In the recent phase III trial MPACT the combination of gemcitabine and nab-paclitaxel (Gem/NabP) showed increased overall survival compared to gemcitabine alone in the treatment of advanced pancreatic ductal adenocarcinoma (aPDA). Until now there has been limited information on the clinical benefit and toxicity of the combination regimen in a real world setting. In addition the value for patients with locally advanced rather than metastatic aPDA has been unclear, since the former category of patients was not included in the MPACT trial.A multicentre retrospective observational study in the South Eastern Region of Sweden was performed, with the first 75 consecutive patients diagnosed with aPDA  who received first-line treatment with Gem/NabP.In the overall population median progression free survival (PFS) and overall survival (OS) were 5.2 (3.4–7.0 95% CI) and 10.9 (7.8–14.0 95% CI) months, respectively. Patients with metastatic disease displayed a median OS of 9.4 (4.9–13.9) and a median PFS of 4.5 (3.3–5.7) months whereas the same parameters in the locally advanced subgroup were 17.1 (7.6–26.6) and 6.8 (5.2–8.4) months, respectively. Grade 3–4 hematologic toxicity was recorded: Neutropenia, leukopenia, thrombocytopenia, and anaemia were observed in 23, 20, 5, and 4% of patients, respectively. Dose reductions were performed in 80% of the patients.This study confirms the effectiveness and safety of first-line Gem/NabP in both locally advanced and metastatic PDA in a real world setting.The online version of this article (10.1186/s12885-018-5244-2) contains supplementary material, which is available to authorized users. Pancreatic cancer is a severe disease with increasing incidence and high mortality, being the fourth most frequent cause of cancer related death in Sweden , Europe,Since the ground breaking trial by Burris et al. in 1997 , where gThe situation was altered by the recent phase III MPACT trial that showed nearly two months of increased median OS, from 6.7 to 8.5 months, with the nano albumin bound (nab)-paclitaxel/gemcitabine (Gem/NabP) combination regimen compared with gemcitabine alone . On the A retrospective observational study was conducted in the South East Region of Sweden, covering a population of approximately 1,050,000 citizens, involving the oncology departments of Jönköping, Kalmar, and Linköping. The study was approved by the Regional Ethics Review Board in Linköping (diary number 2017/110–31). At the participating sites all prescription of chemotherapy is covered by the digital software CSAM Cytodose . Therefore all eligible patients were identified with this software. Inclusion criteria were aPDA, including locally advanced as well as metastatic disease, and the administration of first line palliative treatment with Gem/NabP, with treatment starting between the approval of the regimen in December 2014 and March 2017. Histology based diagnosis was preferred but, when absent, diagnosis based on combinations of cytology, imaging, and/or serum tumour marker (CA19–9) was accepted. Treatment pause and follow up with slight progression allowing for restart of the same therapy was not considered as second line therapy. Neither was toxicity related de-escalation to single drug regimen (i.e. Gem/NabP to gemcitabine) considered second line therapy, unless clinical and/or radiological signs of progressive disease were evident. Concomitant treatment with other modalities and treatment beyond progression with other chemotherapy regimens were allowed. Patients that received Gem/NabP in other settings than first line palliative treatment or with other histology than PDA were excluded.2 and 125 mg/m2 for gemcitabine and nab-paclitaxel, respectively. Bone marrow toxicity was monitored with blood samples prior to each dose and treatment response was usually evaluated with computed tomography every 2–3 cycles.At the three centres Gem/NabP was administered in a similar way by intravenous infusion at days 1, 8, and 15 in a four week cycle at doses of 1000 mg/mPatient and tumour characteristics, baseline biochemistry, performance status, and CA19–9 as well as treatment data were extracted from medical records using a standardised case report form. All patients’ medical records were followed until date of death, or no later than 27th of Sep 2017, when follow-up period was closed. Primary endpoint was OS, which was defined as time from start of treatment until date of death or last follow-up. Secondary endpoints were median progression free survival (PFS), occurrence and grade of bone marrow toxicity, number of treatment cycles, dose intensity, and presence of second line therapy. PFS was defined as time from start of treatment until progression, either radiological or clinical, or death, whichever came first.http://www.strobe-statement.org/).The present study was conducted and reported in accordance with the STROBE guidelines . Patient characteristics and tumour and treatment data are reported as frequencies and percentage for categorical variables and median with 95% confidence interval and range for continuous variables. Median OS and PFS for the whole cohort and subgroups  were estimated using Kaplan-Meier survival analyses and the significance of the difference in estimates of median survival with 95% confidence interval was calculated using log rank test. Cox regression analysis was used to evaluate hazard ratios for the same subgroups. Analyses were performed using SPSS v24 (IBM Corp. Armonk NY).A total of 92 unique patients treated with Gem/NabP were identified. Of these patients 17 were excluded based on the exclusion criteria, leaving a total cohort of 75 patients representing the final study cohort  and the median OS was 10.9 months  as a surrogate for PFS and database persistence as a surrogate for OS, in a Celgene corporation funded study including 122 patients , and repThis study provides additional value since it was performed on a reasonably large population, with robust recognition of clinico-pathological and treatment parameters and sufficient follow up times to analyse measured (and not estimated or surrogate) primary and secondary endpoints. Mature data on the locally advanced subgroup, that was not included in the MPACT trial, is further provided and encourages further usage of the combination regimen in this subgroup. The Swedish general and public funded health care system means that all individuals regardless of socio-economic status, insurance status, or comorbidities are offered similar treatments and follow-up programs, and the present study therefore adds key information about the outcome and tolerability of Gem/NabP in a truly ‘real world’ setting.The patients in the present study experienced less hematologic grade 3–4 toxicity than in the MPACT study, and in the lower interval of that reported in previous studies , 16, 17.This study confirms the effectiveness and safety of Gem/NabP in both locally advanced and metastasised PDA in a real world setting. Less treatment related haematological toxicity than expected was observed. The vast majority of patients underwent dose-adjustments. Biomarkers predicting effects and toxicity of Gem/NabP are still lacking, and prospective trials investigating and validating serum- or tissue markers are needed in order to establish tools for personalised treatment decisions.Additional file 1:Figure S1. Kaplan-Meier diagrams showing PFS (A) and OS (B) for subgroups according to the occurrence of prior curative intent pancreatic resection. (PDF 124 kb)Additional file 2:Table S1. Baseline patient characteristics for subgroups divided according to previous chemotherapy treatment and previous curative intent surgery, respectively. (DOCX 19 kb)Additional file 3:Figure S2. Kaplan-Meier diagram showing OS for the entire cohort with or without the inclusion of four patients who received unconventional/experimental treatment  in addition to Gem/NabP. (PDF 100 kb)Additional file 4:Table S2. Survival data in months (95% CI) for the entire cohort compared to patients that received standard treatment only. HR = hazard ratio calculated using Cox regression analysis. Experimental treatments were RFA (n = 1), HIPEC (n = 1), and IRE (n = 2). (DOCX 14 kb)


Arteriovenous malformations (AVMs) in the pediatric population are rare, yet they form the most frequent cause of hemorrhagic stroke in children. Compared to adults, children have been suggested to have beneficial neurological outcomes. However, few studies have focused on other variables than neurological outcomes. This study aims to assess the long-term functional and educational outcomes of children after multimodality approach of treatment for intracranial AVMs.All children treated in our center between 1998 and 2016 for intracranial AVMs were reviewed. Patient characteristics, as well as AVM specifics, were collected. Functional outcomes were compared using the modified Rankin scale (mRs). Educational levels, using the International Standard Classification of Education (ISCED), were compared to the age-matched general population of the Netherlands.n = 11) or were stable (n = 13). At follow-up, all patients scored a stable or improved mRs compared to discharge, with 23 children (92%) scoring mRs 0 or 1. These 23 children followed regular education during follow-up without specialized or adapted schooling. No significant differences in educational level with the age-matched general population were found.In total, 25 children were included at mean age of 10 years (range 2–16 years). Nineteen patients (76%) presented with intracranial bleeding. Mean follow-up was 11.5 ± 5.3 years (range 4.1–24.4). Four (16%) of patients were treated with embolization, three (12%) with microsurgery, and 18 patients (72%) received a combination of different treatment modalities. Altogether, 21 (84%) were embolized, 14 (56%) were treated with microsurgery, and eight (32%) received stereotactic radiosurgery. One child had a worse mRs at discharge compared to admission; all others improved (This retrospective review shows positive long-term results of both functional and educational outcomes after multidisciplinary treatment of pediatric brain AVMs. Arteriovenous malformations (AVMs) in the pediatric population are rare, yet they form the most frequent cause of hemorrhagic stroke in children . BesidesBecause not all AVMs are symptomatic, real incidence rates remain unclear. Prevalence of symptomatic AVMs is estimated to be between 0.06 and 0.11% in the total population . Due to Upon bleeding, up to one third of children are comatose, and approximately half of patients have a neurological deficit . The posChildren have been suggested to have beneficial neurological outcomes , especiaAll children who underwent any form of treatment at our center for an intracranial AVM between 1998 and 2016 were reviewed. This specific timeframe was chosen so that a meaningful assessment of long-term follow-up could be done. Data were collected by using the digital medical records and by telephone call contact with patients or their parents. All patients who met the following criteria were included: 1) any form of treatment for intracranial AVM; 2) age below 18 at time of first treatment. No exclusion criteria were used.We collected patient age (years), gender, and presenting symptoms. Additionally, AVM size, localization, and the presence of deep venous drainage were obtained, and each AVM was graded following the Spetzler-Martin grading system (SMG) . All treAs a control group for highest achieved educational levels, we used the general population of the Netherlands, matched by age. This data was collected from a public database  . Howevert test was used for all continuous variables. To reveal potential predictors, univariate analysis was done by using logistic regression. For all tests, p values below 0.05 were considered as statistical significant.Statistical analysis was conducted using IBM SPSS statistics 24. To determine significant differences in the distribution of baseline characteristics we used the following tests: 1) Fisher tests for binary variables if the expected value was below five; 2) chi-squared tests if the expected value was five or higher; 3) and Student’s n = 3), seizures (n = 1), and a defect in the skull (n = 1), and one patient was a coincidental finding. Four (16%) patients were treated in the acute setting on the same day as the hemorrhage. Mean follow-up was 11.5 years (range 4.1–24.4 years) (SD 5.3). Mean age at follow-up was 22.0 years (range 12.4–33.7 years) (SD 5.8).In total, 25 children were included at mean age of 10 years (range 2–16 years). Demographics, Spetzler-Martin grades, and mRs at admission are summarized in Table One patient was diagnosed with Wyburg-Mason syndrome. Another patient had unspecified congenital psychomotor impairment and diagnosed early in life with an accompanying AVM. Furthermore, two patients had family history of multiple family members with subarachnoidal hemorrhage or AVMs, yet without a syndromal diagnosis. Finally, one patient had accompanying Marfan syndrome.Embolization, surgical resection, and stereotactic radiosurgery (SRS) using a linear accelerator or gamma-knife were used to treat the AVMs Table . Embolizp = 1.00). However, AVMs with a SMG lower than 3 had higher occlusion rates (p < 0.001) and more often complete obliteration (p = 0.04). Upon treatment, two (8%) children experienced temporary increase of neurological deficits, and one (4%) had a new mild coordinative difficulty in one leg. Furthermore, three (12%) patients experienced post-treatment hydrocephalus. In two of these, this was due to the intracranial hemorrhage. In the third case, besides the patients preexisting psychomotor retardation, no other explanation was found. All patients with hydrocephalus needed an additional ventriculoperitoneal shunt. Re-bleeding occurred in three (12%) patients, of which two could be seen as AVM recurrence after initial complete AVM obliteration. All needed additional treatment. In the total cohort, no major complications or deaths occurred.Post-treatment angiography showed complete occlusion of the AVM in 19 of 25 patients (76%). The mean obliteration percentage of the total cohort was 93.4% (SD 15.5%). Acute treatment as well as the form of primary treatment was not associated with lower occlusion rates (both n = 10) and 5.3% (n = 1) scored worse. Of the patients with unruptured AVMs, 16% improved (n = 1) and none scored worse. At follow-up, five patients had persisting and stable neurological deficits, and three children had persisting but improved deficits. Six of these patients had mild deficits like visual field deficits (n = 5), diplopia (n = 1), and the previously mentioned coordinative difficulties (n = 1) with no interference in their daily life. In the other two patients, remaining deficits were hemiparesis (n = 1) and mental retardation (n = 1). The patient with mental retardation was known with these disabilities prior to treatment. Finally, in follow-up, all patients scored a stable or improved mRs compared to discharge. Of the children, 91.3% had either mRs 0 or 1, whereas the other two scored mRs 2 and mRs 3 respectively. The mRs scores at admission, discharge, and follow-up are arranged in Fig. Comparing mRs at admission and at discharge, 52.6% of the children with a ruptured AVM improved n = 0 and 5.3During follow-up, 23 of the 25 children (92%) followed regular education without specialized or adapted schooling. One patient needed adjustments for physical disabilities with normal educational levels. Only one patient, who was known to have mental retardation previous to treatment, was in need of adaptive education. Furthermore, eight (34.8%) patients attained tertiary education, which we scored as high education. Of two children, specific educational levels were not obtainable, and only a simple assessment could be made. Therefore, we did not implicate these children in the comparison with the general population. The distribution of different educational levels of this case series in comparison to age-matched peers is shown in Fig p > 0.05). Although patients with SMG below 3 more often had complete obliteration, this association was not significant in logistic regression (p > 0.05). Reckoning the fact that re-bleeding only occurred in three cases, incomplete occlusion of the AVM was not associated with re-bleeding (p = 1).In this cohort, there was no significant relationship between unfavorable neurological or functional outcome  and bleeding of the AVM, size (grouped following the SMG), or location (eloquent versus non-eloquent of the AVM, modified Rankin scale at admission, recurrence or re-beeding, and used treatment modalities (all This retrospective review shows positive long-term results of both functional and educational outcomes after a multidisciplinary approach of treatment in pediatric brain AVMs. This study investigated educational achievement, as this both reflects cognitive function and is particularly important for the quality of life of these patients. Almost all patients were able to continue their normal lives with minimal to no remaining neurological disabilities or cognitive restrictions. In comparison to age-matched peers, the educational levels of these children showed no difference.Since the long-term functional outcome of the cohort was very good, it was impossible to find a predictor for unfavorable outcome. Because of the same reason and because less than a third of the patients were treated with a single strategy, no single treatment strategy was found superior in this case series. These results may confirm the importance of a multidisciplinary approach of this rather serious condition in the pediatric population. To our knowledge, there currently are no studies available comparing results of different treatment modalities and combinations. However, combining modalities is suggested only to improve clinical outcomes , 17. A rAt presentation, prognosis may seem unclear to parents and clinicians. AVM bleeding can be life threatening and sometimes even requires emergency treatment. This study shows that in our cohort, neurological as well as educational outcomes are promising after multidisciplinary approach of treatment. Analysis of patient- and AVM-related factors did not result in significant associations with differences in outcomes. This is in contrast with some of the existing literature, in which associated aneurysms, deep venous drainage, and partial occlusion are suggested to be associated with higher chance of re-bleeding and worse outcomes . FurtherDue to the low incidence rates, true evaluation of the effects of any form of treatment and neurological and cognitive outcomes remains difficult. With 25 patients in as many years and multiple options for treatment, we still depend largely on retrospective analyses, which is also the main limitation of this study. The fact that some of the previously described factors related to changes in outcomes were not found significantly associated, might very well be based on the small size of this cohort. Presumably, a larger population may have demonstrated a wider range of outcomes. In addition, the use of a subjective scoring system should also be noted as a possible bias. The modified Rankin scale is a validated adult scoring system and commonly used in clinical trials , 23. AltThis retrospective review shows satisfactory long-term results of both functional and educational outcomes after multidisciplinary treatment of pediatric brain AVMs. Unfortunately, it was impossible to find predictors for either better or worse outcomes. However, these findings might contribute to the existing belief that clinical outcomes seem to be favorable among children as compared to adults.


Firmicutes, Actinobacteria and underrepresented Bacteroides, of Indian and Chinese gut bacteria. This community structure closely matched with the gut bacterial composition of the Russian population. Therefore, we hypothesized that enrichment of these bacterial clades is supported by high consumption of starch-rich diet such as rice, potato, refined grains. The dominance of genus Bifidobacterium due to carbohydrate-rich diet is another notable feature of this study. Moreover, Indian gut bacteria are significantly represented by Bacteroidetes (p = 0.001) and Prevotella (p = 0.002) in contrast to Chinese, which could be associated with whole grains and plant-based vegetarian diet of Indians. The gut bacterial population of Indian adults were as diverse as Chinese adults (p > 0.1), but significant difference was noticed in gut bacterial composition and relative abundance between two populations . Partial least squares discriminant analysis and non-metric multidimensional scaling plots showed dietary habit wise clustering of subjects. Thus, the present work confirms an important role of diet in determining gut bacterial composition. LEfse analysis revealed genera Prevotella, Megasphaera, Catenibacterium, Lactobacillus, Ruminococcus and species Prevotella copri, Lactobacillus ruminis as the potential biomarkers of diet.The interaction between diet and gut microbiota, and ultimately their link to health, has turned into the concentration of huge research. However, this relationship still needs to be fully characterized, particularly in case of the Asian population. We compared the fecal bacterial diversity and composition of healthy Indian and Chinese adults, ages 22–35 years, using next-generation sequencing analysis on IlluminaHiSeq 2500 platform. Our analysis revealed unique community structure, dominant The online version of this article (10.1186/s13568-018-0632-1) contains supplementary material, which is available to authorized users. The human gut is a host of trillions of bacteria. The entire gut microbiota is estimated to contain 150-fold more genes than our host genome  characterize the prevalent bacterial taxa to define the community structure in gut samples from healthy Indian and Chinese adults, (2) compare the bacterial diversity distribution within and between the two groups studied, and (3) discover possible relationship of gut bacteria with diet and identify gut bacterial biomarkers of diet which distinguish Indian and Chinese adults. The gut bacterial profiling of 16 healthy Asian adults, including 11 Indians and 5 Chinese, obtained using next-generation sequencing. We further explored the influence of dietary habits on the composition of gut microbiota.A total of 16 healthy adults, including 11 Indians and 5 Chinese, were recruited for the current study . The fecal water was removed and samples were stored at − 80 °C freezer prior to DNA extraction.All participants were asked to refrain from smoking, eating, drinking for at least 1–2 h prior to samples collection. Study participants were provided with two different containers: a sterile pot and a 50 mL sterile centrifuge tube. The volunteers were asked to transfer fresh feces from the sterile pot to the tube immediately after defecation. The samples were anonymized in the same order with the questionnaire . Samples were homogenized, 10 g of feces were taken in 50 ml falcon tube and centrifuged . The concentration of DNA sample was measured using NanoDrop and purity was monitored on 1% agarose gel. The concentration of DNA sample was diluted to 1 ng/µL in sterile water.For each sample, 16S rRNA genes of V4 region were amplified. The primer set corresponding to primers 515F-806R with the barcode was used for amplification. All PCR reactions were performed with Phusion high fidelity master mix (New England Biolabs). The same volume of loading buffer containing SYBR green was mixed with PCR products. Gel electrophoresis with 2% agarose was performed for detection. Samples with the bright main strip between 400 and 450 bp were selected for facilitating experiments. PCR products were mixed in equidensity ratios and afterward purified with Qiagen gel extraction kit .Sequencing and analysis were performed by NovogeneAIT (Singapore). Sequencing libraries were generated using TrueSeq DNA PCR-free sample preparation kit  according to manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Then, sequencing was performed on an Illumina HiSeq 2500 platform and 250 bp paired-end reads were generated.Samples were assigned paired-end reads and reads were merged using FLASH  hierarchical clustering was performed to interpret the distance matrix using average linkage. All these analyses were conducted by QIIME software (Version 1.7.0) and R software (Version 2.15.3). Non-metric multidimensional scaling (NMDS) based on Bray–Curtis dissimilarity was applied with Analysis of similarities (ANOSIM) and Multi-response permutation procedure (MRPP) to analyse the difference in microbial composition among subjects and test the significance of the difference Ramette .Metastats analysis , Clostridia (class), Clostridiales (order), Lachnospiraceae (family), Bifidobacterium (genus) at different taxonomic levels.Amplicon sequencing on IlluminaHiSeq 2500 platform covering the V4 region of bacterial 16S rRNA of 16 fecal DNA samples representing 11 Indian and 5 Chinese produced on an average of 85,752 high quality reads (average read length 413.12 nt) in each sample   and Cyanobacteria (p = 0.003) were significantly higher in Indian group at the phylum level. Particularly, the abundance of Bacteroidetes in Indians (16.39%) was almost 4 times higher as compared to Bacteroidetes in Chinese (4.27%). The abundance of Firmicutes (68.08%), Actinobacteria (25.48%), in Chinese were not significantly different and slightly higher, as compared to Firmicutes (60.5%), Actinobacteria (20.57%) in Indians. It is noticeable that Firmicutes has outnumbered Bacteroidetes in both the populations  between two groups were Bacteroidia, Bacilli, Gammaproteobacteria, and Chloroplast. The bacterial orders (p < 0.05) accounting for significant variation between Indians and Chinese were found to be Bacteroidales, Lactobacillales, Bacillales, Streptophyta. The bacterial families (p < 0.05) Prevotellaceae, Lactobacillaceae, Leuconostocaceae, Carnobacteriaceae showed significantly higher abundance in Indians whereas Porphyromonadaceae was higher in Chinese.The bacterial classes that showed a significant difference (p < 0.05) Prevotella, Megasphaera, Catenibacterium, Lactobacillus, Mitsuokella, Carnobacterium, Lachnospira were significantly higher in Indians as compared to Chinese. The genera, Bifidobacterium (15.2%) and Prevotella (13.07%) were almost equally dominant in Indian population  and Blautia (13.19%). Previous studies on different populations have reported that human gut microbiome could be classified based on one of the four distinct communities, namely Prevotella, Bacteroides, Clostridiales, and Bifidobacterium  is highly abundant as opposed to very low percentage of Prevotella in Chinese (0.58%)  were performed at all taxa levels Fig. a, b. PLSp > 0.1 for all the indices.Alpha diversity is widely used for the analysis of microbial community diversity. It reflects the richness and diversity of microbial community by using a series of statistical indices, species accumulation curve, and species richness curve. As can be seen from Fig. p < 0.02) between Indian and Chinese groups, as shown by two-tailed t test and Wilcoxon two-sample test. It reveals that two groups can be distinguished based on both community composition and relative abundance of gut bacteria. The beta diversity indices box plots which describe differences of species diversity between groups are shown in Fig. The alpha diversity indices were found to be similar in both the groups, now to estimate the extent of variation between gut bacterial profiles of Indians and Chinese, we proceeded to analyse beta diversity using unweighted and weighted unifrac distances. The unweighted unifrac distance is calculated according to phylogenetic relationships of OTUs and weighted unifrac distance is based on the OTU’s abundance information. The beta diversity indices were significantly different (p = 0.001), and multi-response permutation procedures (MRPP) (p = 0.003). R > 0.5 implies that separation between groups is good and intergroup variation is significantly greater than intragroup variations.Non-metric multi-dimensional scaling (NMDS) plot of the gut bacterial profiling data indicates the tendency of dietary habit wise grouping of subjects Fig. a. The stOur analysis has revealed a noteworthy contrast in gut bacterial profiles of Indian and Chinese adults. It suggests that different dietary habits could be the major drivers towards distinction in gut microbiota. This led us to examine gut bacterial biomarkers of diet. LEfse [linear discriminant analysis (LDA) effect size] analysis, which couples statistical significance with biological consistency and effect size estimation, detected 15 bacterial clades (LDA score > 4) showing marked differences between Indian and Chinese adults Fig. a. The hiPrevotella, Megasphaera, Catenibacterium, Lactobacillus, Ruminococcus and species Prevotella copri, Lactobacillus ruminis can potentially serve as biomarkers of diet to distinguish Indian and Chinese adults.The discriminating features at higher taxonomic levels are unlikely to be an effective biomarker, as they incorporate a diversity of genera and species. In this regard, genera Bacteroidetes.The colours in the cladogram represent the branch of the phylogenetic tree more significantly represents a certain group. In this case, Indians are significantly represented by Firmicutes and Actinobacteria and underrepresented Bacteroides, of Indian and Chinese gut bacteria closely matches with the gut bacterial composition of eastern Russia and rural regions consuming starch-rich diet  ratio was 14:1 in Chinese adults and the ratio was 3.7:1 in the Indian adults  and fermented products containing Lactobacillus.In our study, Catenibacterium, Megasphaera, and Mitsuokella were also found to be enriched in Indian gut. Higher abundance of these three genera was noted in Egyptian children when compared to US children . In comparison to Indians, starch-degrading genera, namely, Ruminococcus, and Blautia, were more abundant in the stools of Chinese, feasibly due to the high preponderance of starch as a dietary polysaccharide in their diet. It concurs with De Filippis et al. ), while a positive correlation with Faecalibacterium Dialister, Lactobacillus (0.641–0.775). The correlation of Prevotella with these bacteria is in agreement with previously reported results .Additional file 3: Table S2. The summarized information of amplicon sequencing of 16 fecal DNA samples.Additional file 4: Table S3. Metastats analysis showed gut bacterial clades with significant variation between Indian and Chinese adults.Additional file 5: Table S4. Alpha diversity indices.Additional file 6: Figure S1. Between groups T test analysis. Figure S2. Between group variation of alpha diversity indices. Figure S3.Firmicutes to Bacteroidetes (F/B) ratio in Indian and Chinese adults. Figure S4. Relative abundance of Firmicutes and Actinobacteria.


Enterococcus faecium on the outcome of patients with BSI after orthotopic liver transplantation (OLT).The relevance of vancomycin resistance in enterococcal blood stream infections (BSI) is still controversial. Aim of this study was to outline the effect of vancomycin resistance of Enterococcus faecium (VSE) was compared based on data extraction from medical records. Multivariate regression analyses identified risk factors for mortality and unfavourable outcomes (defined as death or prolonged intensive care stay) after 30 and 90 days.The outcome of OLT recipients developing BSI with vancomycin-resistant (VRE) versus vancomycin-susceptible n = 39) and VSE- (n = 138) group after 30 (p = 0.44) or 90 days (p = 0.39). Comparable results occurred regarding unfavourable outcomes. Mean SOFANon-GCS score during the 7-day-period before BSI onset was the independent predictor for mortality at both timepoints . Timely appropriate antibiotic therapy, recent ICU stay and vancomycin resistance did not affect outcome after adjusting for confounders.Mortality was similar between VRE- (Enterococcus faecium bacteraemia after OLT. Only underlying severity of disease predicted poor outcome among this homogenous patient population.Vancomycin resistance did not influence outcome among patients with DRKS00013285).This study was registered at the German clinical trials register (DRKS-ID:  For decades, enterococci were considered to be harmless inhabitants of the human gastrointestinal tract, only rarely causing opportunistic infections in critically ill patients. Currently, they have been studied to be among leading pathogens of nosocomial infections, and thus they are a major international health burden –3. EnterEnterococcus faecium isolates proved to be vancomycin-resistant  and unfavourable outcomes [Odds Ratio (OR)], respectively. For all analyses, a two-sided E. faecium BSI in our study centre; 177 of them were adult liver transplant recipients and were included in the final analysis  were VSE cases. The baseline characteristics of the patients included are presented in Table During the 11-year observation period, we identified a total of 1250 patients with p = 0.05)]. However, the average number (mean) of interventions per patient to increase the arterial perfusion of the graft within 30 days before BSI was higher in VRE than in VSE patients. The proportion of BSI caused by more than one pathogen was low (17 of 177 cases) and similar in both groups (p = 0.29). See Additional file There was no difference between groups regarding median time from last OLT to first enterococcal BSI or meanSOFANon-GCS scores from day − 7 to day − 1 before BSI. During the week prior to BSI onset, VRE patients tended to spend more time on the ICU than VSE patients , followed by tigecycline given in 6 of 39 (15.4%) patients. Vancomycin was the most applied antibiotic in the VSE group [88/138 (63.8%)], followed by linezolid [31/138 (22.5%)]. Details are given in Additional file : Table S2. Adequate antibiotic treatment according to the susceptibility testing of the detected E. faecium isolate within 48 h post-BSI onset was established in 21 of 39 (53.9%) patients in the VRE group and 83 of 138 (60.1%) patients in the VSE group (p = 0.48). All patients who did receive antibiotic therapy during the study period were treated with an appropriate agent according to resistance testing. See Additional file p = 0.64). The number of performed interventions from BSI onset until day 30 did not differ significantly between the two study groups (p = 0.20); more details are presented in Additional file In the VRE and the VSE group, 6 of 39 16.2%) and 22 of 138 (17.3%) patients, respectively, were treated empirically with effective antibiotics before receipt of the microbiological result (6.2% and p = 0.44). See Fig. p = 0.39).Table Non-GCS score (from day − 7 to day − 1) was an independent and strong predictor for mortality either on day 30 or 90 post-bacteraemia onset. The relative increase of the mortality risk was 32.1%  14.1–53.1%) (day 30 post-BSI onset) and 17.9% (day 90 post-BSI onset), respectively, for each additional point in the SOFANon-GCS score. A similar finding was obtained for the combined endpoint, with an elevated risk for an unfavourable outcome at day 30 and 90 of 24.8 and 17.9%, respectively. As already shown in the univariate analysis, even after adjustment for confounders, vancomycin resistance was not identified as an independent risk factor for mortality or unfavourable outcome after 30  were considered by multivariate logistic regression analysis, we could not outline a difference of the mortality risk between the VRE and VSE groups. In contrast, the mean SOFAIn this multivariate retrospective analysis on the outcome of patients with VRE- or VSE-BSI after OLT, vancomycin resistance was not identified to be a risk factor for mortality and was not associated with an increased 30- or 90-day mortality. This lack of an effect was confirmed even after adjustment for various potential confounders. Likewise, there was no association with the combined endpoint of an unfavourable outcome, which refers to death or prolonged need for ICU treatment.p = 0.391).To focus on this group of patients is relevant because of their notoriously high risk for VRE and it allows outcome analysis in a more homogenous cohort than in several other investigations, which included patients with broadly varying underlying diseases –27. MortE. faecium and E. faecalis and even other enterococcal species. A German study [E. faecium compared to E. faecalis BSI even after adjusting for underlying disease and vancomycin resistance. Similarly, even after adjustment for effective antibiotic treatment, Hayakawa et al. [E. faecium compared to vancomycin-resistant E. faecalis strains. Prematunge et al. [E. faecium; however, their number of patients was very small and information about the time to adequate antibiotic therapy was lacking.To the best of our knowledge, three meta-analyses in this field evaluated the effect of VRE bacteraemia within the last two decades and attributed an increased risk of death to vancomycin resistance , 13, 32.an study  reporteda et al.  observede et al.  analysedThe meta-analysis of Prematunge et al. , who excNon-GCS Scores from day − 7 until the day before BSI onset were collected. Multivariate analysis confirmed the overwhelming impact of the acute state of illness on the patients‘outcome. Similarly, Cheah et al. [Most studies, that examined the underlying severity of illness in this field, relied on scores on the day of BSI onset. This approach is problematic because values assessed on the first day of bacteraemia might already reflect the effects of the BSI rather than the patients’ acute state of illness independent from and prior to bacteraemia. To circumvent this problem in our study, SOFAh et al. , identifh et al. . This waStaphylococcus aureus [Time to the application of an effective antimicrobial substance is crucial for successful BSI treatment. Recent analyses of enterococcal blood stream infections showed worse outcome when adequate antibiotic treatment was delayed , 43, 44;s aureus .E. faecium BSI onset, was associated with death and an unfavourable outcome. Given these results, one could put contact isolation policies in question. The body of evidence is steadily increasing, that –provided the compliance with standard hygiene and routine cleaning procedures is high- isolation measures due to VRE can be suspended without harm and even reduce other non-infectious adverse events [After carefully adjusting for confounding factors, our results suggest that not vancomycin resistance itself, but the severity of illness before e events , 48. Inte events , 50.Enterococcus faecium isolates was nearly the same in the VRE- and the VSE-group . Nevertheless, vancomycin-resistance is a main-driver of linezolid use contributing to selection pressure in favour of linezolid-resistant isolates. Therefore, another important part of preventive strategies are antibiotic stewardship programs.The proportion of linezolid resistant E. faecium strains. Researchers who focus on the influence of different genotypes state that the VanB genotype is associated with increased length of stay compared to the VanA genotype [This study has several limitations. First, it was a retrospective single-centre study. Therefore, the generalisability of our findings to other settings or patient cohorts must be considered cautiously. Second, because breakpoints for daptomycin susceptibility testing of enterococci are not provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the clinical isolates in this study were not routinely tested. Therefore, we have no data on potential therapeutic alternatives beyond linezolid to treat patients with VRE bacteraemia. Recent studies and meta-analyses that compared daptomycin and linezolid could not provide a definitive answer to this question, but some studies tend to favour daptomycin –53. Thirgenotype . This toNon-GCS scores before BSI onset as the only strong and independent predictor of mortality. Each additional point increased the likelihood of death after 30 days by 32.1% (95% CI 14.1–53.1%) and also the potential for an unfavourable outcome by day 30 and 90 post-BSI. In contrast, in an era of broadly available effective treatment, vancomycin resistance had no impact on mortality or a poor outcome in patients with E. faecium BSI after OLT.In summary, our multivariate analysis identified the mean-SOFAAdditional file 1: Table S1. Polymicrobial blood stream infections and identified Co-pathogens in study subjects. Table S2. Antibiotic therapy used to treat the study subjects Table S3. Interventions from blood stream infection (BSI) until day 30 Table S4. Risk factors for death within 90 days after Enterococcus faecium bacteraemia (Cox regression). Table S5. Factors associated with unfavourable outcomes 90 days after Enterococcus faecium bacteraemia (logistic regression). Table S6. Source of infection and related outcome in patients with vancomycin-resistant Enterococcus faecium (VRE) Table S7. Source of infection and related outcome in patients with vancomycin-susceptible E. faecium (VSE) bacteraemia.


Limnonectes fujianensis Brevinvin (LFB) has been identified in the skin secretion from the Fujian large headed frog, Limnonectes fujianensis. The cDNA sequence was cloned from a skin secretion library and the predicted mature peptide was identified through MS/MS fragmentation sequencing of reverse phase HPLC fractions on the same sample. LFB was predicted to be an amphipathic, hydrophobic, alpha helical, and beta turn peptide that inserts into a lipid bilayer in order to kill the cells. In antimicrobial assays, a synthetic replicate of this novel antimicrobial peptide demonstrated significant activity against the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the yeast, Candida albicans. This novel peptide was highly potent (MIC 4.88 uM) against Gram-negative bacterium, and also has the ability to inhibit the growth of human cancer cell lines with IC50 values ranging from 18.9 μM down to 2.0 μM. These findings help to enrich our understanding of Brevinin-like peptides. Moreover, the data presented here validate frog secretion as a source of potential novel antimicrobial peptides, that also exhibit anti-tumor properties, that could be useful for the treatment of cancer.Amphibians are a natural source of abundant antimicrobial peptides and thus have been widely investigated for isolation of such biomolecules. Many new antimicrobial peptide families have been discovered from amphibians. In this study, a novel antimicrobial peptide named  In nature, frog’s skin has an important function to act as a crucial chemical and physical barrier to microbes. Follow any injury and/or sympathetic nervous stimulation, within the dermal granular glands of the frog’s skin antimicrobial peptides (AMPs) are synthesized, stored and secreted ,2,3; theXenopus laevis  and [U-251 MG cell line human (09063001-1VL)] were purchased from the American Type Culture Collection (ATCC) USA and Sigma-Aldrich UK respectively. The cells were cultured using Dulbecco’s Modified Eagle’s Medium (DMEM) medium or RPMI-1640 medium (Invitrogen) depending on different cell lines. In general, the medium was supplemented with 0.1% (w/v) gentamicin and 10% (v/v) fetal bovine serum (FBS) . The cells were then seeded into 150 cm2 tissue culture flasks  for culturing.Human cancer cell lines [NCI-H460 [H460]  was added in to experiment groups, respectively. The fresh medium contained equal amounts of PBS, used as a negative control. At least three replicates were included in each group. Then after 24 h of treatment, we measured the cell viability according to the procedure performed in our previously study . In the Cells were treated with LFB ranging from 100 μM to 1 μM for 24 h. LDH Cytotoxicity Assay Kit  was employed to detect the cytotoxicity of LFB according to the manufacturer’s instructions. For details, please kindly refer to the previous study .Cancer cells were treated with LFB at 100 μM to 1 μM for 2 h. Then, the Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit  was employed to detect apoptosis according to the manufacturer’s instructions. Flow cytometer BD Accuri™ C6 was used for the analysis and blank control group was treated with equal volume of PBS.v/v) suspension of red blood cells was prepared from defibrinated horse blood . Red blood cell suspension samples (200 μL) were incubated with a range of LFB concentrations (1–512 mg/L) for 60 min at 37 °C. Lysis of red cells was assessed by measurement of optical density at λ = 550 nm using an ELISA plate reader . Negative controls employed consisted of a 2% (v/v) red cell suspension and sodium PBS in equal volumes; and positive controls consisted of a 2% (v/v) red cell suspension and an equal volume of PBS containing 2% (v/v) of the non-ionic detergent, Triton X-100 .A 2% .Statistical analysis was performed using Version 6.0 software of GraphPad Prism. The L. fujianensis was subjected to RP-HPLC and the resultant chromatogram is shown in The lyophilized skin secretion of A single cDNA encoding the precursor of the novel peptide was consistently and repeatedly cloned from the library using the protocol described. The nucleotide and translated open reading frame amino acid sequences and the domain architectures were shown in The helical wheel plots and secondary structures of LFB were predicted with online analysis tools. It also had a hydrophobic face consisting of V, L, V, V, V, L, F, which were illustrated in S. aureus at concentrations at and above 16 mg/L, against the Gram-negative bacterium E. coli at concentrations at or above 32 mg/L and against the potentially-pathogenic yeast, C. albicans, at concentrations at or above 64 mg/L  and the ICCancer cell HCT116 was treated with LFB and incubated in the IncuCyte live cell imaging system. As shown in −/PI−), early apoptosis (Annexin V+/PI−), late apoptosis (Annexin V−/PI+) and dead (Annexin V+/PI+). From the results in Annexin V and Propidium Iodide (PI) staining along with flow cytometer analysis were employed in order to determine if LFB inhibits cancer cells via cell apoptosis or by other modes of actions. The readouts from flow cytometry divided the cells into four groups: normal , this structural modification could lead to a considerable reduction of its hemolytic activity without loss of any antibacterial activity [According to the studies in the last two decades, there are many different antibacterial mechanisms have been reported. However, the most well recorded one is classified as a non-specific interaction with the membrane with different models, such as the Barrel–Stave model, Carpet model, and Toroidal model . In one activity ,43,44,45activity .L. fujianensis for the first time. LFB demonstrated broad spectrum and highly potent antimicrobial activity against Gram +ve bacteria, Gram −ve bacteria, and fungi. Furthermore, LFB also showed the ability to inhibit the growth of several types of cancer cell lines. With the purpose of developing LFB as a new antimicrobial prototype drug, structural-functional modification should be seriously taken into consideration for reducing its hemolytic activity. In general, the current study suggests that LFB is a promising drug candidate for the development of novel antibiotic class, and these findings may pave a new insight into natural antimicrobial prototype drug design.In summary, a newly reported bioactive peptide LFB was identified from the frog skin secretion of


STRC, the causal gene for DFNB16. Various techniques are used clinically for CNV detection, and droplet digital PCR (ddPCR) provides highly precise absolute quantification of DNA copy number. We aimed to validate the feasibility and efficiency of ddPCR in combination with long-range PCR (LR-PCR) in identifying CNVs and mutations in STRC. Additionally, we determined the frequency of CNVs and mutations in STRC in Japanese patients with mild-to-moderate hearing loss. We evaluated 84 unrelated Japanese patients with mild-to-moderate bilateral idiopathic or autosomal recessive nonsyndromic sensorineural hearing loss. The ratio of STRC copy number to the copy number of the internal control RPP30 ranged from 0.949 to 1.009 (0.989 ± 0.017) in 77 patients; it ranged from 0.484 to 0.538 (0.509 ± 0.024) in five patients and was 0.000 in two patients, indicating heterozygous and homozygous deletions, respectively. The copy number deletion prevalence rates were 7.7% and 0.9% in the patients and healthy controls, respectively. In combination with LR-PCR, ddPCR revealed that at least three patients (3.6%) had STRC-related hearing loss. Detecting STRC CNVs by ddPCR was rapid, precise, and cost-effective and facilitated the identification of STRC CNVs.Copy number variations (CNVs) are commonly reported in  STRC, a gene required for function of the sound-detecting hairs in the inner ear, can cause hearing loss. Compared to detecting mutations, counting gene copies had been very challenging. Taku Ito at Tokyo Medical and Dental University, Japan, and colleagues applied a new technology that divides samples into thousands of droplets, then measures the genetic information in each droplet, allowing precise gene copy counting. Studying samples from nearly 100 Japanese patients with hearing loss, they were able to determine that hearing loss in several patients was caused by deletion of one or both copies of STRC. This method will speed diagnosis of STRC-related hearing loss, and has potential for application to other disorders.Detecting a common genetic cause of hearing loss is now easier, thanks to a new method. Loss of one or both copies of  Until recently, real-time quantitative PCR (qPCR) assays and microarray hybridization were the two main methods used to determine CNVs in the genome. Conversely, droplet digital PCR (ddPCR) allows for high-precision absolute quantification of DNA copy number. During ddPCR, each sample is partitioned into several tens of thousands of nanoliter-sized droplets prior to amplification. PCR amplification proceeds to the reaction endpoint, which is followed by the determination of whether every droplet in a well is positive or negative for both the target and the reference. The fluorescence intensity in each droplet is measured, and those above the threshold are counted as positive, thus providing a digital yes/no result. ddPCR overcomes several limitations of qPCR and microarray techniques for CNV analysis.Copy number variations (CNVs), defined as the presence of variable copy numbers of specific DNA segments, can be associated with a wide range of disorders. The most recent study has estimated that CNVs constitute 4.8–9.5% of the human genome and comprise a greater fraction of nucleotides than single nucleotide variants (SNVs). Specifically, in bilateral sensorineural hearing loss (SNHL), CNVs were identified in ~20% of all deafness-causing genes and found in ~15% of patients undergoing genetic testingSTRC (MIM: 606440) encodes the extracellular structural protein stereocilin, which is necessary for proper hair cell function and is the causal gene for DFNB165. Mutations in STRC, commonly reported as CNVs, were described mainly in association with bilateral mild-to-moderate hearing loss7. With ~1–1.6% of individuals in the general population carrying a heterozygous STRC deletion10, the estimated incidence of STRC-associated hearing loss is 1 in 16,0009. Numerous studies suggest that DFNB16 constitutes a significant proportion of the genetically heterogeneous SNHL, and STRC is increasingly recognized as one of the most significant contributors to autosomal recessive nonsyndromic SNHL.STRC contains 29 exons encompassing approximately 19 kb and is tandemly duplicated with the coding sequence of the second copy, the pseudo-STRC (pSTRC) gene4. The first half of the functional STRC copy is 100% identical to pSTRC. Over the entire locus, STRC and pSTRC are 98.9% homologous across exons and introns7. Therefore, pathogenic SNVs identified by targeted sequences across the entire STRC coding region should always be differentiated from those derived from the amplification products of pSTRC. Conventional direct PCR and short-read massively parallel sequencing (MPS) are unable to provide consistently reliable data on STRC. In fact, some variants previously reported as pathogenic likely reflect codetection with pseudogenes rather than causal mutations in the STRC coding sequence4. In this study, we present rapid, cost-effective, and precise methods for the diagnosis of STRC-related hearing loss by ddPCR and long-range PCR (LR-PCR).11. None of the patients were from consanguineous families. A total of 107 Japanese individuals with normal hearing were included as controls. Based on Sanger sequencing results, we previously excluded pathogenic mutations in the coding sequences of GJB2 and Mt-155511. This study was approved by the research ethics committee of Tokyo Medical and Dental University.We analyzed archival DNA samples from 84 unrelated Japanese patients  with mild-to-moderate SNHL who visited the Department of Otolaryngology at Tokyo Medical and Dental University Medical Hospital between April 2000 and September 2010 and who were diagnosed with idiopathic or autosomal recessive nonsyndromic bilateral SNHLTM ddPCR system . Briefly, the master mix for ddPCR included 1 × ddPCR supermix for probes , 1.0 μM primer, and 0.25 μM probe , together with 20 ng sample DNA. Primers and a probe were designed for intron 23 of STRC . Droplet generation oil for probes (Bio-Rad) was then added to the cartridge, which was placed into the QX200 Droplet GeneratorTM (Bio-Rad). After droplet generation, the droplets were carefully transferred to a twin-tec semi-skirted 96-well PCR plate , and the plate was sealed for 5 s at 180 °C using a PX1 PCR plate sealer (Bio-Rad). Subsequent amplification was performed in a C1000 Touch thermal cycler (Bio-Rad) with a ramp rate of 2 °C/sec. First, the enzyme was activated at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s and 55 °C for 1 min. The enzyme was deactivated at 98 °C (10 min), and the reaction was held at 4 °C. Droplets were read using a QX200 droplet reader TM (Bio-Rad), and the ddPCR data were analyzed using the Quantasoft software program version 1.7.4 (Bio-Rad). Manual thresholds were applied for both STRC and RPP30.DNA integrity and quality were verified by a NanoDrop spectrophotometer and agarose gel electrophoresis. The ratio of absorbances at 260 nm and 280 nm for each DNA sample was ≥1.8, and all samples were diluted at a concentration of 10 ng/μl by pure water. CNVs were detected by ddPCR with standard protocols using the QX100STRC but not pSTRC were obtained from the literature12. LR-PCR was performed with the Qiagen Long-Range PCR Kit  using cycling profiles. Amplification products were loaded and run on a 0.6% agarose gel, and the fragments of appropriate DNA sizes were extracted from the gel using NucleoSpin® gel and a PCR clean-up kit . Nested PCRs and sequencing continued after LR-PCR products for a portion of STRC exon 16, which were used for verification of amplification from only the STRC sequence. PCR products were purified using a QIAquick PCR Purification Kit (Qiagen) and were directly sequenced using the Applied Biosystems Prism BigDye Terminator Cycle Sequencing Ready Kit and an ABI Prism 3130xl Genetic Analyzer.To exclude pseudogene sequences, two LR-PCR products were generated for the subsequent nested PCR. Sequences of primers that specifically amplify STRC CNVs in 84 patients with SNHL and 107 healthy controls. Among the patients with SNHL, the STRC/RPP30 copy number ratio ranged from 0.949 to 1.009 (0.989 ± 0.017) in 77 patients and from 0.484 to 0.538 (0.509 ± 0.024) in five patients; the ratio was 0.000 in two patients. The ratios of 0.509 and 0.000 likely indicated heterozygous and homozygous deletions, respectively .First, we performed ddPCR to identify STRC sequencing in five patients carrying the heterozygous STRC deletion. LR-PCR-based sequencing successfully detected SNVs without pSTRC contamination, which was observed in conventional direct sequencing  were considered to exhibit hearing loss associated with STRC dysfunction. All patients were recognized as sporadic cases.We evaluated the CNVs and SNVs in Japanese patients with mild-to-moderate SNHL using ddPCR in combination with LR-PCR. The prevalence of copy number deletion was 7.7% among the patients. In addition to the homozygous deletion of 14, and 4.8%–9.5% of the human genome can be classified as CNVs15. Among these, large repeats, such as whole gene repeats, are generally detected and identified by fluorescent in situ hybridization, array comparative genomic hybridization, single nucleotide polymorphism or oligo arrays, and paired-end mapping. Recently, MPS has also been used to detect CNVs with high genomic resolution. Indeed, MPS is used in the clinic and can efficiently detect CNVs in most Japanese patients with hearing loss10. These techniques are suitable for searching the whole genome at once but require a considerable amount of work, time and cost. Conversely, ddPCR is insufficient for global identification; however, it provides rapid and easy completion at less than $10 per sample, and the total assay time is 2.5 h, illustrating its efficacy in determining the copy number of specific DNA areas16. Additionally, the results are highly reproducible, with minimal variability among DNA samples that does not require strict sample preparation and that is accurate and acceptable for homogenous and heterogeneous statuses. Considering the relatively high prevalence of STRC deletion in the Japanese population and challenges in diagnosing inheritable causes of hearing loss, screening with ddPCR is a potential approach before evaluation of patients by MPS.Recent studies indicate that approximately two-thirds of the entire human genome is composed of repeatsSTRC is garnering increasing attention. Our results reconfirm that CNVs in STRC are a common phenomenon in mild-to-moderate SNHL, found in more than 7% of the patients with a carrier rate of less than 1% in healthy Japanese controls, which is consistent with previous reports10. However, no mutations in the exons of the translated regions of the opposite allele were detected in four of the five patients with heterozygous gene deletion. It is possible that these patients might harbor mutations in genes other than STRC, such as GJB2 and Mt-1555. Alternatively, the significantly high prevalence of CNVs in the patients compared to the healthy controls suggests undetectable mutations that might be present in the intronic, promoter, or enhancer regions of STRC. Further studies are warranted to verify this hypothesis.As a causal gene for mild-to-moderate idiopathic SNHL, STRC coding sequence have been reported thus far. However, direct PCR or MPS cannot distinguish whether such variants originate from STRC or pSTRC. Indeed, the c.4057 C > T, Q1353X variant is coincident with the reference sequence for exon 20 from the pSTRC17. Therefore, novel nonsense variants that are detected in patients with hearing loss should be verified to ensure that they are a pathogenic variant in the STRC coding sequence and not a pseudogene contamination. We recommend LR-PCR-based sequencing as an excellent technique that might be necessary for comprehensive screening for STRC-related hearing loss.Numerous pathogenic SNVs across the entire STRC copy number deletion was detected in approximately 1% of the Japanese healthy controls, whereas STRC was the causal gene in more than 3% of patients with mild-to-moderate hearing loss in Japan. The detection of CNVs using ddPCR is a rapid, precise, and cost-effective method for relatively prevalent deafness genes, such as STRC. The combination of ddPCR and LR-PCR-based sequencing is a reliable approach to detect patients with STRC-associated hearing loss.In conclusion,


Silver nanoparticles (AgNPs) have attracted great attention due to their outstanding electrical, optical, magnetic, catalytic, and antimicrobial properties. However, there is a need for alternative production methods that use less toxic precursors and reduce their undesirable by-products. Phyto-extracts from the leaves of olive and rosemary plants can be used as reducing agents and (in conjunction with Tollens’ reagent) can even enhance AgNP antimicrobial activity.Escherichia coli and Salmonella enterica) and Gram-positive bacteria (Staphylococcus aureus), by using both the Kirby–Bauer and broth microdilution methods on Mueller–Hinton (MH) agar plates.Conditions for the proposed hybrid synthesis method were optimized for olive leaf extracts (OLEs) and rosemary leaf extracts (RLEs). The resultant AgNPs were characterized using UV–visible spectroscopy, an environmental scanning electron microscope, and Dynamic Light Scattering analysis. An atomic absorption spectrophotometer was used to measure AgNP concentration. Fourier transform infrared spectroscopy (FTIR) was used to determine the specific functional groups responsible for the reduction of both silver nitrate and capping agents in the leaf extract. Additionally, the antimicrobial properties of the synthesized AgNPs were assessed against Gram-negative bacteria (Escherichia coli and Staphylococcus aureus with minimum inhibitory concentrations (MIC) of 9.38 and 4.69 μl/ml for OLE-AgNPs and RLE-AgNPs, respectively. The MIC for Salmonella enterica were 18.75 μl/ml for both OLE-AgNPs and RLE-AgNPs. Furthermore, our results indicated that the RLE-AgNPs exhibited a stronger antibacterial effect than OLE-AgNPs against different bacteria species. These results contribute to the body of knowledge on nanoparticle production using plant-mediated synthesis and performance. They also offer insights into the potential for scaling up this production process for commercial implementation.A simple, feasible, and rapid method has been successfully developed for silver nanoparticle synthesis by reducing Tollens’ reagent using leaf extracts from olive and rosemary plants (widely available in Jordan). Scanning electron microscopy images showed that the method produces AgNPs with a spherical shape and average core sizes of 45 ± 2 and 38 ± 3 nm for OLE and RLE, respectively. A negative zeta potential (ζ) of −43.15 ± 3.65 mV for OLE-AgNPs and −33.65 ± 2.88mV for RLE-AgNPs proved the stability of silver nanoparticles. FTIR spectra for AgNPs and leaf extracts indicated that the compounds present in the leaf extracts play an important role in the coating/capping of synthesized nanoparticles. The manufactured AgNPs exhibited an antibacterial effect against  Silver nanoparticles (AgNPs) have attracted great attention due to their outstanding electrical, optical, magnetic, catalytic, and antimicrobial properties . In 2015Silver nanoparticles are produced using a wide variety of physical and chemical methods. Most physical methods require high energy consumption, a large space, and/or lengthy time periods. This is due to the need to achieve thermal stability while raising the environmental temperature around the source material in the tube furnace to ensure stable operating temperatures. On the other hand, common chemical methods utilize hazardous reducing chemicals, such as sodium borohydride, hydrazine, or hydrogen, that can have adverse effects on the environment and human health .Chemical methods involve the reduction of silver salts with a reductant such as citrate acid and a solvent like sodium borohydride. Additionally, a stabilizer is needed to prevent agglomeration of the nanoparticles . Since tGreen synthesis encompasses the use of less toxic precursors, a low number of reagents, and benign solvents such as water at close to room temperature—with the expectation of fewer byproducts and waste streams as compared with conventional processes . Some prPlant-mediated synthesis is one of the most common approaches used in green synthesis: extracts from various plant components, such as leaves and roots, are used as reducing and stabilizing agents . This prCinnamon zeylanicum (Mentha piperita (Lamiaceae)  , olive (miaceae) , maple (miaceae) , Eupreno procera , and Alo procera . The mos procera .3)2]+ as a source of Ag+ and aldehyde as a reducing agent, produces AgNPs with a controlled size in a one-step process , silver hydrosols (20–50 nm), and AgNPs of different shapes (3)2]+ with glucose, galactose, maltose, and lactose (Tollens’ synthesis method using Tollens’ reagent [Ag(NH process . On the t shapes . In this lactose . To incr lactose .3)2+ (aq) into AgNPs. OLE and RLE have been used effectively to reduce silver salts directly into nanoparticles that display antimicrobial properties (This work presents the development of a hybrid synthesis method in which rosemary leaf extract (RLE) and olive leaf extract (OLE) were used instead of saccharides to reduce the Tollens’ reagent Ag(NHoperties . In thisoperties .Olea europaea) and rosemary  were selected because of their ubiquity, economic efficiency, and well-documented nutritional and medicinal applications , polyvinylpyrrolidone , nitric acid , nutrient agar, nutrient broth, ammonium hydroxide aqueous solution , and sodium hydroxide pellets  were used without any further purification. Deionized (DI) water was used for all experiments. Lastly, all glassware was periodically washed with diluted nitric acid (25%) and then dried in a hot-air oven overnight at 40 °C.Staphylococcus aureus (ATCC 25923) and two Gram-negative strains of Escherichia coli (ATCC 12900) and Salmonella enterica (CIP 104220).Antibacterial tests were then performed using three representative pathogenic species obtained from Princess Haya Biotechnology Center, Jordan University of Science and Technology (JUST) : one Gram-positive strain of Leaves of olive and rosemary were collected in June 2014 from the campus of the JUST in Irbid, Jordan. The collected leaves were transported to the laboratory and left to dry at room temperature (25–30 °C) for 10 days, following the procedure described by 3)2+ (aq) into AgNPs.Silver nanoparticles were prepared using the well-known Tollens’ method. OLEs and RLEs, rather than saccharides, were used as reducing agents  to reduc3 (10−3 M), sodium hydroxide (1.25 × 10−2 M), and PVP (8.4 × 10−5 M) were prepared. To prepare AgNP dispersions, a tin-foiled 250 ml Erlenmeyer flask container was used as a reaction vessel and placed on a stirrer plate while a syringe pump apparatus was fitted to feed the vessel with the reducing agent (PLEs). Initially, 12.5 ml AgNO3 stock solution was added to the vessel at a stirring speed of 600–700 rpm. Then 38.5 μl of ammonia solution was added dropwise, followed by 12.5 ml of PVP stock solution. Finally, 25 ml of 1:1 PLE:NaOH mixture was slowly added to the vessel at a rate of 75 ml/h using an automatic dosing syringe through a plastic slip. The synthesized AgNP suspension was then ultrafiltrated using a 10 kDa nominal molecular weight cut-off membrane via ultrafiltration stirred cell  for concentration, purification, and pH adjustment. Any nitrate, PVP, Ag+, or PLE not bounded to the nanoparticles was therefore removed from the solution. The ultrafiltration process consumed up to 600 ml of DI water for each sample, in order to achieve a final purified concentrate of 50 ml in volume. The AgNP nanosuspension was kept in tin-foiled covered containers at 4 °C for a period of 6 months for further characterization and bactericidal experiments.In addition to the previously prepared extracts, stock solutions of AgNOd), size distribution, polydispersity index (PDI) and zeta potential (ζ) were determined by dynamic light scattering analysis (DLS) using a Malvern Zetasizer . Measurements were determined three times for each sample, and uncertainties are given as standard deviations. Raw data were subsequently correlated to the mean hydrodynamic size by cumulants analysis (Z-average mean), according to ISO 22412:2017 (−1. Fourier transform infrared spectroscopy (FTIR) measurements were carried out to identify representative functional groups of possible molecules on the surface of the nanoparticles.Optical properties of the prepared AgNP nanosuspension were determined using a UV–Vis spectrophotometer . The shape and size of the AgNPs produced were identified using an environmental scanning electron microscope (ESEM) Quanta 450 FEG-USA/EEU that operated at an electron gun power of 30 kV. In order to obtain the ESEM images, the synthesized AgNP dispersions were sonicated for 10 min in an ultrasonic bath (100 watts) that generates ultrasonic waves at a 35 kHz frequency . Then the sonicated dispersions were diluted  and re-sonicated for 30 min. After that, a drop of this dilute nanosuspension was placed on an ESEM pin stub specimen mount for measurement. Average silver core diameter was calculated by averaging 50 particles from the ESEM image. Average hydrodynamic diameter . The nanosuspensions were centrifuged at a maximum relative centrifugal force of 4,185×g  for 15min . The centrifuge separates the AgNPs at the bottom of the centrifuge tube, leaving the remaining Ag+ in the supernatant. After proper dilution, the obtained supernatants were analyzed. The difference in Ag+ concentration between the supernatant of the AgNP suspension and the standard AgNO3 stock solution thus represents the amount of silver transformed to AgNPs  and minimum bactericidal concentration (MBC), whereas the Kirby–Bauer method was used to assess the sensitivity of bacteria to nanoparticles.6 CFU/ml. After well-mixing, the inoculated 96-well microtitration plate was incubated at 37 °C for 24 h. Both the MIC and MBC were then detected during this incubation period. Mueller–Hinton broth was used as a negative control.A stock of the AgNP suspension was prepared at a concentration of 100 mg/l and, as recommended by Clinical and Laboratory Standards Institute (CLSI) guidelines , was tes5 CFU/ml were spread out on MH agar plates. The cultured MH agar on each plate was perforated with holes (wells) using a sterilized glass tube and labeled appropriately. Each well was filled with 50 μl of either OLE-AgNPs or RLE-AgNPs, and MH agar plates were incubated at 37 °C for 24 h. Finally, the zones of inhibition (ZI) were measured and reported. In all tests, silver nitrate was used as a positive control and a blank agar plate was incubated to detect any contamination that might have occurred during testing.The Kirby–Bauer method  was usedOptical spectra shown in + is consumed for the formation of nuclei that grow into controlled nanoparticles  for OLE-AgNPs and RLE-AgNPs were 53% and 48%, respectively.At favorable conditions, the nanosuspensions were analyzed for AgNP concentration by AAS. The measured concentrations for OLE-AgNPs and RLE-AgNPs were approximately 50 and 45 mg/l, respectively. The percentages of AgZ-average size) were 70.27 nm for OLE-AgNPs and 64.16 nm for RLE-AgNPs. The PDI was found to be 0.295 and 0.424 for OLE-AgNPS and RLE-AgNPs, which are both less than 0.7 indicating monodispersed particles  of −43.15 ± 3.65 mV for OLE-AgNPS and −33.65 ± 2.88 mV for RLE-AgNPs that indicate a high colloidal stability of the particles . This ne−1) and the fingerprint region . The intense absorption peaks for both the extracts and the nanoparticles were used to evaluate the surface of the AgNPs produced. Because of the adsorption and modifications occurring at the surface, the functional group region is the most informative. Therefore, our analysis focused on that region for both the extracts and the synthesized AgNPs. The IR spectra of OLE and RLE show only minor differences . −1 is due to C–H stretching and indicated the presence of alkanes, while the peaks at  and , and (690) cm−1 corresponded to C–H stretching, C=C bond aromatic, and C–H bending respectively, and implied the presence of aromatic compounds. The peak at 1,687 cm−1 also could correspond to amide C=O stretching. The observed O–H and C=O modes for the OH and C=O groups might be attributed to oleuropein, apigenin-7-glucoside and/or luteolin-7-glucoside which, as suggested by The spectra for both RLE-AgNPs and OLE-AgNPs  were alsTo explore the antibacterial activity of OLE- and RLE-AgNPs, both the broth microdilution method (as recommended by CLSI protocol) and the Kirby–Bauer method were used. The MIC and MBC of OLE- and RLE-AgNP values against selected bacteria are listed in Staphylococcus aureus and Escherichia coli bacteria were 9.38 μl/ml, with MBC of 12.5 μl/ml. The MIC and MBC against Salmonella were 18.75 and 25 μl/ml, respectively. On the other hand, the MIC results for RLE-AgNPs against Staphylococcus aureus and Escherichia coli bacteria were 4.69 μl/ml, with MBC of 6.25 μl/ml; against Salmonella the MIC was 18.75 μl/ml and MBC of 25 μl/ml.Escherichia coli and Salmonella) and gram-positive (Staphylococcus aureus) bacteria, as shown in 3. The PLEs had a negligible inhibitory effect, most likely due to the low concentrations used  was well-known for its antimicrobial properties even during ancient times (0 (AgNPs) slowly releases silver ions via oxidation under aerobic conditions .These results offer insights into the potential of scaling up the production of AgNPs using leaf extracts from olive and rosemary plants for commercial implementation.The antimicrobial mechanisms of AgNPs are still not completely understood. One mechanism proposed is that AgNPs are able to interact with the bacterial cell wall, alter its properties via decaying lipopolysaccharide molecules, and form “pits” that increase wall permeability . In addiIn conclusion, AgNPs were successfully manufactured through the reduction of Tollens’ reagent in conjunction with OLE and RLE. Rapid formation time of AgNPs was achieved in approximately 3 h. The synthesis conditions such as extract concentration, temperature, and pH highly affected the synthesis. We were able to optimize the synthesis to obtain a smaller AgNP core size, an approximately spherical shape, and stability. The functional groups present in the RLE-AgNPs and OLE-AgNPs played a major role in the production and stability of AgNPs.3, and better or equal to other reported biosynthesis approaches. Finally, further investigations are recommended to analyze the antimicrobial mechanisms and to explore the potential of scaling up the proposed methodology.The results showed that the synthesized AgNPs provided inhibition properties comparable to the control solution of AgNO10.7717/peerj.6413/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.6413/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.6413/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.6413/supp-4Supplemental Information 4Click here for additional data file.


Echinococcus multilocularis larvae in the liver. It is a chronic and potentially lethal parasitic disease. Early stage diagnosis for this disease is currently not available due to its long asymptomatic incubation period. In this study, a proton nuclear magnetic resonance (1H NMR)-based metabolomics approach was applied in conjunction with multivariate statistical analysis to investigate the altered metabolic profiles in blood serum and urine samples obtained from HAE patients. The aim of the study was to identify the metabolic signatures associated with HAE.Hepatic alveolar echinococcosis (HAE) is caused by the growth of P < 0.001) in the blood serum obtained from the HAE patients than it was in the healthy patient group.A total of 21 distinct metabolic differences between HAE patients and healthy individuals were identified, and they are associated with perturbations in amino acid metabolism, energy metabolism, glyoxylate and dicarboxylate metabolism. Furthermore, the present results showed that the Fischer ratio, which is the molar ratio of branched-chain amino acids to aromatic amino acids, was significantly lower  contains supplementary material, which is available to authorized users. Echinococcus n ~ acquisition) was used to acquire spectra of blood serum samples with an echo time (τ) of 250 μs and a free relaxation duration (2nτ) of 100 ms. For urine samples, nuclear overhauser effect spectroscopy  was implemented with a 2 s water suppression and mixing time (tm) of 120 ms. Free induction decays (FIDs) were recorded with 64 scans at a spectral width of 10 kHz. The FIDs were zero-padding to 32 k data points prior to fast Fourier transformation.1H NMR spectra  was performed using MestReNova v.8.1.2 software . For peak alignment, the TSP signal was set as δ 0.00 for urine samples, and left split of the doublet of lactate signals was set as δ 1.336 for serum samples. Residual water signals , urea resonances (δ 5.70–6.40) and peak-free regions were selectively excluded for further analyses. The remaining spectra over the ranges of δ 0.8–8.5 for blood serum and δ 0.8–9.5 for urine were segmented into bucketed data using self-adaptive integration [1H NMR spectra were assigned based on previously published studies [Data pre-processing for the acquired egration , and theegration  method t studies , 29, the studies  and the  studies . The relR2  and Q2 (model predictability) were applied to validate the constructed models [P-value provided another venue of further model validation. The variable importance projection (VIP) and absolute correlation coefficient (|r|) calculated based on the OPLS-DA analysis were used as parameters for differential selection of metabolites. The VIP parameter, which was denoted as a unitless number, delineates the contribution of each predictor variable to the model and presents the influence of each predictor on the response variables. A larger VIP corresponds to a greater discriminatory power for the metabolites. In addition, metabolite sets were also analysed using Student’s t-test, which constitutes a simple statistical analysis to determine statistically significant metabolic variations in univariate analysis with the transformed P-value [Multivariate analyses of the pre-processed data were performed using SIMCA v.14.1 software . The data were examined by the non-supervised principal components analysis  and orthogonal partial least squares-discrimination analysis . A seven-fold cross-validation  and permutation test (200 permutations) were performed and the obtained values of d models , 33. An  P-value . For a p10  on the vertical axis and log2 (fold change) on the horizontal axis with circles of different sizes and colours for displaying VIP and |r| values, respectively.In the present study, the results of multivariate statistical analysis were visualized and integrated with volcano plots to identify metabolites with significant difference between the HAE and control groups , 36. In post-hoc power analysis with specified significance level α, sample size, and effect size was executed with the online statistics software G*power v.3.1 (http://www.gpower.hhu.de/) [http://www.metaboanalyst.ca/) [Additionally, a hhu.de/) , 38 to dyst.ca/) . The areP > 0.05). All HAE patients were classified into six categories according to the PNM classification by WHO/IWGE, as stated in methods. Among the participants, patients in the P3N1M1 and P4N1M1 classes showed lung metastasis.The metadata of the participants  are summarized in Table 1H NMR spectra of biofluids collected from the HAE patients and healthy volunteers are shown in Fig. Typical P-values by CV-ANOVA were also used to assess the statistical significance of metabolic differences between the control and the HAE groups. Moreover, the OPLS-DA models were found to be robust with good explained variations R2Y and predictive powers Q2 following permutation tests (200 permutations).To identify HAE-induced metabolic changes, we performed multivariate analyses on the processed NMR data. First, the data were analysed by principal component analysis (PCA), of which the resulting score plots are shown in Fig. r|) > 0.5, − log10  > 2 (i.e. P < 0.01), and absolute log2 (fold change) > 0.20. Generally, identified metabolites with significant changes  by multivariate statistical analyses tended to locate at the upper-left or upper-right zones of the enhanced volcano plot with larger circle shapes and warmer colours, as marked with abbreviations in Fig. To identify metabolic markers that differentiate the HAE patients from the healthy controls, we next presented the data using a four-dimensional enhanced volcano plot which may also offer intuitive data visualization , as showγ-aminobutyrate (GABA), 3-methylhistidine (3-MH), and p-hydroxyphenylacetate (p-HPA), as well as lower concentration of creatinine (Cn) , glutamate (Glu), tyrosine (Tyr), formate (For), and lactate (Lac), together with decreases of valine , leucine (Leu), isoleucine (Ile), lysine (Lys), serine (Ser), glutamine (Gln), betaine (Bet), creatine (Cr) and 1-methylhistidine (1-MH) Fig. a. Using Cn) Fig. b.post-hoc analysis of the achieved statistical power calculation yielded an average value of 0.92 (SD = 0.09) under the given condition of significance level α = 0.05, sample size n = 18 and specified effective sizes  values. In principle, an AUC value close to 1 indicates an excellent diagnostic power, and an AUC of 0.5 suggests no diagnostic power. Therefore, metabolites with high AUC values have a better predictive power for identifying HAE patients. In serum samples, ROC curves showed that glutamate (AUC = 0.924), leucine (AUC = 0.920), valine (AUC = 0.920) and phenylalanine (AUC = 0.914) have a high discriminatory ability for HAE. Overall, 10 metabolites had an AUC > 0.8. In urine samples, glutamate, creatinine and In addition, we used the 21 characteristic metabolites to build a PLS-DA model  ≥ 5 and considered as the most relevant pathways in HAE  [In the present study, HAE was found to result in significant changes in amino acid metabolism. This is evidenced by decreased levels in lysine, serine, glutamine and branched-chain amino acids , together with increased levels of glutamate and aromatic amino acids  Figs. , 7. Tyro (BCKDC) . The BCK (BCKDC) . BCAA ar (BCKDC) . The hosIn addition, increased urinary concentrations of GABA and 3-methylhistidine were found in the HAE patients. GABA is known to have relaxant effects on muscle tone while 3-methylhistidine is an indicator of muscle catabolism . Taken tBeing large neutral amino acids, BCAA and AAA can travel across the blood-brain barrier into the brain through the same type of neutral amino acid transporter LAT-1 . TherefoP < 0.001) indicated that HAE may have triggered hepatic failure in these patients. Therefore, the Fischer ratio may emerge as a reliable index for diagnosing HAE. Although the present 1H NMR data do not provide absolute concentrations of AAA and BCAA, the Fischer ratio calculated using normalized data was found to provide good discrimination between the HAE patients and the healthy individuals.Previously, Fischer et al.  reportedAlveoli, host metastasis and massive immune response due to HAE are known to lead to the generation of a large amount of unstable reactive oxygen species (ROS) , 57. OveIt should be noted that the present study has several limitations. First, a larger number of participants are needed to further increase the reliability and accuracy of the present results. Secondly, an integrated application of NMR with LC–MS or GC–MS will extend the metabolite coverage, and multiple -omic technologies (e.g. genomics and proteomics) can cross-validate and better support the experimental results. Thirdly, data for early stage HAE are currently not available due to the limitation of sample number. Data from different stages of HAE are important for the early detection, prediction and diagnosis of HAE. Finally, a discriminating method that can distinguish HAE from other types of hepatic disease is not available in the present study. If developing such a method, it is crucial to prevent clinical misdiagnoses as HAE exhibits similarities (e.g. lower Fischer ratio) with other hepatic diseases. The possibility of using the Fischer ratio and a unique metabolic signature for HAE to distinguish between different hepatic diseases warrants further study.The attractive properties of the NMR-based metabolomics approach, i.e. excellent repeatability, short detection duration and multiple metabolite coverage in a single measurement, makes it suitable for the HAE studies. A systematic exploration of multiple metabolites of small molecules helps to reveal the comprehensive metabolic variations induced by HAE infection. The early stage HAE diagnosis may be possible with a combination of both metabolomics and imaging techniques. For instance, chemical-exchange weighted magnetic resonance imaging techniques can be utilized to yield maps weighted by metabolites of interest –62. The 1H NMR-based metabolomics approach was used to investigate metabolic perturbations of HAE. Multivariate statistical analyses have highlighted several characteristic metabolites for HAE, e.g. decreased BCAA , increased AAA , increased lactate and decreased creatine, to list a few. The altered Fischer ratio together with specific metabolic changes identified in the present study may provide new insights into the molecular mechanism of HAE, and provide some clues to therapeutic intervention for HAE.In this study, a Additional file 1: Figure S1. Typical abdominal CT images of echinococcosis lesions in patients. Echinococcosis lesions are indicated by arrowheads. a Hepatic alveolar echinococcosis; CT image reveals dense calcification with irregular and indistinct margins in the lesion. b Hepatic cystic echinococcosis; CT image shows a sharply defined homogeneous cyst with multiple daughter cyst.Additional file 2: Figure S2. Predicted power profile with sample size per group .Additional file 3: Figure S3. The main window of the post-hoc power analysis specification in Gpower v.3.1 and the “effect size” drawer. Take as an example the metabolite of 1-methylhistidine which is different between control and HAE groups.Additional file 4: Figure S4. Univariate ROC curve analyses of metabolites in serum for discrimination of HAE patients from healthy individuals.Additional file 5: Figure S5. Univariate ROC curve analyses of metabolites in urine for discrimination of HAE patients from healthy individuals.Additional file 6: Figure S6. PLS-DA scores plot (left panel) and permutation test (right panel) of PLS-DA model comprising 21 identified characteristic metabolites.