Traffic-related air pollution (TRAP) is a major source of ambient air pollution in urban areas. Shopkeepers of heavily trafficked roadside shops are persistently exposed to high levels of TRAP.To estimate the prevalence of respiratory morbidity in shopkeepers of shops in heavily trafficked roadside areas in Bhopal city (India) and to determine any association with long term exposure to TRAP.This cross-sectional study was conducted among 251 shopkeepers working in roadside shops of three major traffic corridors in Bhopal city. The demographic profile and prevalence of respiratory morbidity were collected by administering a validated questionnaire. The total exposure period (TEP) to TRAP was calculated for each individual by multiplying their work duration (in years) and average working hours per day. Odds ratios were calculated to estimate the association of TEP with respiratory morbidity.The age of the study population was 44.8±13.5 years old and 95% were male. Nearly 55% of the shopkeepers reported at least one respiratory symptom. The prevalence of bronchial asthma, chronic bronchitis, breathlessness, and cough was 3.6% : 1.9–6.7), 13.9% (95% CI: 10.2–18.8), 41.8% (95% CI: 35.9–48.0), and 18.3% (95% CI: 14.0–23.6), respectively. The adjusted risk ratios of bronchial asthma 2.17 (95% CI: 0.35–13.41), chronic bronchitis 1.42 (95% CI: 0.58–3.48), breathlessness 1.71 (95% CI: 0.94–3.11), and cough 0.97 (95% CI: 0.47–2.03) for those with a TEP over 100.Shopkeepers working in heavily trafficked roadside shops suffer from respiratory morbidity and the risk increases with higher TEP. Total exposure period is a valuable indicator to estimate the effects of long-term TRAP exposure.ObtainedThe study was approved by the Institutional Ethics Committee of the National Institute for Research in Environmental Health .The authors declare no competing financial interests. Outdoor air pollution accounted for 6% of the total disease burden in India in 2016.3The adverse health effects of TRAP are usually greater than industrial pollutants, as TRAP is released near ground level and remains there for longer periods, especially where roads are enclosed by high-rise buildings. Epidemiological studies have established the adverse health effects of TRAP exposure and its association with higher cardiorespiratory morbidity and mortality.7The aim of present study was to estimate the prevalence of respiratory morbidity in shopkeepers working in heavily trafficked roadside shops in Bhopal city and to determine its association with duration of work, a surrogate for long-term exposure to TRAP.Figure 1). Area I was located around Hamidia Road, a well-known congested and busy road of old Bhopal city. The intercity bus terminal and rail station are situated on this road. The vehicles on Hamidia Road include passenger cars and heavy vehicles and this area has an estimated traffic volume of 72,000 passenger car unit (PCU).A cross-sectional descriptive study was carried out in Bhopal. The study was approved by the Institutional Ethics Committee of the National Institute for Research in Environmental Health . Bhopal city is divided into two distinct parts: the old and new city. The roads of the old city are narrow, congested and have a high volume of heterogeneous traffic. Remote sensing and geographic information system-based mapping demonstrate that major traffic corridors of Bhopal city are responsible for poor ambient air quality.CIConfidence intervalCPCBCentral Pollution Control BoardINSEARCHIndian Study on the Epidemiology of Asthma, Respiratory Symptoms and Chronic BronchitisIQRInterquartile rangePCUPassenger car unitPMParticulate matterTEPTotal exposure periodTRAPTraffic-related air pollutionWHOWorld Health OrganizationShopkeepers from the three study areas were approached to participate in the present study. We enrolled only shop owners who were actively involved in the management of their shops, to ensure that this would reflect actual exposure. Other shop employees were excluded as they often change jobs, thus their actual exposure to TRAP could not be determined. Restaurant owners were also excluded as they might have additional exposure to kitchen fumes. The nature of the study was explained and verbal consent was obtained from each participant prior to data collection.Information on various respiratory symptoms was collected using a pre-validated Hindi translation of the Indian Study on the Epidemiology of Asthma, Respiratory Symptoms and Chronic Bronchitis (INSEARCH) questionnaire.A previous study from Bangalore (India) reported a 12.4% and 32.2% prevalence of cough and breathlessness in roadside shopkeepers, respectively.2* P * (1 - P) / d2, where Z was the level of confidence (95%), P was the expected prevalence (20%), and d was the allowable error (5%).With a sample size of 251 subjects and a margin of error set at 5%, we used the formula n = ZAll baseline variables and outcomes were summarized as percentages, median with interquartile range (IQR), and mean ±standard deviation. The prevalence of respiratory symptoms with 95% confidence interval (CI) was calculated based on the Wilson score method. The association of respiratory symptoms with location was assessed using the chi-square test and Fisher's exact test based on the data distribution. To standardize the duration of TRAP exposure, we calculated the total exposure period (TEP) of each individual by multiplying their work duration (in years) and working hours per day. Longer periods of exposure to TRAP are necessary to perceive health impacts. Thus, TEP measurements were categorized into three categories  based on the TEP distribution of the study population to examine the prevalence of respiratory morbidity in each TEP measurement. Univariate logistic regression was performed to identify the association of TEP, smoking, and age with different respiratory morbidities . Multivariate logistic regression analysis to estimate the effect of TEP on respiratory morbidity was carried out using TEP ≤ 100 (i.e. worked for 10 hours per day for 10 years) as a reference. The odds ratios with 95% CI were presented. Stata 13.1 software for windows  was used for the statistical analysis. For all analyses, a two-tailed P value <0.05 was considered to be statistically significant.The present study was carried out between September 2017 and January 2018. A total of 485 shopkeepers were approached for recruitment and 251 (52%) participated. The main reason shopkeepers gave for refusing to participate was that they were preoccupied with customers. The number of shopkeepers recruited from area I, II, and III were 85 (33.9%), 68 (27.1%) and 98 (39%), respectively. All participants resided in houses that were at different locations from their shops. The demographic characteristics of the study population did not differ between study areas. The education level of study participants was as follows: 34% had a high school graduate level or higher, 50% had a middle to high school education, and 16% had a primary school level education or lower. The majority of the study population was male (97%) and their mean age was 44.8±13.5 years old. The mean working hours per day was 10.4±2.0 and median work duration was 16 years (IQR: 10–26). The median TEP of the study population was 165 (IQR: 84–300). Smoking history longer than one year was reported by 15.5% of study participants. The prevalence of previously diagnosed diabetes and hypertension among the participants was 10% and 17.9%, respectively.The prevalence of different respiratory symptoms in the three different areas is summarized in Table 2). The association of different respiratory symptoms with TEP, age, and smoking are presented in Figure 2). Multivariate analysis showed an adjusted odds ratio of chronic bronchitis 1.42 (95% CI: 0.58–3.48), bronchial asthma 2.17 (95% CI: 0.35–13.41), breathlessness 1.71 (95% CI: 0.94–3.11), and cough 0.97 (95% CI: 0.47–2.03), respectively, for those with a TEP greater than 100.The prevalence of respiratory morbidity increased with higher TEP and the highest risk was observed for breathlessness  and the CPCB standard are 20 μg/m3 and 60 μg/m3, respectively), the average SO2 ranged from 2 to 5 μg/m3 , and average nitrogen oxides ranged from 9.6 to 29.9 μg/m3 .3, SO2 ranged from 8.5 to 16.5 μg/m3, and nitrogen oxides ranged from 7.8 to 25.2 μg/m3.2.5 and other important air pollutants known to have adverse health effects such as black carbon, fine and ultrafine particles, and volatile organic compounds have never been monitored. Based on satellite data, the average annual concentration of PM2.5 in Bhopal city during 1998 to 2014 was 49.9 ± 6.7 μg/m3, above the prescribed upper limits of the WHO (10 μg/m3) and CPCB (40 μg/m3) standards .16–18In India, real-time ambient air pollution data are available for a few cities under the National Ambient Monitoring Program, operated by the Central Pollution Control Board . Prior to 2018, air pollution in Bhopal was monitored by manual stations operated by the Madhya Pradesh Pollution Control Board. From 2011 to 2016, the average concentrations of respirable suspended PM (PM of aerodynamic diameter <10 μm) in Bhopal City ranged from 101.0 to 354.6 μg/m1 decline have been observed in adult females chronically exposed to higher levels of PMs from automobile exhaust.23The risk of cardiopulmonary mortality is higher for those residing near major roads and experiencing long-term exposure to PMs from TRAP.Ramesh et al. evaluated 121 shopkeepers in India working for more than a year in shops located within 100 m radius of the national highway of Bangalore and observed a prevalence of cough, breathlessness, and asthma of 32.2%, 31.4%, and 12.4%, respectively.The INSEARCH questionnaire used in the present study was developed to assess the overall burden of chronic respiratory diseases in India. The original INSEARCH study reported the prevalence of asthma and chronic bronchitis in adults as 2.05% and 3.49% respectively.10In the present study, the prevalence of all respiratory symptoms, including bronchial asthma and chronic bronchitis in shopkeepers was higher compared to the INSEARCH study.The present study demonstrated that shopkeepers working in congested and heavily trafficked roadside shops suffer from respiratory morbidity and the risks are higher for those with longer TRAP exposure. In addition, it showed that TEP can be a valuable indicator to assess the cumulative effects of TRAP exposure. There is a need to increase the awareness of individuals working or living near heavily trafficked roads of the adverse health effects of TRAP exposure and to promote measures such as glass doors on road-facing fronts to reduce direct exposure. Finally, measures to reduce traffic congestion, widen urban roads, promote the use of cleaner fuels, and prohibition of polluting vehicles can help to reduce ambient TRAP.


Streptococcus (GBS) remains a leading cause of illness and death among infants globally. We conducted prospective and retrospective laboratory-based surveillance of GBS-positive cultures from infants <3 months of age in 18 hospitals across China during January 1, 2015–December 31, 2017. The overall incidence of GBS was 0.31 (95% CI 0.27–0.36) cases/1,000 live births; incidence was 0–0.76 cases/1,000 live births across participating hospitals. The case-fatality rate was 2.3%. We estimated 13,604 cases of GBS and 1,142 GBS–associated deaths in infants <90 days of age annually in China. GBS isolates were most commonly serotype III (61.5%) and clonal complex 17 (40.6%). Enhanced active surveillance and implementation of preventive strategies, such as maternal GBS vaccination, warrants further investigation in China to help prevent these infections.Invasive group B  Streptococcus (GBS), the gram-positive Streptococcus agalactiae bacterium, is a leading cause of illness and death among infants, including those in high-income countries. After a series of systematic reviews and a meta-analysis, a compartmental-model simulation estimated ≈319,000 GBS cases, including ≈90,000 deaths, worldwide in 2015 (<3 months of age in 2015 was estimated to be 0.49 (95% CI 0.43–0.56) cases/1,000 live births; the case-fatality rate was estimated at 8.4% (95% CI 6.6%–10.2%) .In 2015, China had 12.4% (17.8 million) of the 143 million global births. Systematic reviews noted the paucity of data on GBS from Asia, including China, as a major data gap  for cases occurring within 0–6 days of birth and late-onset disease (LOD) for cases occurring within 7–90 days of birth  on June 13, 2016, under registration no. NCT02812576.The Medical Ethics Committee of Guangzhou Women and Children’s Medical Center, Guangzhou, China, served as the central institutional review board for all facilities and approved this study . Each participating hospital had the option of using this approval or obtaining approval at their institution. For the prospective component of the study, we obtained written informed consent from parents or guardians of infants with invasive GBS disease. For the retrospective study, the review board waived the need for informed consent. We registered this study in the US National Library of Medicine clinical trials database (https://www.biomerieux.com) or BACTEC  culture bottles and analyzed with VITEK 2 COMPACT (bioMérieux) or BD Phoenix 100 (Becton Dickinson). GBS strains were grown at 37°C in 5%–10% CO2 in trypticase soy agar supplemented with 5% sheep’s blood for 18–24 h, according to the manufacturer’s instructions. All GBS isolates were stored at –70°C and shipped on dry ice in standardized skim milk–tryptone-glucose-glycerol storage medium to the laboratory of Guangzhou Women and Children’s Medical Center, which is certified by Joint Commission International (https://www.jointcommission.org), for further analysis.Each local hospital laboratory performed GBS isolation, cultivation, and identification by using the following protocol. Sterile samples were inoculated in French  rapid latex agglutination test kit to cross-check the PCR results  according to the allelic profile of the 7 amplicons. We compared alleles and STs of all GBS isolates with those in the S. agalactiae MLST database . We assigned GBS isolates to the same group when they shared identical alleles at 6 of the 7 loci with >1 other member of the group. We also assigned isolates to different clonal complexes (CCs).We used multiplex PCR for Lancefield serotyping on all isolates  and used BioNumerics 5.1  to create minimum spanning trees.We calculated 95% CI by using the Wilson interval method and analyzed categorical data by using χ<24 hours after birth  EOD and 158 (52.0%) LOD; 123 (64 EOD and 59 LOD) were identified during the retrospective study and 181 (82 EOD and 99 LOD) during the prospective study. Of the 146 EOD cases, 82 (56.2%) were identified er birth  Table 1.er birth  Table 3.S. agalactiae was 100% susceptible to cephalosporins, ampicillin, vancomycin, meropenem, and linezolid. We observed high prevalence of reduced susceptibility to tetracycline (80.1%), erythromycin (78.3%), and clindamycin (68.2%).Most (83%) case-patients, received an initial combination therapy of 2 antimicrobial drugs, including 54.2% who received empiric treatment with third-generation cephalosporins. Of 304 GBS cases, 199 occurred in infants born at sentinel hospitals, 115 EOD and 84 LOD cases. Sentinel hospitals reported 634,531 live births during the study period, 388,005 during the prospective period and 246,526 during the retrospective period . The calGuangdong Women and Children’s Hospital in the south reported the highest incidence rate, 0.76 (95% CI 0.54–1.08) cases/1,000 live births, along with Hubei Maternal and Child Health Hospital in the central region , and Guangzhou Women and Children’s Medical Center in the south  , panel A<3 months of age in China would be 13,604 among the country’s birth cohort of 17.9 million. Using the global case-fatality rate of 8.4%, we estimate China would have 1,142 GBS-associated deaths annually  was most pervasive, along with Ib (28.7%), and we saw fewer instances of Ia (5.7%) and V (2.9%), and 1 case (0.4%) each of serotypes IV and VI, as well as 1 nontypeable isolate , panel AWe performed MLST on all 244 serotyped isolates and identified 27 different sequence types (STs); 83.2% of which were ST17, ST19, ST10, ST12, and ST23. ST17 was most prevalent , along with ST19 , ST10 , and ST12 . Of ST17 isolates, 98% were serotype III, as were 88.2% of ST19 isolates; 2.2% of ST17 and 2.9% of ST19 were serotype Ib; and 8.8% of ST19 isolates were serotype V. Most ST10 (93.4%) and ST12 (93.8%) isolates were serotype Ib and 66.7% of ST23 isolates were serotype Ia.Twenty-seven STs clustered into 10 CCs . The mos<3 months of age in China. We reported an overall GBS incidence rate of 0.31 (95% CI 0.27–0.36) cases/1,000 live births, which is lower than the estimated worldwide incidence rate of 0.49 (95% CI 0.43–0.56) cases/1,000 live births, but similar to estimates from elsewhere in Asia   than observed globally in a meta-analysis , which could reflect early detection of suspected GBS and timely management with antimicrobial drugs. Also, we found that only 5.6% of surviving infants across all participating hospitals had neurologic sequelae at discharge. However, this value could be an underestimate because we did not use a standard assessment across sites and sequelae might not be obvious in infants. Further investigation in longer term follow-up studies in China are warranted to quantify the residual burden of neurologic sequelae in infants with invasive GBS disease.Given that maternal GBS colonization is the primary risk factor for EOD, the US Centers for Disease Control and Prevention issued guidelines recommending intravenous intrapartum antibiotic prophylaxis (IAP) during labor to prevent EOD in infants born to women colonized with GBS or with risk factors (Vaccination of pregnant women against GBS is a promising strategy to prevent invasive GBS disease in their infants (<24 hours after birth. Findings might differ in primary and secondary settings because of differences in populations and clinical practice, so these settings should be included in future studies. Second, the retrospective collection of data during January 2015–May 2016 did not include all isolates and only 46.3% were available for strain analysis. Last, rates of GBS could be underestimated because our study did not account for GBS in infants treated at nonstudy hospitals after discharge from study hospitals.Our study has several limitations. First, all participating hospitals were large urban tertiary hospitals. We did not include secondary hospitals and primary care institutions mainly because they lacked adequate laboratory facilities for GBS culture and identification of invasive GBS disease, which might have led to delayed confirmation of EOD cases. We found 56.2% of EOD cases were identified In summary, our large generalizable study on invasive GBS disease in infants in China informs the current clinical and molecular epidemiology of GBS. Despite the low rates of fatality and detectable neurologic sequelae at discharge, clinicians, laboratory technicians, and epidemiologists should be aware that GBS is prevalent, especially in the south and central regions of China. Future work and continuous surveillance are warranted to better understand the heterogeneity of incidence across regions, identify risk factors, and conduct long-term follow-up to further assess the prevalence of invasive GBS disease on neurologic development in surviving infants.Streptococcus infections in infants <3 months of age, China.Additional information on invasive group B


Piaractus mesopotamicus) is a Neotropical fish with remarkable productive performance for aquaculture. Knowledge of genetic resources in Neotropical fish is essential for their applications in breeding programs. The aim of this study was to characterize the genetic diversity of seven farmed populations of pacu which will constitute the basis for a broodstock foundation for coming breeding programs in Brazil. Analysis of one wild population (Paraná River) was used as a reference to compare genetic parameters in the farmed populations. The analyses were performed using 32 single-nucleotide polymorphisms (SNP) and 8 simple sequence repeat (SSR) markers. No significant differences in genetic diversity between populations estimated through the number of alleles and allelic richness, observed heterozygosity, expected heterozygosity, and minimum allele frequency were detected (p > 0.05). Low genetic diversity was observed in all farmed stocks and the wild population. Moreover, we detected low genetic structure when comparing farmed and wild populations for SNPs  and SSRs . Analysis of molecular variance (AMOVA) demonstrated that genetic variation was mostly within populations. Kinship analysis showed that most fish farms included related individuals at a proportion of at least 25%. Our results suggest that the basal broodstock for pacu breeding programs should be founded with individuals from different fish farms for higher genetic diversity and to avoid inbreeding risks.The pacu ( Piaractus mesopotamicus) is a Characiform fish with a wide natural distribution throughout La Plata basin which covers an area over five South American countries: Brazil, Uruguay, Bolivia, Paraguay, and Argentina. Wild populations of pacu are threatened by overfishing since this species is considered to have high commercial value, with large-scale catches occurring by industrial and recreational fisheries [The pacu  and 8 SSRs to assess the genetic diversity, structure, and kinship as a support to designing a suitable pre-breeding program for this species.In this study, seven farmed stocks and one wild population of This study was conducted in strict accordance with the recommendations of the National Council for Control of Animal Experimentation (CONCEA)  and was approved by the Animal Use Ethics Committee (CEUA), protocol no. 22.255/15. The present study was performed under authorization no. 33435-1 issued through the Chico Mendes Institute for the Conservation of Biodiversity, Brazilian Ministry for the Environment (ICMBio). Fin clips were collected from each fish under benzocaine anesthesia and all efforts were made to minimize suffering. Fin samples were stored in 95% ethanol at −20 °C.Individuals of seven different broodstocks from fish farms in Brazil (São Paulo State) were collected (FF1–FF7) . Markers were genotyped in a 3730XL DNA analyzer (Applied Biosystems) in Macrogen (Korea). Allele scoring was performed with the Peak Scanner program (Applied Biosystems) using the GeneScan 500 LIZ Size Standard.SSR genotyping was performed using eight microsatellite markers  in two Multiplex PCR reactions according to Posner  and prevSNP genotyping was carried out using 32 markers obtained from the liver transcriptome . Analysinull) and allelic dropout in microsatellite loci were tested using MICRO-CHECKER 2.2.3 [a), number of private alleles (Np), and observed (Hobs) and expected (Hexp) heterozygosity were estimated using CERVUS 3.0 [p < 0.05) were performed using GENEPOP 4.0.11 [r) and inbreeding coefficient (FIS) were estimated using FSTAT 2.9.3.2 software [The presence of null alleles  was estimated by the linkage disequilibrium method in NeESTIMATOR V2.01 [The effective population size   using STThe partitioning of variation at different hierarchical levels was calculated by analysis of molecular variance (AMOVA) in ARLEQUIN version 3.5.2.2  using 10xy) [xy < 0.125) corresponding to unrelated individuals; intermediate values 0.125 ≤ rxy ≤ 0.375 were considered half-siblings; and rxy ˃ 0.375 were considered full siblings [Kinship coefficients (rxy)  and potexy) . Parentasiblings .p values of HWE and FIS values are shown in The parameters of genetic variability for pacu populations determined by 8 SSR and 32 SNP markers are shown in For SSR loci, a total of 44 alleles were detected in the analyzed populations. The average number of alleles per locus was 3.84 ± 1.16, with numbers ranging between 2 and 7. Allelic richness ranged from 2.000 to 6.140, with average population values ranging between 3.331 ± 0.868 (FF1) and 3.975 ± 1.189 (FF7). Private alleles were detected in individuals belonging to FF2, FF4, FF7, and WILD.Monomorphic SNPs were found more frequently in FF2 (six monomorphic SNPs) and FF4 (four monomorphic SNPs). By contrast, FF1, WILD, FF6, and FF7 showed only polymorphic SNPs and the highest MAF average values . On the other hand, FF3 (0.269 ± 0.138) was the fish farms with the lowest MAF average values.obs was 0.502 ± 0.219 for SSRs and 0.391 ± 0.173 for SNPs. When the average Hexp was estimated, 0.525 ± 0.170 was found for SSRs and 0.379 ± 0.133 for SNPs. The genetic diversity values observed in the fish farms  were not significantly different from the WILD values , which showed Ar = 3.420 ± 1.0005, Hobs = 0.441 ± 0.213, and Hexp = 0.434 ± 0.214 for SSRs and MAF = 0.275 ± 0.132, Hobs = 0.385 ± 0.171, and Hexp = 0.370 ± 0.130 for SNPs (The average Hfor SNPs .p = 0.001), even when populations were subjected to global tests for all loci. By contrast, some SSR loci departed from HWE after Bonferroni correction (p = 0.006), such as locus Pm4  and locus Pm5 (FF4). Null alleles were detected in the locus Pm4  and locus Pm11 (FF2) (p < 0.05) when applying global tests for SSRs. However, only FF4 presented significant Hardy–Weinberg disequilibrium after Bonferroni correction (p < 0.006).All SNP loci were in accordance with HWE after Bonferroni correction (11 (FF2) . NeitherFIS values varied similarly between SSR (−0.113 and 0.212) and SNP loci (−0.082 to 0.056). However, no significant deviations from zero of FIS values were found in the fish farms when Bonferroni correction was applied for SNPs (p = 0.001) and SSRs (p = 0.006). Additionally, FIS values were not considered significantly different from zero over all loci for both markers (p > 0.05), presenting low 95% confidence interval values for SSRs (−0.022 to 0.135) and SNPs (−0.075 to 0.011) when bootstrapping over loci analyses were performed.Overall population p < 0.001). For SSR markers, LD was detected in FF4 (5 pairs), FF2 (2), FF1 (1), FF5 (1), and WILD (1) (p < 0.01). However, in both markers, the observed LD was not homogeneous among populations or loci , FF7 (5), WILD (4), FF5 (2), FF3 (1), and FF4 (1)  and in WILD for SNPs (0.02) and the highest values in FF4 for SSRs (0.07) and in FF1 for SNPs (0.22). Evidence of recent reductions in population size was found in all fish farms (M-ratio < 0.68) by considering SSR analysis . N8 in FF6 . The ratanalysis  characteAfter the detection of reduced effective population sizes, kinship evaluation showed that most of the fish farms had related individuals  in a proportion of at least 25%, as estimated from both marker sets , with siFST values suggested low genetic differentiation among populations when using SSRs  or SNPs . Pairwise FST values were also calculated for fish farms and significant differentiation was found between most pairs when considering SNP and SSR sets   and ranged between −0.002 and 0.204. The highest significant FST values (p < 0.006) were observed between WILD and FF4 (FST = 0.204) and FF2 and FF4 (FST = 0.143). Conversely, the lowest genetic differentiation was detected between FF3 and FF6 . Regarding SNPs, the highest genetic differentiation (p < 0.001) was between FF2 and FF4 (FST = 0.146). Conversely, WILD and FF7 (FST = 0.032) registered the lowest genetic differentiation (p < 0.001).Global ctively) . Low to FST analyses.To evaluate the level of admixture among samples, Bayesian model-based clustering analyses were performed based on the ΔK distribution by examining SSR and SNP loci. The selection of the estimated number of clusters in the dataset was based on the number of analyzed fish farms (K = 1 to 8). According to the analysis based on the Evanno method , the hypp < 0.001) and 91.1% by SSRs (p < 0.0001). The estimated variation between groups (FCT) was only 2.2% (p = 0.01) for SNPs and 3.9% (p = 0.02) for SSRs. In addition, the variation among populations and within groups (FSC) presented 5.3% of the genetic variation (p < 0.001) for SNPs and 5.0% (p < 0.0001) for SSRs.Despite the genetic structure among populations being confirmed by STRUCTURE analysis, AMOVA analysis based on groups supported by the results of STRUCTURE showed that the higher percentage of genetic variation was assigned to differentiation within populations: 92.4% by SNPs . Therefore, our hypothesis is that the WILD population may be threatened due to the high level of exploitation imposed by commercial and/or recreational fishing, since the region where the WILD population was collected is a famous fishing spot in Brazil [Considering SSR loci, the analysis of genetic diversity parameters estimated on pacu broodstocks showed low values for the number of alleles (ranging around 4) and heterozygosity (ranging around 0.500). It was expected that cultivated stocks would present low genetic diversity values, as herein observed; these low values characterize populations with genetic drift events due to low effective population sizes and, consequently, recent bottleneck effects/founder events, similar to other studies in a related Neotropical species, chypomus . Howeverchypomus  that repn Brazil . Moreoven Brazil ,42 whichobs and Hexp) and MAF indicated low genetic variability in the farmed populations studied. This hypothesis was based on similar values found in WILD and sampled fish farms, considering that wild populations of pacu have no expressive heterozygosity values when using microsatellite loci. In addition, the diversity values herein observed were similar to those in other fish studies involving SNP analyses [Although there is no comparative research of the genetic diversity of pacu populations using SNP markers, the values of heterozygosity  must receive special attention, since our results will serve to find a base population of pacu for upcoming breeding programs.In general, farmed stocks have the tendency to show reduced genetic variability over generations due to artificial selection and a reduced number of breeders in the initial base population ,46,47,48Kinship analysis has been an essential tool in genetic pre-breeding programs of fish to reduce inbreeding rates by directing the mating of unrelated individuals ,50,51,52Impacts related to insufficient individuals used in hatchery productions and their interference with the genetic diversity of cultivated populations have been studied for important species cultivated worldwide, such as Atlantic salmon . SimilarFST estimates indicating genetic differentiation (driven by genetic drift and isolation), as detected in FF4, similar to in previous studies carried out in other related species such as P. brachypomus [FST values suggested significant genetic differentiation of 0.067 by SNPs and 0.080 by SSRs , indicating low differentiation between the farmed stocks. This may be explained using two hypotheses: (i) stock foundation based on sharing of breeders among fish farms, resulting in genetic similarities between broodstocks, and/or (ii) stock foundation in the fish farms based on the capture of wild breeders, which are characterized as belonging to a panmictic unit due to the lack of genetic structure in natural populations [In the present study, our initial hypothesis was based on the fact that pacu farmed stocks did not have gene flow because the broodstocks are geographically isolated and producers frequently do not perform exchange of breeders among the fish farms. Therefore, we would expect higher values of chypomus . Howeverulations ,17, partThe STRUCTURE analysis revealed differences between SNPs and SSRs, showing three and two clusters for the pacu stocks, respectively. In this study, the dataset of SNPs originated from the pacu liver transcriptome and these SNPs were annotated mostly in genes related to productive traits, including SNPs classified as non-synonymous mutations . TherefoThe results of this study are aimed to provide initial knowledge about the genetic profile of pacu stocks in different fish farms, considering the importance of pacu to South America aquaculture and the necessity to offer subsidies for the development of its production. The results provide information relevant to one of the most important cultivated Neotropical species. The genetic variability and differentiation of stocks and fish farm profiles considering the risks of inbreeding and the necessity of directed matings of the stocks should be known in order to take appropriate actions for the creation of the base population. In conclusion, the SNP and SSR sets showed their applicability in a pre-breeding program, particularly in delineating the formation of the best families in terms of genetic variability and genetic structure.


Shigella species are a major cause of dysentery and may attribute for death worldwide. Currently antibiotic resistance became the critical challenges for management of infectious disease. The aim was to conduct a systematic review and meta-analysis of Shigella species and its drug resistance pattern in Ethiopia.2 test statistic were used to test heterogeneity across studies. The Pooled estimate of Shigella species and its drug resistance pattern was computed by a random effects model.A comprehensive literature search was conducted through internet searches using database of MEDLINE, PubMed, Google scholar, EMBASE, HINARI, Cochrane Library and reference lists of previous prevalence studies from January 1999 to November 2018. Results were presented in forest plot, tables and figures with 95% CI. The Cochrane Q test and IShigella species in Ethiopia was 6.6% (95% CI 4.7–8.8). In the subgroup analysis, the highest prevalence was observed among patients in Health facility  whereas the lowest prevalence was observed in Community based studies . In addition, Shigella species were highly resistant to ampicillin, amoxicillin, erythromycin and multi-drug resistant (MDR) with the pooled resistance proportions of 83.1% (95% CI 75.7–88.6), 84.1% (95% CI 75.6–90.1), 86.5% (95% CI 70.9–94.4) and 83.2% (95% CI 77.1–87.9), respectively. On the other hand, comparably low resistance pattern was reported for ciprofloxacin 8.9% (95% CI 6.0–12.8), ceftriaxone 9.3% (95% CI 3.9–20.5), and norfloxacin 8.2% (95% CI 3.8–16.6) and gentamycin 17.3% (95% CI 11.2–25.9). Subgroup analyses indicated that study years were associated with a decreasing Shigella prevalence over time (p = 0.002).The pooled prevalence of Shigella infection and its high proportion of drug resistance pattern to ampicillin, amoxicillin and erythromycin in Ethiopia. Therefore, initiating and scale up of performing drug susceptibility test for each shigellosis case, educate the community and health care providers on appropriate use of antibiotics need to be considered and strengthened.The pooled estimate showed high burden of The online version of this article (10.1186/s12941-019-0321-1) contains supplementary material, which is available to authorized users. Shigella species are a major cause of dysentery disease and may attribute for death worldwide. Shigellosis is a major public health threat in developing countries like Ethiopia, people living with poor sanitation and overcrowded condition. Annual number of Shigellosis episodes globally estimated to be about 165 million, more than half (100 million) of episodes occurs in low and middle income countries, and attributed for more than 1 million deaths per year. Shigellosis is more prevalent among under five children, and causes dysentery and under five mortality [ortality .Shigella species is one of the eight dangerous drug resistance bacteria. Worldwide, there are 700,000 deaths as a result of antimicrobial resistance (AMR) every year. The experts suggest that this figure will rise to 4.2 million in Africa and 10 million globally by 2050, if nothing is done [Antibiotic resistance becomes a critical public health problem around the globe in recent years. Based on 2016 WHO report,  is done –5.Shigella significantly vary from area to area of the world in relation with the practice of widespread use of antimicrobial agents [The treatment of Shigellosis has currently become more challenging due to the emergence of drug resistant species and associated with a variety of biological, pharmacological and societal variables with the worst combinations in low and middle income countries –8. Multil agents , 9.Shigella species [Antibiotic resistance is a natural phenomenon that occurs whenever antibiotics are in use. However, there are human behaviors that contribute to the rapid development and spread of bacterial antibiotic resistance. Availability and use of broad spectrum antibiotic without prescriptions facilitate the development of resistance by  species .Shigella species. However, there is no summarized prevalence data of this bacterial infection and its drug Susceptibility pattern at country level to help in the formulation of appropriate intervention methods. Therefore, the present study is the first of its kind and aimed to determine the pooled prevalence pooled prevalence and drug susceptibility pattern of Shigella species in Ethiopia.Different studies have been carried out in different parts of Ethiopia at different times to document the epidemiology of and drug Susceptibility pattern of shigella species and its antimicrobial resistance pattern among Ethiopia population. Potentially relevant studies were identified through a literature search of Medline, PubMed, Google scholar, HINARI and Cochrane Library. The search was based on the combination of the following special index search terms  and Boolean operations: “Shigella” AND “Prevalence “OR “Epidemiology” AND “Drug Resistance, Microbial” AND “Dysentery, Bacillary/epidemiology” AND “Ethiopia” “title and abstract” from January 1st 1999 to November 20th 2018. Articles search were focused on published studies with epidemiological and/or clinical data. All records were managed in Endnote version X7 . The search was carried out from April 5th, 2016 to November 20, 2018. The limit of language was English and the limit of study group was human.A comprehensive literature search was conducted on the prevalence of We reviewed abstracts from initial search using defined inclusion and exclusion criteria.Shigella species and its drug resistance pattern 4, literatures published in the English language and 5. Published articles were considered.Studies were selected for systematic review and meta-analysis, 1, if they were conducted in Ethiopia 2, study design been cross-sectional 3, studies reported the prevalence of Studies, which were not fully accessed after reading the titles and abstracts were excluded since we are unable to assess the quality of each article in the absence of their full texts.Shigella species, medical treatment type, and resistance pattern of Shigella species. Disagreement on data extractions between researchers were resolved through discussion and consensus.The data extraction was done by three researchers  independently from included studies using a standardized and pretested format prepared in Microsoft Excel. The data abstraction format included first author, study design, region in the country (study site in the country), publication year, sample size, population characteristics, age group of study participants, prevalence of The quality was assessed using 9 point Joanna Briggs Institute (JBI) critical appraisal tools, the following criteria is established: sample frame appropriate to address the target population, study participants sampled in an appropriate way, adequate sample size, study participants sampled in an appropriate way, study subjects and the setting described in detail, data analysis conducted with sufficient coverage of the identified sample, valid methods used for the identification of the condition, the condition measured in a standard and reliable way for all participants, appropriate statistical analysis and adequate response rate. Individual studies were assigned a score that was computed using different parameters in line with the review objectives. The responses were scored 0 for “No and not reported” and 1 for “Yes”. Total scores ranged between 0 and 9. Studies with medium  and high quality were included for analysis   was presented.The data entry and analysis were done using Comprehensive Meta-Analysis (version 3.1) software. The original articles were described using forest plot, figures and tables. Since there was heterogeneity among studies, random effect model was used to compute the pooled prevalence and antimicrobial susceptibility of Sub-group analysis was performed based on Region; , age group; , study population  and year of study; .2 statistic. The I2 provides an estimate of the percentage of the variability in effect estimates that is due to heterogeneity rather than sampling error or chance differences. So, the existence of heterogeneity was verified using Cochran’s Q test  and I2 test that measures level of statistical heterogeneity between studies. I2  [Statistical heterogeneity was evaluated by Cochran’s Q test and Ictively) , 13. Begctively) . As the Shigella species and the remaining five were excluded due to unavailability of full text articles. Finally, 29 studies were found to be eligible and included in the meta–analysis , which is indicative to use random effects model. The distribution of the studies using funnel plot . Of this, 7571 study participants were included in the meta-analysis of Shigella species drug resistant pattern. A total of twenty-nine studies were considered eligible for quantitative syntheses. Twenty-five studies were used to estimate the proportions of drug resistant for Shigella species  from Oromia region, 1545 (18.13%) from southern region, 425 (4.99%) from Central Ethiopia (Addis Ababa), 244 (2.86%) from Harare region and 717 (8.41%) were from Tigray region. Cross sectional study design was used in all studies. The study population varied from 24 to 1200, and were conducted between the years 1999–2018 [Shigella species . The total study populations involved for the estimation of 999–2018  (Table 1Shigella species in Ethiopia was 6.6% (95% CI 4.7–8.8)  in Amhara, 4.1% (95% CI 1.0–14.5) in Oromia, 7.7% (95% CI 2.7–20.1) in Southern Ethiopia, 2.2% (95% CI 0.1–40.5) in Addis Ababa, 7.0% (95% CI 4.4–11.0) in Hareri and 8.8% (95% CI 5.6–13.6) in Tigray. In year of study 26.9% (95% CI 14.8–43.8) in 1999–2003, 12.6% (95% CI 6.6–22.7) in 2004–2008, 8.1% (95% CI 5.1–12.8) in 2009–2013 and 4.9% (95% CI 3.3–7.0) from 2014 to 2018 .The analysis of 29 studies, according to the Der Simonian–Laird random-effects model, revealed that the pooled prevalence of .8) Fig. . Pooled Shigella species were 83.1% (95% CI 75.7–88.6) for ampicillin, 84.1% (95% CI 75.6–90.1) for Amoxicillin and 86.5% (95% CI 70.9–94.4) for erythromycin. Comparatively low resistance pattern was reported in ciprofloxacin 8.9% (95% CI 6.0–12.8), ceftriaxone 9.3% (95% CI 3.9–20.5), gentamycin 17.3% (95% CI 11.2–25.9), and norfloxacin 8.2% (95% CI 3.8–16.6). However intermediate resistance were recorded for augmentin (Amox + clav) 59.1% (95% CI 19.6–89.5) and co-trimoxazele 59.4% (95% CI 49.3–68.8). Moreover, resistant to more than one drugs or MDR were reported 83.2% (95% CI 77.1–87.9)  is widely growing public health threat worldwide, particularly in resource limited countries including Ethiopia, where infectious diseases are widespread. It is now occurring across the world .Shigella species is highly antibiotic resistant among eight drugs resistance bacteria. Regarding antibiotic resistance, if nothing is done, a person will die every three second by 2050 [ by 2050 .shigella species in Ethiopia using 29 studies. According to the results of this meta-analysis, the pooled prevalence was 6.6% (95% CI 4.7–8.8). This finding is in agreement with 6.6% (95% CI 3.4–9.7) Shegilla prevalence in systematic review among US military and similar populations [This meta-analysis determined the pooled prevalence of ulations .Shigella species among regions was also calculated, hence a higher prevalence of Shigella species (8.8%) was reported in Tigray, which was nearly four times higher than the finding from Central Ethiopia (2.2%), even though the studies conducted and included in this review and meta-analysis from this region were small. The variations in prevalence estimates may be due to differences in the study populations and year of study.Regional prevalence of Shigella species by study year (26.9%) was reported in 1999.2003 and (4.9%) in 2014–2018. This finding is inconsistent with the study done in China [Shigella prevalence over time. The decreased in prevalence through time might be due to decrease in poverty, increase quality of life, rise of awareness on sanitation and hygiene, prevention and control strategy of communicable disease through deployed of health extension workers at community level across the country.Pooled prevalence of in China . This reShigella cause diarrhoeal morbidity among infants and young children than adults [Shigella in children compared to adults suggests a higher vulnerability of children to Shigella infection, this may be due to the unhygienic food handling practices, compromised sanitation, malnutrition and the ability to cause disease with a small inoculum and facilitating spread from person to person [In this study, the pooled prevalence of shigellosis in children was (7.0%), while in adult population (2.2%). This finding confirms that n adults  and the n adults . The higo person .Shigella species showed high resistance to amoxicillin, ampicillin, and erythromycin. This finding is in line with the study done in Germany [Based on the data obtained from twenty-five published articles;  Germany .In Ethiopia the drug of choice on shigellosis treatment is norfloxacin, ciprofloxacin, and ceftriaxone for adult. This is similar with guideline USA . HoweverShigella isolates resistant to two or more drugs were high (83.2%). This increment may be due mobile genetic units  [Furthermore, the occurrences of sposons) , inadequsposons) –54.Shigella identification. This estimate appears to be less sensitive method than molecular methods and may underestimate the actual occurrence of this bacteria [In this review, included studies primarily used stool culture for bacteria .Shigella infection and its high proportion of drug resistance pattern to ampicillin, Amoxicillin and Erythromycin in Ethiopia. Therefore, initiating and scale up of performing drug susceptibility test for each shigellosis case, educate the community and health care providers on appropriate use of antibiotics need to be considered and strengthened.The pooled estimate provides high burden of Shigella infection were not tested at species level.The patient data were collected from urban and rural populations of the country that took service from hospitals but urban dwellers have better access to health care setting. Therefore, the pooled estimates are more applicable to patients in urban areas than rural. Resistance pattern of shigellosis was not reported for money drugs by some studies and in most studies Additional file 1: Table S1. Study design and quality assessment of the studies included in systematic review and meta-analysis of shigella species.Table S2. Subgroup meta-analysis of Shigella species prevalence estimation in Ethiopia from 1999 to 2018. Table S3: Microbiological methods used to isolate and identify Shigella species in Ethiopia from 1999 to 2018. Table S4. Prevalence of drug resistance (95% CI) for Shigella from 1999 to 2018.Additional file 2: Figure S1. Egger regression intercept of analysis of included studies reporting on the prevalence of Shigella species in Ethiopia. Figure S2. Begg and Mazumdar rank correlation of analysis of included studies reporting on the prevalence of Shigella species in Ethiopia.


This article has been corrected: Due to errors in image selection, Figure 5A shows an inadvertent duplicate of Donor 2 for M81. Figure 7D shows the Actin control from Donor 2 that was inadvertently duplicated from the Donor 2 Actin control shown in Figure 4 (which is the same extract and donor sample). Figure 4 is correct and unchanged. The corrected Figure 5 and Figure 7 are shown below. The authors declare that these corrections do not change the results or conclusions of this paper.7248-7264. https://doi.org/10.18632/oncotarget.14540Original article: Oncotarget. 2017; 8:7248–7264.


Streptococcus mutans biofilms were treated with chitosan dispersion (CD) or chitosan microparticles (CM). (2) Methods: CD at 0.25% and 1% were characterized by texture analysis, while CD at 2% was spray-dried to form CM, which were characterized with respect to particle size distribution, zeta potential, and morphology. After determining the minimum inhibitory and bactericidal concentrations, S. mutans biofilms were grown on glass slides exposed 8×/day to 10% sucrose and 2×/day to CD or CM at 0.25% and 1%. Biofilm viability and acidogenicity were determined, using appropriate control groups for each experiment. (3) Results: CD had high viscosity and CM were spherical, with narrow size distribution and positive zeta potential. CM affected bacterial viability and acidogenicity in mature S. mutans biofilms more strongly than CD, especially at 1%. (4) Conclusions: Both chitosan forms exerted antimicrobial effect against mature S. mutans biofilms. CM at 1% can reduce bacterial viability and acidogenicity more effectively than CD at 1%, and thereby be more effective to control the growth of mature biofilms in vitro.(1) Background: The effectiveness of chitosan to improve the action of antimicrobial compounds against planktonic bacteria and young biofilms has been widely investigated in Dentistry, where the biofilm lifecycle is a determining factor for the success of antibacterial treatment. In the present study, mature  The use of natural antimicrobial products for the control and prevention of several oral diseases has been extensively studied . ChitosaSeveral approaches have been reported to improve the antimicrobial activity of this natural polysaccharide, including formation of micro- and nanoparticles . RecentlStreptococcus mutans figures as the most common microorganism  as vehicle control for microparticles; d) 0.25% and 1% chitosan dispersion (CD); and e) 0.25% and 1% chitosan microparticles (CM), using 48% EtOH as solvent .Treatments. From the third to the fifth day, biofilms were exposed to one of the following treatments, 2×/day for 1 min (n = 4): a) 0.12% chlorhexidine digluconate solution (CHX), as positive control; b) saline solution (NaCl), as negative control; c) 48% ethanol (EtOH) [96% ethanol  as an indicator of biofilm acidogenicity . 2, the number of colonies grown were counted  and the results were expressed in CFU/cm2 glass slide.Bacterial viability. On the sixth day, glass slides containing biofilms were washed three times in 0.9% NaCl to remove loosely adherent cells and were individually transferred to microcentrifuge tubes containing 1 mL of 0.9% NaCl. Next, biofilms were sonicated  for 15 s pulses at 20% amplitude to improve homogenization . BiofilmThe statistical analysis was carried out using the SAS software . The assumption of equality of variances and normal distribution of errors was checked, and data were analyzed by one-way ANOVA followed by the Tukey post-hoc test  or the Tukey–Kramer post-hoc test (biofilm acidogenicity and viability), with the significance level fixed at 5%.


In brief, when compared to previously published protocols, this article presented an improved protocol that requires shorter experimental time and less laborious approach for higher yield of intact single myofibers for downstream analyses.Satellite cells are myogenic cells responsible for muscle growth shortly after birth and muscle repair/regeneration during adulthood. Therapies based on satellite cells hold promise for treating muscular dysfunctions. Studying satellite cells is technically challenging owing to their low abundance, small size and anatomical dispersed location between the basal lamina and the sarcolemma of myofibers. In this article, we present three improved protocol strategies for studying the properties of satellite cells of the mouse during the different stages of muscle regeneration: (1) immunostaining of freshly isolated single myofibers to facilitate the study of quiescent satellite cells, (2) cultivation of single myofibers on Matrigel Skeletal muscle is a highly complex and heterogeneous tissue that plays essential roles in voluntary body movements, insulating internal organs, and producing heat to maintain core body temperature, as well as acting as a major energy storage depot. Human have approximately 639 to 640 skeletal muscle units, which together account for around 38% and 30% of total body mass in men and women, respectively .in situ [Skeletal muscle has a robust and effective regenerative capacity. Muscle function can be restored even after the tissue is removed, minced and replaced in situ . This rein situ . Activatin situ . Since se.g., fibroblasts, endothelial cells, interstitial cells, and blood vessel-associated cells), in order to obtain a pure population of satellite cells. These factors have limited the study of satellite cells. Therefore, an approach known as single myofiber isolation aims to isolate satellite cells while retaining them within their niches was established by Dr. Richard Bischoff and his colleagues in 1975 [Studying satellite cells can be technically challenging. Their scarcity, small size, dispersed location under the basal lamina of myofibers and their non-random distribution along the length of individual myofibers makes them difficult to be identified and manipulated ,4. Addit in 1975 . Their p in 1975 .in situ position as well as examining their progeny after migrating from the myofibers. For examples, the freshly isolated myofibers can be either immunostained immediately to characterize the quiescent satellite cells [in vitro [Methods for single myofibers isolation provide essential tools for studying the biological properties of satellite cells during the different stages of myogenesis. It allows studying satellite cells in their te cells  or cultuin vitro .Previously published protocols incorporate more complex and time-consuming procedures to isolate single myofibers. It is essential that the isolation duration to be performed as short as possible. The prolonged isolation time will induce the activation of satellite cells and eventually leading to the inaccuracy of the results . It is c®, St. Louis, MO, USA; cat. #C0130)Collagenase type 1 Matrigel®, Paisley, Scotland, UK; cat. #11995-073)Dulbecco’s Modified Eagle Medium with 4500 mg/l L-glucose, 4.0 mM L-glutamine, and 110 mg/l sodium pyruvate (DMEM) (GibcoStandard and non-heat inactivated horse serum (HS) ®, Paisley, Scotland, UK; cat. #10270-106)Standard and non-heat inactivated fetal bovine serum (FBS) ®, Paisley, Scotland, UK; cat. #15140-122)Penicillin-Streptomycin with 10000 units/ml penicillin and 10000 μg/ml streptomycin  ®, St. Louis, MO, USA; cat. #P6148)Crystalline paraformaldehyde (PFA) powder Triton X-100 reagent Tween-20 reagent Goat serum Mouse monoclonal anti-myogenic differentiation 1 (MyoD) Goat anti-mouse Alexa Fluoro 488 Ig G1 secondary antibody ® Gold Antifade reagent containing 4, 6-diamidino-2-phenylindole (DAPI) ProLongSuperFrost™ Plus microscope slides Liquid repellent PAP pen Dissolve 12 mg of collagenase type 1 powder in 6 ml of DMEM. Filter-sterilize the mixture using 0.22 μm syringe filter.CAUTION: It is recommended to use DMEM with sodium pyruvate throughout all of the procedure to ensure myofiber survival.Supplement 49.5 ml of DMEM with 500 μl of penicillin-streptomycin.Dissolve 10 g of chicken embryonic extract powder in 10 ml of 1× PBS solution. Filter-sterilize the mixture using 0.22 μm syringe filter.Supplement 44.5 ml of DMEM with 5 ml of HS (10% v/v) and 500 μl of penicillin-streptomycin.Supplement 34 ml of DMEM with 10 ml of FBS (20% v/v), 5 ml of HS (10% v/v), 500 μl of chicken embryo extract (1% v/v) and 500 μl of penicillin-streptomycin.® stock solution with 9 ml of ice-cold DMEM.Dilute 1 ml of MatrigelNOTES: The 4% PFA solution must be prepared in a fume hood.Dissolve 4 g of crystalline PFA powder with 100 ml of sterilized water and subsequently incubate the mixture in a 55°C water bath until the PFA powder is fully dissolved. Adjust the pH value to pH 7.4 with sodium hydroxide. Filter-sterilize the mixture using a 0.22 μm filtration unit.Supplement 49.75 ml of 1× PBS with 250 μl of Triton X-100 reagents.Supplement 9.9 ml of 1× PBS with 100 μl of goat serum.Supplement 49.975 ml of 1× PBS with 25 μl of Tween-20.Supplement 320 μl of 1% (v/v) of blocking buffer with 80 μl of mouse monoclonal anti-Pax7 stock solution.Supplement 398 μl of 1% (v/v) of blocking buffer with 2 μl of mouse monoclonal anti-MyoD stock solution.Supplement 999 μl of 1% (v/v) of blocking buffer with 1 μl of Alexa Fluoro 488 Ig G1 secondary antibody stock solution.2 incubator  Standard humidified COTissue culture hood Diamond pen Standard 9-inch glass Pasteur pipettes ®, St. Louis, MO, USA; cat. #Z270318)Bunsen burner  Micro-dissecting forceps  ®, St. Louis, MO, USA, cat. #S3146)Micro-dissecting scissors  3 cc disposable plastic syringes 2 plastic culture dishes 60 mm1.5 ml round-bottom microcentrifuge tubes 24-well plastic tissue culture dishes Stereo microscope ® FIVE acquisition software)Fluorescence microscope Preparation prior to muscle harvesting1.1. Pasteur pipettes of different sizes for single myofibers isolationFig. 1). The bore pipette with the widest diameter is used to transfer digested muscle from dish to dish . The bore pipette with a narrower diameter is used to triturate digested muscle in order to release single myofibers . The finest bore Pasteur pipette is used to isolate healthy myofibers . The procedures for the generation of Pasteur pipettes set are as detailed below:A set of Pasteur pipettes with bore diameters ranging from 1 mm to 3 mm is needed for different stages of the myofiber isolation procedure (1.1.1. Cut the glass Pasteur pipette using a diamond pen and snap away the cutting end to create a set of pipettes with different bore diameter sizes.1.1.2. Flame polish the sharp ends in the hottest part of a Bunsen flame (non-luminous flame near the tip of the inner blue cone) until the sharp edges are softened and smoothened.1.1.3. Autoclave the flame-polished glass Pasteur pipettes.1.1.4. Coat the pipettes with HS by passing undiluted filtered HS through them several times.1.2. HS-coated 1.5 ml round-bottom microcentrifuge tube for the immunocytochemistry staining of the freshly isolated myofibers.1.2.1. Transfer 1 ml of HS per 1.5 ml round-bottom microcentrifuge tube and swirl the tube gently to ensure even coating.1.2.2. Allow the tubes to sit for 5 min at room temperature.1.2.3. Discard HS and air dry the tubes for at least 30 min at room temperature.1.3. HS-coated 60 mm2 plastic Petri dishes for single myofibers culture1.3.1. Transfer 1 ml of HS per 60 mm2 plastic Petri dish and then swirl the dishes gently to ensure even coverage with serum.1.3.2. Incubate the dish for 5 min at room temperature.1.3.3. Discard the HS and allow the dishes to air-dry for at least 30 min at room temperature.1.3.4. Transfer 6 ml of washing media into each dish. Keep the dishes with washing medium in the CO2 incubator prior to use.HINTS: HS-coated plastic and glasswares should be used throughout the isolation procedure to prevent adhesion of single myofibers to the walls of those plastic and glasswares prior to plating or immunocytochemistry staining. Alternatively, FBS can be used as a coating agent.1.4. Matrigel®-coated 24-well culture dishes for myofiber culture.1.4.1. Pipette 500 μl of 10% (v/v) Matrigel® solution into each well of ice-chilled 24-well dishes.1.4.2. Gently swirl the 24-well dishes to ensure an even coating with Matrigel®.1.4.3. Allow the dishes to sit for 20 min at 4°C.1.4.4. Discard the Matrigel® solution. Incubate the dishes in the CO2 incubator for at least 30 min prior to use.HINTS: Matrigel® will solidify at temperatures > 10°C. Therefore, throughout the coating procedure always keep the Matrigel® solution chilled. The plastic pipette tips, conical tubes and 24-well dishes should also be pre-chilled to prevent premature gelling.Animal handling and euthanasiaad libitum. All mice were euthanized by cervical dislocation after being anesthetized in a chamber containing 2.5% isoflurane in 100% oxygen.NOTES: In this experiment, all mouse involved were adult male C57BL/6 mice at postnatal (P) days 90. The enzymatic digestive conditions (collagenase concentration and incubation time) stated in this, step 4 serves as a reference only. The experimental conditions are optimal for the EDL muscle derived from the mouse with the same background. The researcher may optimize the enzymatic digestive conditions by taking into consideration on the age, gender, size and the strain of the mouse. The outlooks of a well-digested EDL and the important points for the enzymatic digestive procedures are detailed in step 4.All experiments involving animal breeding and handling were approved by the Universiti Putra Malaysia Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with institutional regulations on experimental animals (Reference number: UPM/IACUC/AUP-R003/2014). All animals involved in this study were adult male C57BL/6 mice at postnatal (P) days 90 and they were bred under controlled environmental conditions of a 12 h light/dark cycle at 21°C–23°C with a relative humidity of 55%. A standard pellet diet and water were available Harvest the EDL using a tendon to tendon approach3.1. Sterilize all working area surfaces and dissection tools with 70% ethanol.3.2. Sterilize the entire mouse body with 70% ethanol and place the mouse in a supine position on the dissection board. Subsequently, secure all limbs to the dissection board using big head pins .3.3. Make a small incision in the skin at the anterior knee area. Extend the incision vertically towards the paws . Retract the skin lateral to expose underlying musculoskeletal structures .3.4. Identify the distal TA and EDL tendons under a stereomicroscope and cut them using sharp micro-dissecting scissors .3.5. Grip the distal TA and EDL tendons with curved tip micro-dissecting forceps and gently lift them upwards. The EDL is located underneath the TA. Separate the EDL from the TA by pulling the individual tendons in opposite directions .3.6. Cut the proximal end of the EDL tendon and transfer the tissue into a Petri dish containing 6 ml of washing media (washing dish) .CRITICAL STEP: It is important to isolate the muscle from tendon to tendon in order to maintain myofiber integrity. A well dissected/undamaged EDL should be long and slender .Enzymatic digestion4.1. Transfer the EDL to a Petri dish containing 6 ml of 0.2 % (w/v) collagenase type 1 solution.4.2. Incubate the muscle inside the humidified CO2 incubator for 90 min. Gently swirl the dish every 10–15 min during the digestion. The muscle should be examined regularly under a microscope to avoid over-digestion that may lead to myofiber hyper-contraction.CRITICAL STEP: Collagenase digestion is a critical step in determining the success of the single myofibers isolation procedure. The collagenase concentration and incubation time should be adjusted based on the age, gender, size and the strain of the mouse. Longer digestion times will be required for male, larger and/or more aged muscle.HINTS: A well-digested EDL looks less defined and slightly swollen, and has many hairlike single myofibers loosened from the edge . Single myofibers can be detached from the muscle with gentle shaking.4.3. When digestion is complete, use the widest bore Pasteur pipette to transfer the digested EDL to another Petri dish containing washing media.4.4. Transfer the digested EDL to a second, third and then fourth Petri dish containing washing medium to further dilute any residual collagenase and wash away any remaining debris.4.5. The digested EDL can be used for different downstream analyses (1) immediately fixed, triturate and immunostaining for Paired box family of transcription factors (Pax7) to study quiescent satellite cells (step 5.1), (2) triturated and cultured on a substrate such as Matrigel®-coated dish to investigate in vitro myogenesis events (step 5.2) and (3) triturated and cultured in floating conditions for at least 48 h before fixation and immunostaining for MyoD to investigate the activated satellite cells or doubling time of satellite cells in myofibers (step 5.3).Downstream analyses5.1. Liberation and immunocytochemistry staining of freshly isolated single myofibers—To investigate quiescent satellite cells5.1.1. After enzymatic digestion and several times of washing, carefully transfer the digested EDL bundle into a HS-coated 1.5 ml round-bottom microcentrifuge tube using the widest bore glass Pasteur pipette. Subsequently, fixed the digested EDL bundle with 800 μl of 4% (w/v) PFA solution for 10 min.5.1.2. After 10 min of fixation, carefully remove 4% (w/v) PFA solution and gently rinse the EDL bundle with 1 ml of 1× PBS (3× for 10 min each) to remove residual PFA.5.1.3. Transfer the PFA-fixed EDL bundle into a 60 mm2 plastic Petri dish and subsequently triturate the muscle bundle gently with the widest bore glass Pasteur pipette to release individual myofibers. After 5 min of trituration, an uncountable number of single myofibers would be liberated from the muscle bulk.HINTS: The diameter of the muscles will decrease as time goes. Consequently, repeat the titration process with a narrower bore Pasteur pipette until the required amount of myofiber was obtained.5.1.4. Next, with the aid of a stereomicroscope, select and transfer the straight and non-fragmented myofibers into a HS-coated 1.5 ml rounded bottom microcentrifuge tube using a HS-coated Pasteur pipette with a small aperture in the middle.5.1.5. Place the tube in an upright position to allow the myofibers to sink to the bottom of the tube. Later, carefully remove the 1× PBS from the tubes using the finest bore Pasteur pipette inserted just below the liquid surface. Maintain the 1× PBS to a height of 5 mm above the myofibers.CAUTION: Ensure that the myofibers sink to the bottom of microcentrifuge tubes before aspirating and changing the reagents during the immunostaining procedure. A sufficient amount of solution (5 mm above the level of the myofibers) is needed to prevent the loss of myofibers during the immunostaining procedure.5.1.6. Permeabilize the myofibers with 800 μl of 0.5% (v/v) Triton X-100 detergent for 8 min at room temperature. Then, discard the detergent and gently rinse the myofibers with 1 ml of 1× PBS (3× for 5 min each) before immuno-blocking with 800 μl of 1% (v/v) blocking buffer for at least overnight at 4°C with gentle agitation .5.1.7. Remove the blocking solution and subsequently incubates the myofibers with 200 μl of the primary antibody (pre-diluted to 1:5 in 1% (v/v) blocking buffer) for overnight at 4°C with gentle agitation .5.1.8. On the next day, remove the antibody and rinse the myofibers with 1 ml of 0.05% (v/v) Tween-20 washing solution (3× for 5 min each). Next, incubate the myofibers for 1 h in a dark room with 200 μl of Alexa Fluoro 488 Ig G1 secondary antibody (pre-diluted to 1:1000 in 1% (v/v) blocking buffer).5.1.9. Discard the secondary antibodies and rinse the myofibers with 1 ml of 0.05% (v/v) Tween-20 washing solution (3× for 5 min each).5.1.10. Use a PAP pen to outline a liquid repelling barrier on the SuperFrostTM Plus microscope slide.NOTES: The use of SuperFrost™ Plus microscope slides is strongly recommended to prevent myofiber detachment.5.1.11. Add 500 μl of 1× PBS into microcentrifuge tubes containing immunostained myofibers and gently pour the mixture into a Petri dish pre-coated with horse serum. Subsequently, rinse the microcentrifuge tubes with another 500 μl of 1× PBS to remove remaining myofibers.5.1.12. Collect myofibers under a stereomicroscope and transfer them onto the SuperFrost™ Plus microscope slide with a drop of ProLong® Gold Antifade Reagent with DAPI. Finally, cover the slide gently with a glass cover slip.NOTES: This transfer step should be performed as quickly as possible to prevent photo-bleaching.5.2. Liberation and culture of single myofibers—To study myogenesis programs initiated by satellite cell activation5.2.1. Prepare seven Petri dishes containing washing media and label as “1st trituration dish”, “2nd trituration dish”, “collection dish”, “washing dish 1”, “washing dish 2”, “washing dish 3” and “incubation dish”. Pre-warm these dishes in a CO2 incubator at 37°C.5.2.2. Transfer the digested EDL bundle into the 1st trituration dish and place the dish in the CO2 incubator for 10 min.5.2.3. Gently triturate the digested EDL bundle with the widest bore glass Pasteur pipette to release individual myofibers. After 5 min of trituration, approximately 30 viable single myofibers will be liberated from the digested EDL.5.2.4. Transfer the remaining EDL bundle from the 1st trituration dish into the 2nd trituration dish. Repeat the trituration process with a narrower bore Pasteur pipette. Meanwhile, place the 1st trituration dish containing viable myofibers in the CO2 incubator to keep the myofibers warm.CAUTION: It is crucial to maintain the media containing the myofibers at 37°C as the myofibers will fragment if the temperature of the medium drops below 37°C for an extended period of time.5.2.5. Repeat the EDL transferring and trituration process as described above until the desired number of myofibers are collected.NOTES: Do not over force the single myofibers to be released from the muscle bulk. Excessive force used during trituration will lead to the damage and the fragmentation of myofibers. If the myofibers remain tightly bundled together, the researcher shall digest the muscle bulk again in the freshly prepared 0.2% (w/v) collagenase type 1 solution for another 10–15 min. The morphology of the digested muscle should be examined regularly under a microscope to avoid over digestion.5.2.6. Next, with the aid of a stereomicroscope, carefully select only straight, healthy myofibers and reject partially contracted myofibers.5.2.7. Transfer the selected myofibers from the trituration dish to the collection dish using the finest bore Pasteur pipette with a small aperture.5.2.8. Wash the myofibers at least three times in washing medium by transferring them from the collective dish to the first, second, third washing dish and then incubation dish. This step will remove all dead/short hyper-contracted myofibers and other debris.5.2.9. Incubate those myofibers in a CO2 incubator at 37°C in the incubation dish for at least 1 h prior to switching to a culture medium. This allows myofibers to adjust to the in vitro conditions in the absence of serum.CAUTION: An immediate culture of myofibers in serum rich medium will increase myofibers shrinking.5.2.10. After at least one hour of incubation, transfer a Matrigel®-coated 24-well dish from the CO2 incubator to a laminar-flow hood. Add 250 μl of pre-warmed myofiber culture media into each well of the Matrigel®-coated 24-well dishes.5.2.11. Transfer individual myofiber, taking care to avoid transferring residual medium, and gently release the myofiber in the center of the well.HINTS: Refresh the HS-coated Pasteur pipette by passing undiluted filtered HS through them several times before transferring the myofibers to prevent myofibers from adhering to the pipette wall.5.2.12. Culture the myofibers in the 24-well dishes in a CO2 incubator at 37°C.NOTES: Avoid moving the dish for at least overnight as the myofibers are loosely adhere to the bottom of the well.5.2.13. After three days, add 250 μl of culture medium to the myofibers. After three subsequent days, replace the old medium with 500 μl of fresh culture medium.NOTES: Chicken embryonic extract contains numerous components that have stimulatory and/or inhibitory effects on the cell cycle of satellite cells [te cells . Therefo5.3. Culture of single myofibers in floating conditions—To investigate activated satellite cells or doubling time of satellite cells in myofibers5.3.1. Prepare a 60 mm2 plastic culture dish containing 5 ml pre-warmed HS containing culture medium. Pre-warm that dish in a CO2 incubator at 37°C.5.3.2. Transfer the selected myofibers from the incubation dish to the culture dish using the finest bore Pasteur pipette with a small aperture (Proceed from step 5.2.8). Culture the myofibers in a CO2 incubator at 37°C for 48 h or the longer period prior to immunocytochemistry staining for MyoD.5.3.3. After 48 h or longer period in culture, transfer those myofibers into a HS-coated 1.5 ml rounded bottom microcentrifuge tube using a HS-coated Pasteur pipette with a small aperture in the middle.5.3.4. Immunostain the myofibers with MyoD (pre-diluted to 1:200 in 1% (v/v) blocking buffer).Satellite cells play a distinct role in the routine skeletal muscle maintenance and repair of damaged adult skeletal muscles. A small defect in this population of cells can lead to the delayed, impaired or failure of muscle regeneration responses. Here, we present a reliable and simple technique for the isolation, cultivation, and immunostaining of the viable myofibers to study the characteristics of satellite cells during different stages of the myogenesis. Results and details on the critical point of each step are illustrated below:via the tendon to tendon approach in order to maintain the integrity of muscle. Muscle integrity is essential to determine the success rate of the procedure. An intact muscle facilitates better yield of the intact myofibers while the damaged muscle will lead to the poor yield and fragmented myofibers. Figure 2H is the representative image that shows the appearance of an intact (black arrowhead) and a damaged (green arrowhead) EDL muscle. The intact EDL is fusiform in shape while the damaged EDL appears as tissue chunk with a blunt distal end.In this study, the EDL muscle was harvested Figure 3 are the representative microscopy images that show the morphology of an EDL muscle in three digestive stages, well-digested, under-digested and over-digested. A well-digested EDL looks less defined and slightly swollen and has many hairlike single myofibers loosened from the edge . The under-digested EDL appeared compacted and the myofibers are still tightly bundled together . The over-digested EDL looked slack and will be fragmented easily during handling .Collagenase digestion is another critical step that determines the success of the single myofibers liberation procedure. The under- or over-digestion of muscle tissue can lead to a poor yield of intact myofibers. In this experiment, the EDL muscle was digested with 0.2% (w/v) collagenase type 1 solution at 37°C for 90 min. The collagenase digestion conditions, such as collagenase concentration and incubation time used here were optimized based on the conditions of EDL isolated from adult C57BL/6 male mouse aged postnatal (P) day 90. Ideally, the enzymatic digestion conditions should be adjusted based on the age, size, gender and the strain of the mouse. For example, longer digestion time is required for larger and/or more aged muscle. Therefore, it is highly recommended to monitor the digestion of muscle regularly under a microscope to avoid over-digestion that may lead to myofiber hyper-contraction. 2 incubator to re-equilibrate the liberation temperature. The complexity, tediousness and prolonged isolation procedure will induce activation of the satellite cells. Gnocchi and colleagues suggested that the myofibers should be fixed within 2 h after animal euthanization to preserve the associated satellite cells as near to the quiescent state as possible [Table 1). In addition, the refined protocol also generated a significantly higher yield of intact and viable myofibers (P = 0.003) as compared to the non-fixed muscle . Approximately 400–500 intact myofibers were liberated from the fixed EDL muscle, whereas only 50–100 intact myofibers were harvested from the non-fixed EDL muscle .Myofibers are delicate and satellite cells tend to be activated during the trituration procedures. In the published protocol, single myofibers are liberated from the digested muscle bulk and the liberation condition should be maintained at 37°C to prevent the fragmentation of myofibers ,15. Hencpossible . In thispossible ,11,12, tFig. 4A and 4D). All myonuclei present were revealed by counterstaining with DAPI (blue) .Using the refined protocol, we have identified both quiescent and activated satellite cells using Pax7 and MyoD, respectively. Pax7 and MyoD were expressed in the nuclei of quiescent (white arrow) and activated (yellow arrowhead) satellite cells, respectively. (Table 2).Liberation of single myofibers by trituration using Pasteurs pipette is one of the most critical procedure that determines the yields and quality of myofibers. The inadequate and excessive force used during trituration will lead to poor yields of intact myofibers. This procedure can be technically challenging, especially when handling the muscle with greater mass owing to the lack of exposure of the innermost layer of muscle tissue to the collagenase solution. To overcome this problem, the repetition of enzymatic digestion should be included, when necessary. This strategy allows the collagenase enzyme to further digest the connective tissues between each myofibers in the innermost layer of tissue and, thereby, reduce the compactness of muscle bulk. Consequently, the single myofibers can easily released from the muscle bulk during trituration. Using the improved protocol, we can yield an approximately 200–300 intact myofibers, whereas the published protocols can produce 50–100 intact myofibers from the same mass of muscle tissue . Satellite cells that migrated from the myofibers continued to proliferate, differentiate, and fuse into myotubes .To study myogenesis program initiated by stellite cell activation, single myofibers were isolated and cultured individually in 24-well dishes coated with MatrigelTable 3.Possible problems and their troubleshooting solutions are listed in


Halomonas xianhensis SUR308 (GenBank Accession No. KJ933394) was isolated from multi-pond solar salterns of Odisha, India. Exopolysaccharide (EPS) production by this strain in malt extract yeast extract (MY) medium has been optimized under batch culture system. Among the different media tested, MY medium showed an EPS production of 2.55 g/L, which increased to 2.85 g/L under optimized aeration. An initial pH of 7.5 and incubation temperature of 32 °C were found to be most suitable for EPS production by the isolate under aerobic condition. An EPS production of 3.85 g/L was achieved when the growth medium was supplemented with 2.5% NaCl. Glucose was the most favourable carbon source for EPS production and maximum production (5.70 g/L) was recorded with 3% glucose. However, growth as well as production of EPS was remarkably affected when the growth medium was supplemented with hydrocarbons as sole source of carbon. Among different nitrogen sources, casein hydrolysate at 0.5% level was proved to be the best for EPS production and an initial inoculum dose of 7% (v/v) enhanced the EPS production to 7.78 g/L, while the divalent metal ions were in general toxic to growth and EPS production, EPS synthesis by SUR308 was enhanced with Cr (VI) supplementation.A moderately halophilic bacterium,  Polysaccharides produced by Halomonas eurihalinaH. mauraH. eurihalina has been reported to jellify at acidic pH, while mauran, an anionic, sulfated EPS with high uronic acid composition produced by H. maura are being used for different biotechnological purposes H. ventosae, H. anticariensis and H. almeriensisHalomonas spp. isolated from diverse ecological conditions.Halophilic bacterial strains have been reported to produce exopolysaccharide (EPS) in their natural hypersaline environments as well as under laboratory cultural conditions. Among the wide diversity of halophilic species, members belonging to the moderately halophilic genus H. xianhensis SUR308 (GenBank Accession No. KJ933394) has been reported recently H. xianhensis SUR308 under batch cultivation. For better understanding of the process, the influence of aeration, pH, temperature, NaCl, carbon and nitrogen sources, inoculum dose along with metal ions on the EPS production by the isolate SUR308 have been evaluated.Production and characterization of exopolysaccharide of 2.2.1.Halomonas xianhensis SUR308 (GenBank Accession No. KJ933394), a potent EPS producing moderately halophilic bacterium isolated from solar salterns of Surala, Odisha, India was used throughout this study. The isolate was maintained on slopes of MH agar medium 2.2.2·6H2O, 9; MgSO4·7H2O, 13; CaCl2·2H2O, 0.2; KCl, 1.3; NaHCO3, 0.05; NaBr, 0.15; FeCl3·6H2O, 0.005; glucose, 10; yeast extract, 3; malt extract, 3; protease peptone, 5 (pH 7.2). H. xianhensis SUR308 was grown in MY medium for 24 h at 32 °C under continuous shaking and used as the inoculum. The medium (20 mL/100 mL flask) was inoculated at 2% level with the fresly prepared inoculum and incubated at 32 °C under continuous shaking for 11 days. To establish conditions leading to the maximum EPS production, the isolate was grown under different cultural variables such as aeration, pH, temperature, carbon source, nitrogen source, NaCl concentration, glucose concentration, hydrocarbons and heavy metals. Each experiment was conducted in triplicates and the average ± SE was recorded.Microbial growth and EPS production by the isolate were monitored in modified MY medium 2.3.Growth of the isolate SUR308 was determined by measuring optical density (OD) at 540 nm using an ELICO (CL 157) colorimeter. To determine the dry cell weight (DCW), known volume of culture was centrifuged at 5000 × g for 10 min and the cell mass after thorough washing was transferred into a pre-weighed aluminum foil cup. The cell mass was then dried to constant weight at 80 °C and the difference in weight was used to represent the actual dry weight.2.4.The EPS content of the growing culture was isolated and quantified according to the method as described by Quesada et al. The carbohydrate content was quantified by Dubois method 3.3.1.For screening of culture media suitable for EPS production, the isolate was grown in media of 14 different types supplemented with 5% NaCl. Growth and EPS production were estimated after 11 days of growth under continuous shaking at 32 °C. Results represented in H. stenophila HK30. Similar observation was reported with H. eurihalinaH. eurihalina was depended upon the culture medium used H maura, H. anticariensis and H. ventosae produced 4.28, 0.28 and 0.28 g/L of EPS respectively after 5 days of incubation Amjres et al. 3.2.2 concentration of conical flask H. xianhensis SUR308 required adequate O2 supply for the maximum production of EPS as pointed out by Rehm Aeration is another important environmental parameter for production of both intra- and extracellular metabolites of aerobic heterotrophic organisms. The effect of culture volume on metabolite biosynthesis under shake flask fermentation was reflected in the O3.3.It was observed from At a temperature range of 22–32 °C the isolate was found to produce more EPS along with enhanced biomass production  comparedHalomonas spp.Thus the influence of pH and temperature on the growth and production of EPS by the isolate SUR308 clearly revealed its neutrophilic and mesophilic nature and supported the earlier works of Quesada et al. 3.4.H. maura was 2.5%, while 7.5% salt concentration was proved to be the best for maximum EPS production by H. eurihalina, H. ventosae and H. anticariensis.As a halophilic organism, the isolate SUR308 showed a wide degree of tolerance to NaCl for growth and EPS production was more or less constant in the range of 2.5 to 10.0% NaCl in the medium, where by EPS production varied from 3.85 to 3.54 g/L. It was evident from the results  that the3.5.H. xianhensis SUR308 revealed that addition of glucose, fructose, lactose or glycerol in the medium at 1% level resulted different levels of growth and EPS production by the bacterium. Glucose was most effective in EPS production (3.95 g/L) followed by lactose (2.82 g/L) and glycerol (2.64 g/L). However, compared to glucose and lactose, glycerol exerted maximum influence on the growth of the isolate. Carbon sources like galactose, acetate, fumarate, citrate along with fructose, maltose and sucrose produced poor to moderate EPS, while fructose and galactose were effectively utilized for growth  was tested on growth and EPS production by the isolate, it was evident that the biomass production as well as the EPS accumulation was remarkably ceased with hydrocarbon supplementation in the medium. When the influence of different hydrocarbons as sole source of carbon  was tested on growth and EPS production by the isolate, it was evident  that the3.6.The most common nitrogen sources currently used for EPS production by halophiles include ammonium sulfate, peptone, sodium nitrate, urea and yeast extract but the presence of organic nitrogen sources are known to promote both growth rate and the EPS production To find out the most suitable concentration of casein hydrolysate for EPS production the isolate was grown in media containing 0.1–2% of casein hydrolysate, other conditions of growth were same as in previous experiments. It was found that EPS production by the isolate raised to 6.68 g/L when the medium was supplemented with 0.5% casein hydrolysate . At conc3.7.Since the initial inoculum added to the medium is known to influence the EPS production, MY medium supplemented with 2.5% NaCl, 3% glucose, 0.5% casein hydrolysate at pH 7.5 and with CVF of 1.5:10 was inoculated with freshly grown culture of the isolate at 1–8% (v/v) level and incubated at 32 °C under continuous shaking (120 rpm). As shown in the 3.8.Bacteria resistant to metals often showed production of high amount of exopolysaccharides at heavy metal stressed growth condition Addition of toxic heavy metals in the growth medium showed inhibitory effect on growth and EPS production by the isolate SUR308. Among the metals tested manganese (2–8 mM) did not affect growth as well as EPS production (4.93–6.75 g/L), while lead and nickel inhibited growth and EPS production remarkably. However, induction of EPS production by the isolate was observed when the medium was amended with 2 mM chromium. The highest (6.95 g/L) EPS production was observed in 2 mM chromium supplemented medium .4.H. xianhensis SUR308 at laboratory scale. When the EPS production of H. xianhensis SUR308 were compared with different strains so far available (The present study demonstrated the optimization of media composition along with environmental parameters to scale up the production of EPS by vailable  it showe


We have read with interest the article entitled “Thalidomide for the Management of Bleeding Episodes in Patients with Hereditary Hemorrhagic Telangiectasia: Effects on Epistaxis Severity Score and Quality of Life” .This article highlights the use of thalidomide in the management of patients with hereditary hemorrhagic telangiectasia (HHT) who present with epistaxis. The prevalence of HHT is thought to be between 1.5 and 2 cases per 10,000 people ,4,5,6. TWe carried out a study at our tertiary center for the management of patients with HHT where 10 patients  with genetically confirmed HHT were referred for the management of gastrointestinal-related complications. The impact of small bowel capsule endoscopy (SBCE) and double balloon enteroscopy (DBE) was evaluated. The mean age at first SB endoscopy was 62.6±14.4 years (mean ± standard deviation).Patients had a total of 39 gastroscopies, 16 colonoscopies, and 6 push enteroscopies. Seven patients underwent SBCE: 6 (85.7%) had proximal, 1 (11.1%) had mid, and 3 (33.3%) had distal SBAs. Two patients had a colon capsule that showed angioectasias.Several DBEs were carried out for 6 patients  with a mean of 130.5±133.3 days between DBEs. Fifty-seven SBAs were treated with argon plasma coagulation (APC) on average at each DBE. These procedures take an average of 75 minutes. Mean hemoglobin before and after the procedure was 9.8 and 10.2 g/dL, respectively (p=0.1). Six patients were transfusion-dependent initially but 4 improved following intervention.Need for transfusion resolved in 1 patient when started on lanreotide , regular endoscopy, and APC, and in 2 patients upon starting DBEs and APC. One patient passed away from pneumonia. Another patient was switched unsuccessfully from octreotide to lanreotide. She stopped being transfusion-dependent with regular gastroscopies and APC. Another patient was unwilling to undergo further endoscopies due to multiple comorbidities. He improved on lanreotide. In 2 patients, anemia remains persistently problematic. One of them is also on dalteparin for superior mesenteric venous thrombosis. The other patient has recurrent epistaxis, which makes it harder for him to have further endoscopies.SBCE is a useful screening tool in patients with HHT to assess SBAs. Although classed as invasive endoscopy, DBEs and APC can have a significant impact on mortality and quality of life in patients with HHT. Pharmacotherapy such as somatostatin analogs can additionally help to improve transfusion requirements. They have a good safety profile , unlike


Systemic lupus erythematosus (SLE) is an autoimmune systemic disease and its pathogenesis has not yet been completely clarified. Patients with SLE show a deranged lipid metabolism, which can contribute to the immunopathogenesis of the disease and to the accelerated atherosclerosis. Resolvin D1 (RvD1), a product of the metabolism of the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), acts as a specialized proresolving mediator which can contribute in restoring the homeostasis in inflamed tissues. The aim of the present pilot study is to evaluate plasma levels of RvD1 in patients with SLE and healthy subjects, investigating its potential role as a biomarker of SLE and assessing its relationship with disease activity and laboratory parameters.  Thirty patients with SLE and thirty age- and sex-matched healthy subjects (HSs) have been consecutively recruited at Campus Bio-Medico University Hospital. RvD1 plasma levels were measured by ELISA according to the manufacturer's protocol . RvD1 levels were compared using Mann–Whitney test. Discriminatory ability for SLE has been evaluated by the area under the ROC curve. p = 0.0043). The area under the ROC curve (AUC) for RvD1 was 0.71 (95% CI: 0.578–0.82) and the threshold value of RvD1 for the classification of SLE was <58.4 pg/ml, sensitivity 80% (95% CI: 61.4–92.3), and specificity 63.3% (95% CI: 43.9–80.1), likelihood ratio 2.2 (95% CI: 1.3–3.6).  Lower levels of RvD1, 45.6 (35.5–57.4) pg/ml, in patients with SLE have been found compared to HSs, 65.1 (39.43–87.95) pg/ml ( The present preliminary study allows hypothesizing a dysregulation of RvD1 in patients with SLE, confirming the emerging role of bioactive lipids in this disease. Systemic lupus erythematosus is an autoimmune systemic disease which can involve virtually every organ or apparatus . DespiteOmega-3 polyunsaturated FA can be metabolized in inflamed tissue leading to specialized proresolving mediators (SPMs), which prevent further polymorphonuclear cell (PMN) infiltration, induce efferocytosis of apoptotic bodies in macrophages, and promote tissue repair and healing –22. D-seThe present pilot study is aimed at evaluating plasma levels of RvD1 in patients with SLE and healthy subjects, investigating its potential role as a biomarker of SLE and assessing its relationship with disease activity and laboratory parameters.Thirty patients with SLE, classified according to the 2012 Systemic Lupus International Collaborating Clinics (SLICC) criteria , were coRvD1 plasma levels were measured by ELISA according to the manufacturer's protocol , as validated elsewhere .U test. Fisher's exact test has been used to analyze contingency tables. Receiver operating characteristic (ROC) analysis was used to define the ability of RvD1 to differentiate patients with SLE and HSs; the optimum cutoff value has been identified from the highest Youden's index. Pretest odds, posttest odds, and posttest probability were calculated. Statistical analysis was performed using GraphPad Prism 7  and MedCalc 11.6.1.0 (Belgium).Data were expressed as median (25th–75th percentile). RvD1 levels between patients with SLE and HSs as well as demographic and laboratory parameters have been compared using Mann–Whitney Demographic features and clinical and laboratory characteristics of the patients with SLE and HSs are shown in p = 0.0043), as reported in p = 0.11).Patients with SLE showed lower levels of RvD1, 45.6 (35.5–57.4) pg/ml, compared to HSs, 65.1 (39.43–87.95) pg/mL (p = 0.0012.The area under the ROC curve (AUC) was 0.71 (95% CI: 0.578–0.82), showing a fair discriminatory ability as reported in p = 0.0087, as reported in In the SLE cohort, no relation has been found between RvD1 plasma levels and disease activity scores, SDI, or disease duration. Likewise, no relation has been demonstrated between RvD1 plasma levels and pharmacological therapies. Nevertheless, SLE patients with low C4 levels (<0.1 g/l) also had lower RvD1 plasma levels, 36.05 pg/ml (29.55–42.45), compared to patients with normal (>0.1 g/l) C4 levels of 52.2 pg/ml (43.4–61.8), SLE is a complex autoimmune multisystemic disease, with a huge impact on quality of life and development of organ damage , 35. DesBioactive lipids seem to be a main actor in inflammation, and they could play a pivotal role in immunopathogenesis on many inflammatory diseases , 38, 39.For the first time, our study demonstrates lower levels of RvD1 in plasma of SLE patients compared to HSs. Moreover, the analysis of the ROC curve showed a fair ability of RvD1 to discriminate SLE patients from HSs, providing a preliminary cutoff value of 58.4 pg/ml. Recently, Barden and coworkers demonstrated higher plasma levels of RvD1 in patients with arthritis compared to healthy subjects; therefore, the role of RvD1 in SLE and arthritis appears to be different . In our Several weaknesses of this study should be considered. The sample size was relatively small, and further studies on a larger number of patients are required to better evaluate the role of RvD1 in SLE, especially its potential role as a biomarker. Furthermore, this study did not schedule a follow-up of patients and therefore could not ascertain the role RvD1 in predicting changes in disease activity, damage accrual, or laboratory parameters over time. Moreover, no information about omega-3 polyunsaturated FAs was available. In the present study, we cannot exclude that the difference of RvD1 plasma concentrations between patients with SLE and HSs may partly reflect the use of glucocorticoids or immunosuppressants.In conclusion, this study allows hypothesizing a dysregulation of RvD1 in SLE and confirms the emerging role of bioactive lipids in this disease.