suggests that these two are closely related to each other, the so-called “dyslipidemia-insulin resistance-hyperinsulinemia” cycle. Recently, several new lipid subfractions, such as apolipoprotein (Apo)B, and ApoJ, have been reported to associate with insulin resistance and incident diabetes, which further claim the role of lipid in the pathophysiology of diabetes. Besides, dyslipidemia is also one of the most prevalent diabetic complications. Clinical guidelines have widely recommended lipid management among diabetic patients through lifestyle intervention and lipid-lowering medications, especially statins, to prevent cardiovascular outcomes.
Introduction
Type 2 diabetes mellitus (T2DM) is among the most prevalent chronic disease [1], affecting approximately 463 million people in 2019, and more than 690 million are expected to be diagnosed by 2045 [2]. People diagnosed with T2DM have a 2-4-fold higher risk of developing cardiovascular disease (CVD) [3]. Despite significant advantages in the prevention strategies that lessen related risk factors, CVD remains the leading cause of morbidity and mortality in patients with T2DM [4].
T2DM and CVD both have multi-factorial etiology, and disorders of lipid metabolism is one of the coexistence features sharing by them. For the development of CVD, the cumulation of ApoB-containing lipoproteins in the arterial wall would lead to lipid deposition and an atheroma initiation, resulting in the progression of atherosclerotic plaques, and eventually atherosclerotic vascular disease [5]. Therefore, lipid-lowering drugs, such as statins, have been recommended as front-line therapy for primary prevention of atherosclerotic CVD [6], and the stateof-the-art therapy in dyslipidemia in diabetic patients [3,7,8]. For the potential mechanism between lipid metabolism and diabetes, one meta-analysis reported that lipid parameters, such as triglyceride (TG), and low-density lipoprotein (LDL), can reflect the risk of T2DM [9]. Other lipid subfractions, such as high-density lipoprotein cholesterol (HDL) and lipid-free ApoA-I, could also benefit glycemic control by increasing glucose uptake in skeletal muscle, improving beta-cell function, and decreasing insulin resistance through inhibiting the proinflammatory signal transduction pathways [10]. which in turn aggravates the generation of lipid metabolism disorder [27][28][29]. More recently, Seo et al. found that ApoJ is a novel hepatokine targeting muscle glucose metabolism and insulin sensitivity through low-density lipoprotein receptor-related protein-2 (LRP2)-dependent mechanism, coupled with the insulin receptor signaling cascade. In muscle, LRP2 is necessary for insulin receptor internalization, an initial trigger for insulin signaling, that is crucial in regulating downstream signaling and glucose uptake. Of physiologic significance, deletion of hepatic ApoJ or muscle LRP2 causes insulin resistance and glucose intolerance [30].
Lipid-lowering medication and incident type 2 diabetes mellitus
Lipid-lowering medication plays an essential role in the current healthcare system, not only for the optimization of the lipid profile but also to reduce cardiovascular risk [6,12]. Recently, there is increased awareness of the possibility that lipid-lowering medications may affect glucose control and insulin resistance [31][32][33]. This phenomenon is reported in all classes of lipid-modifying agents, with differential effects of distinct drugs.
Some insights into this question emerged from some recent studies. Barak et al. systematically reviewed the related evidence and reported that both statins and niacin are associated with increased risk of impaired glucose control and development of new-onset diabetes, as opposed to bile-acid sequestrants which display concomitant moderate lipid and glucose-lowering effects, and fibrates (particularly the pan-PPAR agonist bezafibrate) which may produce beneficial effects on glucose metabolism and insulin sensitivity [34]. Another recently published meta-analysis, which included 163,688 nondiabetic patients from thirty-three randomized controlled trials, reported no significant association between 1-mmol/L reduction in LDL cholesterol and incident diabetes for statins or PCSK9 inhibitors. More intensive lipid-lowering therapy (defined as the more potent pharmacological strategy, such as PCSK9 inhibitors, higher intensity statins, or statins) was associated with a higher risk of incident diabetes compared with less intensive therapy (active control group or placebo/usual care of the trial). Meta-regression analysis suggested that these results were mainly driven by a higher risk of incident diabetes with statins, whereas PCSK9 inhibitors were not associated with incident diabetes (P = 0.02 for interaction). Thus, among intensive lipid-lowering therapies, there was no independent association between reduction in LDL cholesterol and incident diabetes [32].
The precise mechanisms for statin-induced diabetes remain unclear. However, several mechanisms have been proposed, including impaired insulin sensitivity, impaired insulin secretion, and compromised beta-cell function via enhanced intracellular cholesterol uptake due to inhibition of intracellular cholesterol synthesis by statins [34]. Recently, genetic studies have added more evidence to this. LDL-lowering alleles in HMGCR, which encodes the statins' molecular target, were associated with a higher risk of T2DM and higher body mass index [35]. A further larger-scale individual meta-analysis concluded that other LDL-lowering alleles, such as NPC1L1, PCSK9, ABCG5/G8, were also associated with a higher risk T2DM [31]. Nevertheless, it has to be stressed here that the cardiovascular benefits of statins far outweigh diabetes risk [3].
Type 2 diabetic dyslipidemia
Diabetic dyslipidemia is a cluster of plasma lipid and lipoprotein abnormalities that are metabolically interrelated among diabetic patients. It is mainly characterized by increased TG levels, low HDL levels, and postprandial lipemia and contributes to the development of vascular complications [36]. Results from the 2010-2012 China National Nutrition and Health Survey (CNNHS) shown that the prevalence of dyslipidemia was 39.9%, 46.8%, and 59.3% in participants with normal glucose, prediabetes, and T2DM [16]. Another study using data from the 2010-2014 Diabetes Mellitus/Hypertension (DM/HT) study, which included 140,557 Thai adults with diabetes, reported that the dyslipidemia prevalence of 88.9% [37]. Despite the heterogeneity between different studies, the prevalence of diabetic dyslipidemia has grown gradually worldwide [4].
The pathophysiology of diabetic dyslipidemia is intricate and has not been fully understood [38]. Briefly speaking, changes in plasma lipoproteins among diabetic patients are affected by insufficient insulin function and hyperglycemia [39]. During the postprandial state, dietary fatty acids (FA) and cholesterol absorbed by the intestinal cells are incorporated as TG and cholesteryl esters into chylomicrons. In the capillary beds of adipocytes (especially in the fed state) and muscle, chylomicrons are the substrate for lipoprotein lipase (LPL), promoting lipolysis of chylomicrons TG and the release of FA. Insulin regulates LPL activity at several levels, including gene expression, protein synthesis, and secretion, and LPL is reduced in insulin-resistant individuals with T2DM with a consequent increase in plasma TG and decrease in HDL [40].
Type 2 diabetic dyslipidemia and cardiovascular disease
Both diabetes and dyslipidemia are important risk factors for CVD development, powered by the dyslipidemia-insulin resistance-hyperinsulinemia cycle [41]. This makes patients with diabetes dyslipidemia much more vulnerable to CVD outcomes. Results from the Multiple Risk Factor Intervention Trial (MRFIT) reported that among men who had diabetes at baseline, the absolute risk of coronary mortality at each level of blood cholesterol (for 20 mg/dL increments in TC starting from 180 mg/dL to >280 mg/dL), was 3-5 times higher in the presence of diabetes [42]. The United Kingdom Prospective Diabetes Study (UKPDS) has provided further evidence of a similarly direct and continuous association of coronary disease risk with LDL concentration. Among newly diagnosed T2DM, one mmol/L increase in LDL was associated with a 57% increased risk of myocardial infarction [43].
Many former studies have widely reported the causal association between dyslipidemia and CVD. Due to the rather complex components of lipid profile, diagnosis of diabetic dyslipidemia is not always revealed by the lipid measures used in clinical practice, as LDL levels may remain within the normal range. Therefore, it is suggested to use non-HDL levels to reflect the whole lipid spectrum [12]. The 2019 ESC/EAS Guidelines stated that ApoB analysis is recommended for risk assessment, particularly in people with high TG, diabetes, obesity, or metabolic syndrome. ApoB can be used as an alternative to LDL-C, if available, as the primary measurement for screening, diagnosis, and management [6].
Since a higher risk of atherosclerotic vascular disease in diabetic patients, lipid management has been recommended by diabetes-related clinical guidelines [3,7,8,44,45]. Consistent data have demonstrated the efficacy of statins, the firstchoice lipid-lowering treatment, in preventing cardiovascular events and reducing cardiovascular mortality in patients with diabetes. A meta-analysis including 18,686 diabetic patients demonstrated that a statin-induced reduction of LDL by 1.0 mmol/L was associated with a 9% reduction in all-cause mortality and a 21% reduction in the incidence of major CV events [46]. The newly released ADA's Standards of Medical Care in Diabetes-2020 has stated that statins should be used DOI: http://dx.doi.org/10.5772/intechopen.96592 for both primary and secondary CVD prevention among diabetes. The detailed guideline are listed according to age groups: for 20-39 years-old diabetic patients with atherosclerotic cardiovascular disease risk factors, statin therapy is highly recommended in addition to lifestyle therapy; for patients with diabetes aged 40-75 years without atherosclerotic cardiovascular disease, use moderate-intensity statin therapy (lowers LDL by 30-49%) in addition to lifestyle therapy; while for patients aged 50-70 years with diabetes, high-intensity statin therapy (lowers LDL by ≥50%) is recommended [8]. The 2013 ACC/AHA guideline emphasized statin therapy recommended for all patients with diabetes 40 to 75 years of ageindependent of baseline cholesterol [47]. Despite the CVD protective effect among diabetic patients, statin therapy has been associated with new-onset T2DM [31,32]. A former study reported that for every 40 mmol/L reduction of LDL by statins, conversion to T2DM is increased by 10% [48,49]. Nevertheless, the benefits in terms of cardiovascular event reduction For patients with diabetes aged 20-39 years with additional atherosclerotic cardiovascular disease risk factors, it may be reasonable to initiate statin therapy in addition to lifestyle therapy.
In patients with diabetes at higher risk, especially those with multiple atherosclerotic cardiovascular disease risk factors or aged 50-70 years, it is reasonable to use high-intensity statin therapy.
In adults with diabetes and a 10-year atherosclerotic cardiovascular disease risk of 20% or higher, it may be reasonable to add ezetimibe to maximally tolerated statin therapy to reduce LDL cholesterol levels by 50% or more.
ESC/EASD
In patients with T2D at moderate CV risk, an LDL-C target of <2.6 mmol/L (<100 mg/dL) is recommended.
Statins are recommended as the first-choice lipid-lowering treatment in patients with DM and high LDL-C levels: administration of statins is defined based on the CV risk profile of the patient and the recommended LDL-C (or non-HDL-C) target levels.
In patients with T2D at high CV risk, an LDL-C target of <1.8 mmol/L (<70 mg/dL) and LDL-C reduction of at least 50% is recommended.
In patients with T2D at very high CV risk, an LDL-C target of <1.4 mmol/L (<55 mg/dL) and LDL-C reduction of at least 50% is recommended.
CDS
The primary goal is to reduce LDL-C to the target (very high risk of ASCVD: <1.8 mmol/L, high risk of ASCVD: <2.6 mmol/L).
Statins are the preferred lipid-lowering drugs. Lipid-lowering therapy should start with a moderate-intensity statin, and the dose should be adjusted according to individual response to medication and tolerability.
LDL-C reduction by ≥50% may be used as an alternative target in the event of high baseline LDL-C and failure to reduce LDL-C to the target after 3 months of standard lipid-lowering therapy.
JDS
The primary goal of antidyslipidemic therapy is to control the LDL-C level to <120 mg/dL in patients without a history of coronary artery disease.
Statins are the agents of choice for hyper-LDL-C in patients with diabetes.
The control goal for fasting triglyceride (TG) is <150 mg/dL.
The control goal for HDL-C is ≥40 mg/dL. Table 2.
Guidelines for the management of diabetic dyslipidemia using statin.
Conclusion
Complex lipoprotein metabolism abnormalities could present both in the development and progression of type 2 diabetes, which indicates that lipid management can prevent cardiovascular complications among diabetic patients and involve in the prevention of diabetes. Epidemiological studies suggest that lipid components could be a marker for diabetes prediction, though it is still uncertain which lipid markers are of the most clinical value. Lipid control using a lipid-lowering medication, such as statins, could reduce CVD risk among the general population also diabetic people. However, it is necessary to consider statin diabetogenicity in clinical practice when the statin is indicated.
Conflict of interest
The authors declare no conflict of interest.
© 2021 The Author(s). Licensee IntechOpen. This chapter is 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. | 2021-05-21T16:57:13.426Z | 2021-04-12T00:00:00.000 | {
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