Supplemental Digital Content is available in the text Patients with cirrhosis are vulnerable to acute hepatic insults and are more likely to develop rapid hepatic deterioration. The aim of this study is to describe the clinical course of patients with cirrhosis and hospitalized for acute hepatic deterioration (AHD).This is a prospective study involving 163 patients with cirrhosis and AHD. The occurrence of organ failures, systemic inflammatory response syndrome (SIRS), and infections during hospital stay were recorded and the relationship between organ failure and death or SIRS/infection was subsequently analyzed.Of 163 patients, 35 did not develop any organ failure during in-hospital follow-ups ; 84 had intrahepatic organ failures  ; and 44 patients developed extra-hepatic organ failures  on the basis of IH-OF with a 90-day mortality of 90.9%. On multivariable analysis by a Cox proportion hazard model, age, WBC, presence of IH-OF, and EH-OF all predicted 90-day death. A logistic regression analysis identified SIRS being associated with the development of EH-OF. Furthermore, IH-OF at admission and infections occurred during the hospital stay were shown to be another 2 potential risk factors.The clinical course of cirrhosis patients with acute hepatic injury was characterized by 3 consecutive stages , which provided a clear risk stratification. The PIRO criteria provided an accurate frame for prognostication of those patients. The systemic inflammatory response syndrome may be a target for blocking the progression to the EH-OF stage. One possible precipitating factor is systemic inflammatory response (SIRS), which consists of heart and respiratory rate, white cell count and temperature. It has been shown that SIRS is associated with hepatic encephalopathy, renal failure, and poor outcome in patients with acute liver failure or cirrhosis.To describe the clinical course of this population of patients, a prospective cohort of 163 patients with liver cirrhosis and acute hepatic deterioration (AHD) were recruited from 2 clinical centers, and the occurrence of organ failures was recorded during the overall in-hospital period. Additionally, the association between SIRS/infection and development of extra-hepatic organ failure was also explored in this study.14 by liver biopsy, endoscopy, radiological examination, or clinical evidence of prior hepatic de-compensation. Acute hepatic injury (AHI) was defined as a rise of alanine aminotransferase (ALT) ≧5 times of the normal upper limit or more than twice of the baseline value within 1 month. Acute hepatic deterioration (AHD) was defined on the basis of AHI and should meet the following criteria as previously described: increase of serum bilirubin ≧85 μmol/L and international normalized ratio (INR) ≧1.5 with a definite hepatic insult.10 As previously described, hepatic-related acute precipitating events included spontaneous flare-up of CHB, HBV reactivation, HAV and HEV superimposed infection, hepatotoxic drug use, and active alcohol drinking. Among them, spontaneous flare-up of CHB was defined as a flare-up of ALT ≧5 times of the normal upper limit, and/or more than twice of the baseline value, with replicating HBV-DNA  within 1 month before admission. HBV reactivation was defined as reappearance of serum HBV-DNA along with a flare-up of ALT ≧5 times of the normal upper limit, and/or more than twice of the baseline value in a person known to have the inactive HBsAg carrier state or resolved hepatitis B. Active alcohol drinking-related AHI should have aspartate aminotransferase (AST) to alanine transaminase (ALT) ratio > 1 (AST/ALT>1), in addition to a flare-up of ALT and excessive alcohol assumption within 1 month.Patients with acute hepatic injury were identified in a registered prospective cohort of patients with liver cirrhosis (ChiCTR-OCH-14005018), who enrolled from May 1, 2014, and February 25, 2015, in hepatic failure ward of First Affiliated Hospital of Zhejiang University and Ningbo No. 2 Hospital. The diagnosis of cirrhosis was described by previous studies:Patients were excluded based on the following criteria: (i) age<18 years; (ii) pregnancy; (iii) hepatocellular carcinoma (HCC) outside Milan criteria or other types of tumors; (iv) previously received a liver transplant; (v) HIV infection.This study complies with the principles of the Declaration of Helsinki and was approved by the ethics committee of both First Affiliated Hospital of Zhejiang University and Ningbo No. 2 Hospital. Written consents were obtained from each participant or their legal representatives.At admission, patients were evaluated for the presence of organ failure and SIRS. For patients suspected for infection, an initial screening by laboratory blood and urine tests, pulmonary CT scan, and abdominal ultrasound was performed, followed by cultures of blood, urine, sputum, or ascites . For in-hospital follow-up, the presence and development of organ failure and SIRS were checked at least every 3 days. In-hospital infection was also screened for suspected patients. In-hospital death was recorded. For those discharged from hospital, 28-day and 90-day prognosis were recorded, which were verified by medical records, telephone contact, or visiting.3.15 The presence of SIRS was confirmed by at least 2 repetitive measurements with at least 2 h interval. Bacterial or fungal infections were diagnosed as previously described:14 (1) pneumonia: new pulmonary infiltrate with fever, respiratory symptoms, findings on auscultation, or WBC count >10,000/mm3 or <4000/mm3; (2) spontaneous bacterial peritonitis (SBP): polymorphonuclear cells in ascitic fluid >250/μL; (3) bacteremia: positive blood cultures without a source of infection; (4) urinary tract infection: urine WBC >10/high power field with positive culture and symptom of urinary irritation; (5) others bacterial infections included skin infection and intra-abdominal infection; (6) fungal infection: positive fungal culture.Organ failure was categorized into either intrahepatic or extra-hepatic identities and the definition was based on the chronic liver failure-sequential organ failure assessment (CLIF-SOFA) (9)  or mechanical ventilation. Patients with coagulation failure received plasma transfusion. Patients with liver failure were treated with ademetionine, ursodesoxycholic acid, glycyrrhizic acid, or artificial liver support. Patients with hypo-albuminemia were given intravenous albumin.Patients with detectable serum HBV-DNA were treated with nucleoside analogs . Patients with bacterial or fungal infection were treated with intravenous albumin plus antibiotic or antifungal therapy empirically or based on the culture test. Patients with overt ascites were treated with diuretics (moderate) or paracentesis plus intravenous albumin (large or refractory). Patients with renal failure were treated with intravenous albumin with or without vasoconstrictors (such as dopamine and terlipressine). Patients with hepatic encephalopathy were treated with lactulose, antibiotics, and U test and nominal variables were compared via chi-square test. The log-rank test was used to compare survival among/between the groups and survival curves were drawn by GraphPad Prism 5 . To identify independent risk factors associated with time-dependent death of patients, a bi-variate analysis using the Cox proportional hazard model was performed to screen candidate variables (P < 0.10), followed by multivariate analysis. A multivariate logistic regression analysis was performed to explore predictors for development of extra-hepatic organ failures. For multivariate analysis, the entry and removal probability for stepwise was set as 0.05 and 0.10, respectively, and variables with P < 0.05 were kept in the final model. All the statistical analyses were performed with SPSS version 16.0 .Variables were expressed as mean ± standard deviation (SD), median with interquartile range, or number/percentage. Continuous variables were compared using Mann–Whitney As shown in Figure It was observed that 117 of 163 patients had organ failures at admission. Among them, most patients (89.7%) had intrahepatic organ failure (liver and/or coagulation failure) alone  and 90-day mortality (90.9%). Similarly, patients who solely had IH-OF, within in-hospital follow-up, also had a considerable 28-day and 90-day mortality at 16.7% and 22.6%, respectively. In contrast, those with no organ failure had a more favorable outcome, with no death reported within 90-day follow-up . According to the admission data, elder age, high WBC count, presence of IH-OF and EH-OF were all independently associated with 90-day mortality on multivariable analysis; however, after combining with the in-hospital data, only elder age, presence of IH-OF and EH-OF were shown to be significant predictors . By using in-hospital data, in addition to SIRS, infection was a potentially significant predictor .Building upon the previous studies,18Our study highlighted several important findings: we observed that organ failures occurred in a chronological order: almost all patients had liver and/or coagulation failure before developing extra-hepatic organ failures. Although abnormality in the coagulation system could be caused by sepsis, it would be reasonable to regard it as another reflection of liver failure in this clinical setting, as it occurred simultaneously with jaundice and independently of infections. Further analysis showed that the presence of liver/coagulation failure was a strong predictor of extra-hepatic organ failures. Moreover, patients with EH-OF had a significantly higher short-term mortality than those with IH-OF alone. Thus, these findings suggest that IH-OF and EH-OF were 2 consecutive stages of disease progression, and such phenomenon was characteristic of cirrhosis patients with acute hepatic deterioration (AHD), which could not be observed in infection-precipitated ACLF patients.20This study also reported that infection in AHD patients with cirrhosis was different from that of the overall population of patients with liver cirrhosis. Infections were seldom acquired from community, suggesting that the acquirement of infections was a late event in the natural history of patients with acute hepatic injury. Secondly, pneumonia but not SBP was the commonest type of infection, perhaps due to most patients having well-compensated cirrhosis previously. The occurrence of fungal infection was not rare, which might be because of long-term antibiotic treatment or immune paracensis.5 The finding was not surprising because the importance of SIRS has been highlighted in patients with cirrhosis, which is associated with renal dysfunction, worsening of hepatic encephalopathy, and poor in-hospital outcome.22 Infection has also been demonstrated to lead to multiorgan failures.24 It should be noted that although infections accounted for a large proportion of SIRS, some cases of SIRS occurred independently from SIRS. Both SIRS with infection (sepsis) and without (sterile inflammation) were associated with EH-OF development (data not shown), suggesting that both sterile and infection-related systemic inflammation might be important precipitants of disease progression. According to our previous study, ACLF patients precipitated by infections had higher occurrence of EH-OF than those precipitated by hepatic insults.10 In line with it, our results indicated a trend toward higher numbers of EH-OF per patient in patients with infections. The findings suggest that secondary infections complicated the natural history of AHD patients with cirrhosis. Of course, the causal relationship between SIRS/infection awaits more definitive confirmation and further studies are required to clarify this issue.In a recent study, our study showed a relationship between SIRS/infection and EH-OF.25 In line with the criteria, 4 predictors of poor prognosis were found to be age, IH-OF, WBC, and EH-OF, which fit well with the corresponding PIRO components . The value of age in predicting the prognosis of cirrhosis patients with hepatic injury has been reported in our previous studies26 and might be linked to the decreased tolerance to damage. WBC, reflecting body inflammatory response to insults, has been confirmed to be a strong predictor of poor prognosis in cirrhosis patients with acute de-compensation.27The PIRO criteria have been successfully applied to predict the prognosis of ACLF patients.28 For patients with IH-OF, LT should be considered for those with concurrent SIRS as these patients are more susceptible to developing EH-OF. Previously, 2 studies showed an encouraging long-term prognosis  in ACLF patients,30 suggesting that liver and coagulation failure had little impact on post-operation mortality. However, some of the patients with uncontrolled infection-induced SIRS would be less likely to receive LT. For patients with EH-OF, the possibility of LT should be considered for all the patients because of the extremely high short-term mortality. Previous studies showed cerebral , kidney, and pulmonary failure were not absolute contraindication for LT, although likely to decrease the success of LT.32Liver transplantation (LT) is the major therapeutic option for patients with acute hepatic deterioration; however, the decision should be made by weighing the need for LT and the risks associated with the procedure.The present study still has certain limitations. Some cases did not progress to EH-OF when discharged from the hospital due to nonmedical reasons. In these patients, the role of EH-OF might be underestimated in the study and a prolonged follow-up is needed. Also, the diagnosis of infections might be underestimated using the current criteria and some cases of SIRS might be attributed to occult infections. In addition, the causal relationship between SIRS/infections could not be established in the study and the majority of our study population had HBV-related cirrhosis and precipitated by spontaneous flare-up of CHB and HBV reactivation.Based on the above findings, the present study proposes a categorization of the natural history of patients with cirrhosis and acute hepatic deterioration by 3 consecutive stages of AHD, IH-OF, and EH-OF. Such categorization effectively described the dynamic disease progression of those patients and provided clear risk stratification, therefore, provides clinical implications for designing therapeutic strategies. For patients in the IH-OF stage, infection control (or prevention) and resolving inflammation might be 2 separate therapeutic targets, and it is crucial to prevent the development of EH-OF. However, once patients developed EH-OF, the possibility of liver transplantation should be immediately considered.


Despite recent progress, pneumonia remains the largest infectious killer of children globally. This paper describes outcomes of not treating community-diagnosed fast-breathing pneumonia on patient recovery.We conducted an exploratory subanalysis of an observational prospective cohort study in Malawi. We recruited children (2–59 months) diagnosed by community health workers with fast-breathing pneumonia using WHO integrated community case management (iCCM) guidelines. Children were followed at days 5 and 14 with a clinical assessment of recovery. We conducted bivariate and multivariable logistic regression for the association between treatment of fast-breathing pneumonia and recovery, adjusting for potential confounders.or worsening of symptoms) was 13% and 23% at day 5 in those who did receive and those who did not receive co-trimoxazole. Non-recovery, when defined as worsening of symptoms only, at day 5 was 7% in treatment and 10% in non-treatment cases. For both definitions, combined co-trimoxazole and lumefantrine-artemether (LA) treatment trended towards protection (adjusted OR (aOR) 0.28; 95% CI 0.12 to 0.68/aOR 0.29; 95% CI 0.08 to 1.01).We followed up 847 children, of whom 78 (9%) had not been given antibiotics (non-treatment). Non-treatment cases had higher baseline rates of diarrhoea, non-severe hypoxaemia and fever. Non-recovery (persistence We found that children who did not receive co-trimoxazole treatment had worse clinical outcomes; malaria co-diagnosis and treatment also play a significant role in non-recovery. Further research into non-treatment of fast-breathing pneumonia, using a pragmatic approach with consideration for malaria co-diagnosis and HIV status is needed to guide refinement of community treatment algorithms in this region. This is an exploratory subanalysis of a prospective observational study, with data on concurrent diagnoses, treatments and outcomes collected in a rural routine care setting.Oral antibiotics and lumefantrine-artemether treatment were not randomised, and therefore the study is subject to bias, although we adjusted for confounders and clustering in the analysis.Pneumonia diagnosis and recovery was based on the WHO integrated community case management guidelines, enhanced with pulse oximetry.Haemophilus influenzae type B and Streptococcus pneumoniae.Pneumonia is estimated to kill 0.9 million children each year, with the highest burden in Africa.The integrated community case management (iCCM) guidelines recommended by the WHO stratify pneumonia severity and subsequent treatment according to clinical symptoms.7–9Treatment failure—the persistence of symptoms or clinical deterioration following antibiotic initiation—14Research is needed into the non-treatment of fast-breathing pneumonia in an African setting, where HIV and malnutrition  and random Global Positioning System (GPS) spot checks by field supervisors of follow-up locations.Briefly, patients were recruited from community-level primary care clinics (village clinics) run by government employed CHWs called Health Surveillance Assistants. Information from the initial clinical assessment (including pulse oximetry with Lifebox) and diagnosis was recorded on case report forms by the CHW, along with their referral or treatment decision. For each CHW, local village-level data collectors (VDCs) conducted follow-up interviews in recruited patients' homes at days 5 and 14, with the day of diagnosis being day 0. VDCs attended village clinics to recruit patients and at follow-up visits conducted a clinical assessment of the child (including pulse oximetry), asked about additional care seeking and antibiotic adherence. Children identified as ‘not recovered’ at follow-up were referred by the VDC back to the CHW for further assessment and if needed, referral or treatment. We conducted cohort was defined as all children aged 2–59 months with CHW-diagnosed WHO fast-breathing pneumonia.The Fast-breathing pneumonia was defined according to the 2013 WHO iCCM guidelines,2) <90%), nasal flaring and head nodding. Wheeze was not specifically assessed or recorded. All children who were severely malnourished (mid-upper arm circumference (MUAC) <11.5 cm) were also referred. We defined moderate malnutrition as a MUAC between 11.5 and 13.5 cm, well nourished as >13.5 cm, and non-severe hypoxaemia as a SpO2 of 90–94%. Malaria diagnosis was based on clinical presentation and presumptive treatment of fever, without the use of rapid diagnostic tests or laboratory confirmation, as this was standard care at the time of this study. HIV testing was not offered by participating CHWs as this service also was not standard care.Complete treatment cases were those who received and completed >80% of the 5-day, twice daily course of co-trimoxazole , according to standard guidelines in Malawi at the time of the study. Non-treatment cases were those who did not receive antibiotics from the CHW at their diagnosis or from any other source, according to caregiver's report and VDC inspection. Non-adherent cases were those who received antibiotics but completed <80% of the recommended doses. There were 80 cases (9%) that were non-adherent and we excluded these cases from the analysis to focus on non-treatment rather than incomplete or partial treatment effects.Children were defined as ‘complete treatment’ or ‘non-treatment’ cases. Non-recovery definitions are presented in relapse (patients who had previously recovered) and non-recovery (classified as not recovered on day 5) were defined using the same parameters.Box 1Non-recovery definition 1 (persistence and/or progression)—presence of any of the following on day 5 of follow-up: fast breathing for age or parameters from non-recovery definition 2.Non-recovery definition 2 (progression)—presence of any of the following on day 5 of follow-up: axillary temperature >37.5°C, lower chest indrawing, any danger sign (as defined in the Methods section), change of antibiotic, hospital admission or death.Relapse/non-recovery—presence of any of the following on day 14 of follow-up in children cured on day 5 (relapse) or not-recovered on day 5 (non-recovery): fast breathing for age or parameters from non-recovery definition 2.2, respiratory rate and malnutrition; age; and gender. These variables were included based on previously published associations with treatment failure,-vce(robust)-, and intracluster correlation coefficients reported. A bivariate analysis, stratified by presence of fever at CHW diagnosis is presented in online All analyses were carried out using Stata SE13. We described the non-treatment and treatment cases. We conducted bivariate and multivariable analysis of outcomes at day 5 (using both definitions of non-recovery) for treatment and non-treatment cases; we did not do this for day 14 outcomes due to the small number of relapse/non-recovery cases. We considered the following variables in the multivariable analysis: clinical malaria diagnosis and lumefantrine-artemether (LA) treatment; other clinical diagnoses and treatments; initial clinical presentation including—temperature, SpO10.1136/bmjopen-2016-011636.supp1supplementary file 110.1136/bmjopen-2016-011636.supp2supplementary file 2Informed verbal consent for follow-up and clinical assessment was sought from the accompanying caregiver.A total of 1542 cases were assessed for eligibility. Of these, 487 (32%) were not eligible , 197 (17%) were lost to follow-up due to respondent unavailability, VDC unavailability or unknown . OverallThe rate of non-recovery (persistence and/or progression—definition 1) for non-co-trimoxazole treatment cases was 23% and for treatment cases was 13%. Children treated with both co-trimoxazole and LA had a non-recovery rate of 12% . Considering progression only (definition 2), non-recovery was 7% and 10% in those who did and did not receive antibiotics, respectively; non-recovery decreased to 5% in children with combined co-trimoxazole and LA treatment . Unadjusted results, including relapse/non-recovery at day 14 are presented in Results of the bivariate and multivariate analysis are presented in Of the patients with malaria diagnosis 81% received LA, while 36% of those who received LA had no malaria diagnosis documented (correlation 0.49)—this incorrect treatment with LA was higher in non-treatment cases (40% vs 31%), as was correct treatment for concurrent malaria diagnosis (84% vs 81%). The association of co-trimoxazole and LA treatment on recovery for both definitions demonstrated protective effects; for definition 1 (persistence/progression) combined treatment showed a 72% decrease in non-recovery . In definition 2 (progression), the effect size was similar for combined treatment  although decreased power led to wider CIs.This paper describes non-recovery in children diagnosed with fast-breathing pneumonia in the community in rural Malawi, specifically looking at the impact of co-trimoxazole and LA treatment. Our results suggest that oral co-trimoxazole treatment may be beneficial at the community level for children with fast-breathing pneumonia as part of routinely delivered community-based care. We found concurrent diagnoses, including malaria, to be important factors in non-recovery, as well as poorer guideline adherence by CHWs when treating children with possible baseline comorbidities, highlighting CHW inconsistencies when implementing iCCM guidelines. The role of malaria and treatment with both co-trimoxazole and LA in this setting needs further research and understanding to refine clinical diagnosis and treatment guidelines.Staphylococcus aureus), especially if the child is malnourished or HIV affected. While not recommended as part of iCCM care and not common in our study (n=5), a CHW may attempt to treat ‘noisy’ breathing in the community with salbutamol tablets. A child can have ‘noisy’ breathing due to many causes including a benign self-limiting viral upper respiratory tract infection, or diseases like bronchiolitis or croup that may lead to slower recovery, or non-recovery, as was observed in both definitions. None of these conditions would necessarily improve with salbutamol treatment, and all could result in a secondary bacterial infection that would benefit from a course of antibiotics. Alternatively the CHW may have diagnosed them with more severe illness and recommended them for referral, with this being misunderstood by the caregiver or not carried out, resulting in non-treatment and poor recovery.Interestingly, we found that the children who did not receive co-trimoxazole treatment had more comorbidities at baseline , and received more alternative treatments such as salbutamol tablets, aspirin and skin creams. These baseline differences could explain the CHW decision to not administer oral antibiotics, if the CHW suspected them of having an alternative diagnosis to pneumonia , despite meeting WHO iCCM criteria—reflecting the lack of specificity of fast breathing for pneumonia. Skin creams were likely dispensed to treat rashes, which could be due to a variety of causes such as self-limiting  or higher risk infections  into Malawi aims to address the issue of stock-outs, although an initial baseline survey found that 27% of CHWs did not have all essential drugs available.The multivariate analysis, for both definitions of non-recovery, demonstrated the association of empiric malaria co-diagnosis with non-recovery, and this was particularly pronounced when using progression of clinical symptoms as the definition of non-recovery. This association remained even once treatment with both co-trimoxazole and LA were considered—potentially a reflection of the poor correlation between the diagnosis and treatment, with almost 20% of malaria cases not receiving LA treatment. These relationships suggests that non-recovery at day 5 is being driven by either co-infection with malaria or initial misdiagnosis (and therefore mistreatment) of malaria as pneumonia. Co-trimoxazole has been shown to prevent malaria infections as part of prophylaxis treatment for HIV patients and to be an effective malaria treatment.The main limitation of this study was that it was not randomised. As a subanalysis of an observational cohort designed to investigate predictors of treatment failure, all children should have received oral co-trimoxazole for home treatment of fast-breathing pneumonia, according to iCCM guidelines. However, considering this was a quasi-programmatic setting, breaches in guideline implementation were seen among some of the CHW—demonstrating considerable inter-CHW variation. This analysis takes advantage of these failures to describe outcomes in non-treatment cases; however, this may reduce the generalisability of the results to other settings. We see that children were not randomly allocated to receive or not receive antibiotics (the same being true for LA treatment), therefore our analysis is subject to selection bias at the CHW level. Owing to the lack of blinding (both of the care giver and CHW) the apparent benefit among treated children could, in part, reflect a placebo effect for some caregiver-reported symptoms if they were pleased to have received treatment, especially if supplies were scarce. Similarly, as the CHWs were not blinded to treatment, their assessment of respiratory rate and other signs and symptoms may have been biased; and this could have been confounded by socioeconomic factors or knowledge of comorbidities such as HIV status. We had a 17% loss to follow-up which may lead to biases in the analysis. However, we have previously published that were only minimal differences seen in nutrition and PCV13 vaccination status between those lost and followed up, therefore it is unclear if this would have affected the results.15We suggest that these results be cautiously interpreted as preliminary data and not conclusive on the impact of non-treatment of fast-breathing pneumonia in a high HIV, malaria endemic setting. As this was a secondary analysis without an a priori sample size, we are aware that our study lacked power to test equivalency; however, we still found a relationship between treatment and outcome. Further research, such as a carefully monitored randomised equivalency study, is needed in the sub-Saharan African setting which accounts for HIV status and laboratory-confirmed malaria co-diagnosis to build on this exploratory analysis. However, caution is needed not to use a study design method which is completely explanatory. As rural CHW settings may not see point-of-care diagnostic tools for malaria and HIV , a study design which removes all of this ‘noise’ may lead to a false-positive finding of equivalence, while in the reality of suboptimal diagnosis and referral, antibiotics could still be beneficial. A balance between pragmatic and explanatory may be more appropriate, an approach which has been highlighted in the revised PRECIS-2 toolOur results suggest that non-treatment of children meeting the current WHO iCCM definition for fast-breathing pneumonia would not be advisable in a rural sub-Saharan African setting with malaria endemicity. Malaria co-diagnosis and treatment play a significant role in non-recovery and so any trial in which placebos are given in a similar setting would need to be implemented under close clinical supervision to avoid adverse outcomes, as well as tease out the complex relationships between antibiotics and LA with recovery. However, we did not record any deaths and only two children who did not receive an antibiotic sought additional care within our 2 weeks of follow-up.


A 10-plex urine-based bladder cancer (BCa) diagnostic signature has the potential to non-invasively predict the presence of BCa in at-risk patients, as reported in various case-control studies. The present meta-analysis was performed to re-evaluate and demonstrate the robustness and consistency of the diagnostic utility of the 10-plex urine-based diagnostic assay. We re-analyzed primary data collected in five previously published case-control studies on the 10-plex diagnostic assay. Studies reported the sensitivity and specificity of ten urinary protein biomarkers for the detection of BCa, including interleukin 8, matrix metalloproteinases 9 and 10, angiogenin, apolipoprotein E, syndecan 1, alpha-1 antitrypsin, plasminogen activator inhibitor-1, carbonic anhydrase 9, and vascular endothelial growth factor A. Data were extracted and reviewed independently by two investigators. Log odds ratios (ORs) were calculated to determine how strongly the 10-plex biomarker panel and individual biomarkers are associated with the presence of BCa. Data pooled from 1,173 patients were analyzed. The log OR for each biomarker was improved by 1.5 or greater with smaller 95% CI in our meta-analysis of the overall cohort compared with each analysis of an individual cohort. The combination of the ten biomarkers showed a higher log OR  than did any single biomarker irrespective of histological grade or disease stage of tumors. We concluded that the 10-plex BCa-associated diagnostic signature demonstrated a higher potential to identify BCa when compared to any single biomarker. Our results justify further advancement of the 10-plex protein-based diagnostic signature toward clinical application. Bladder cancer (BCa) is the second most common genitourinary malignancy in the United States, with 79,030 new cases and 16,870 deaths estimated to occur in 2017 . TherefoBCa detection and diagnosis require cystoscopy and bladder biopsy, which are unpleasant and costly procedures. Although NMIBC can be treated with transurethral resection (TUR) with an excellent survival outcome, this method is associated with an intravesical recurrence rate of approximately 70% within two years after TUR . This isRecent advancements in proteomics technology have promoted discovery of novel protein markers and the number of published reports on urine-based biomarkers has dramatically increased with reported sensitivity ranges from 52% to 97%, and specificity from 43% to 100% for individual biomarkers , PAI1  and IL-8  showed the highest log OR, while MMP10  showed the lowest , followed by support vector machine algorithms , to deveThe urine-based protein biomarkers analyzed in the present study were originally established as a panel of 14 protein biomarkers  using a Currently, published guidelines recommend that patients presenting with hematuria undergo VUC and examination using cystoscopy , 34–36, e.g., prostate cancer, kidney cancer and chronic kidney disease. There was limited overlap of the biomarkers in prostate cancer (only IL-8 was elevated) and kidney cancer (only CA9 and VEGF were elevated). In chronic kidney disease, i.e., GFR < 45 mL/min, significant amounts of proteins were evident in the urine and thus the assay is unable to accurately discriminate if a patient has cancer (data not shown). Urinary tract infection (UTI) is another coincidence that can negatively affect the diagnostic performance of urine-based biomarkers. The present study included 96 subjects with UTI and the 10-plex panel as well as most of the single markers showed better performance with the subjects excluded from the analysis (data not shown). These findings suggest that the 10-plex panel is anticipated to yield an excellent performance in a cohort including those with UTI although it should be applied to those subjects with caution.As for influence of other diseases, the urine-based protein biomarkers have been already assessed in patients with other genitourinary malignancies and renal disorders, Several limitations of this study must be acknowledged. Although targets in all included studies were quantitatively measured, the antibodies used to monitor each urine-based biomarker were not identical among the included studies. The present study did not incorporate detailed data such as race, gender, age, and smoking history, which has been reported to influence diagnostic performance of the multiplex urinary protein panel . Since aIn conclusion, our meta-analysis confirmed significant association between urinary levels of the protein biomarkers and BCa detection. In particular, the combination of the ten biomarkers demonstrated a higher potential for detection of BCa than did any single biomarker. The study has justified further advancement of the multiplex urinary protein biomarker assay toward clinical application as a noninvasive method of detecting BCa in our daily practice. However, further validation steps including analyses of consecutive patients are needed before clinical adoption .An additional search was conducted using Medline and Embase using the following urinary biomarkers for BCa in the search bar: ANG, APOE, A1AT, CA9, IL8, MMP9, MMP10, PAI1, SDC1, and VEGF. The following additional filters were selected: “Publication dates from January 1, 2012 to December 31, 2016” and studies in “Humans.” Studies assessing the biomarker panel in subjects for the purpose of tumor surveillance were excluded. Similarly, studies not describing the 10 biomarkers in a multiplex format for the diagnosis of BCa were excluded. Eventually no article was found in addition to the five studies that we initially selected.vs. no BCa), adjusted for other potential confounders and/or mediators, as appropriate. Note that the weighted least-squares method under the multivariable-pooled analysis can better overcome small-sample-size bias, whereas the random-effect meta-regression model can better overcome between- and within-study heterogeneity. Both methods were applied to generate the most robust results. Statistical analyses were performed using R version 3.2.3 and reviewed by Y.D.We performed a meta-analysis using a random-effect model followed by multivariable-pooled analysis of the molecular data using the weighted least-squares method to account for size effects. Random-effect meta-regression models (linear mixed models) were used to assess the relationship between the estimates and the outcome (BCa


Objective: To examine the impact of genetic, inflammatory, cardiovascular, lifestyle, and neuroanatomical factors on cognitive processing speed (CPS) change over time in functionally intact older adults.Methods: This observational study conducted over two time points, included 120 community dwelling cognitively normal older adults between the ages of 60 and 80 from the University of California San Francisco Memory and Aging Center. Participants were followed with composite measures of CPS, calculated based on norms for 20–30 year-olds. Variables of interest were AD risk genes , markers of inflammation (interleukin 6) and cardiovascular health , self-reported physical activity, and corpus callosum (CC) volumes. The sample was divided into three groups: 17 “resilient-agers” with fast and stable processing speed; 56 “average-agers” with average and stable processing speed; and 47 “sub-agers” with average baseline speed who were slower at follow-up.Results: Resilient-agers had larger baseline CC volumes than sub-agers (p < 0.05). Resilient-agers displayed lower levels of interleukin-6 (IL-6) and insulin (ps < 0.05) than sub-agers, and reported more physical activity than both average- and sub-agers (ps < 0.01). In a multinomial logistic regression, physical activity and IL-6 predicted average- and sub-ager groups. Resilient-agers displayed a higher frequency of APOE e4 and CR1 AA/AG alleles.Conclusion: Robust and stable CPS is associated with larger baseline CC volumes, lower levels of inflammation and insulin, and greater self-reported physical activity. These findings highlight the relevance of neuroanatomical, biological, and lifestyle factors in the identification and prediction of heterogeneous cognitive aging change over time. Cognitive processing speed (CPS) reaches its zenith during early adulthood  and declBiological aging models posit upstream causal factors such as lifestyle variables and genetics interacting with inflammatory status and vascular health to affect downstream factors such as neurodegeneration and cognitive function . Within Less well-understood is which factors drive changes in CPS over time. We reasoned that studying the extremes of CPS change over time provides an opportunity to understand the unique health profile  related to its preservation and decline, some of which may be amenable to change through lifestyle choices . We hypoThis was an exploratory observational study conducted over two time points. We started with a convenience sample of 165 neurologically and functionally intact older adults (aged 60 years and older) from existing research cohorts at the UCSF Memory and Aging Center based on the availability of composite CPS data. Each potential subject underwent a complete history, neurological examination, a functional assessment, and an hour-long neuropsychological screening battery, followed by a consensus conference for the determination of diagnosis and suitability for the study. Subject inclusion was based upon a Mini-Mental State Exam score of >25 , a Clinin = 40; 16 males; age 24 ± 3.1 years; education 16 ± 1.6 years). The average of the 14 z-scores for each older subject yielded a composite z-score, with higher scores denoting slower speed relative to the young adult controls. This comparison of mean reaction times between older subjects and younger controls has previously been validated . Experiments were administered on a standard 15.4″ Dell Latitude D830 laptop. Subjects were presented with stimuli on the display and instructed to make visuospatial judgments. Standard instructions were for subjects to respond as quickly and accurately as possible. All tasks required a binary decision (yes/no or left/right), and subjects were asked to use the index and middle finger of their right hand to press the key on a keyboard corresponding to their answer choice. Tasks commenced with between 5 and 10 practice trials; if accuracy on these trials fell below 70%, an additional round of practice trials was administered before proceeding. The first two experimental trials in each task condition served as ‘cushion’ trials and were not included in the analyses. The median response latency of the subsequent 20 experimental trials of each task condition was recorded for analysis.All task conditions were programmed on a PC using Eprime software   years. Baseline CPS of average-agers and sub-agers was comparable to the larger cohort of older adults from which they were drawn . Demographic variables are summarized in Table 1.We defined three CPS thresholds of change over two time points in order to distinguish both fast and stable CPS, shown below in aterial) . Resilieaterial) . A 0.5 SAPOE was investigated due to the role of the e4 allele as a risk factor for AD, and its association in the slowing of CPS  volume , which has been associated with CPS in older adults  × fasting glucose (mg/dL))/405]. IL-6 was measured using a Quantikine ELISA kit from R&D systems  in the laboratory of Dr. Ralph Green at the UC Davis Medical Center .APOE genotyping (rs429358 and rs7412), and was conducted on an ABI 7900HT Fast Real-Time PCR system  according to manufacturer’s instructions. Sequenom iPLEX Technology  was used for CR1 genotyping (rs6656401). The SpectroAquire and MassARRAY Typer Software packages  were used for interpretation and Typer analyzer (v3.4.0.18) was used to review and analyze data.Genomic DNA was extracted from peripheral blood using standard protocols . Genotyping was performed using either TaqMan or Sequenom genotyping. TaqMan Allelic Discrimination Assay was used for 2 and 1 mm thick slices; matrix 2566240 with 160 slices yielding 1 mm3 isotropic voxels). Structural MR images were analyzed using the FreeSurfer image analysis suite2. After initial automated segmentation through FreeSurfer version 5.1, each subject’s image was individually checked for quality and accuracy of segmentation. Remaining inaccuracies in either gray and white matter segmentation or pial boundaries and residual skull fragments were manually corrected with FreeSurfer’s built-in editing software. These cases were reprocessed and final volumes were recalculated. Intracranial volume (ICV) was calculated based on FreeSurfer’s own eTIV  metric, which uses atlas normalization as well as the relationship between the linear transform to MNI305 space and ICV. Region of interest was the CC , using a protocol that included a T1-weighted 3D MPRAGE sequence . Demographic variables were compared using analysis of variance with chi square for dichotomous variables and Tukey Table 1. Graphical representation of significant health profile variables is presented in Figure 2.There were no group differences in age, sex, or education. The raw mean scores, SD, and effect sizes of test performance by group for all demographic, clinical, and health profile variables are presented in APOE e4 allelic frequency revealed that of the 17 resilient-agers, 10 (59%) had an e4 allele. In contrast, only 18 average-agers (32%) and 11 sub-agers (23%) had an e4 allele. Chi square analysis of APOE e4 allele frequency was significant  with greater frequency of an e4 allele in the resilient-agers than sub-agers. Analysis of CR1 rs6656401 AA/AG allelic frequency revealed that of the 17 resilient-agers, 11 (65%) had AA/AG alleles, versus 6 (35%) with the GG allele. In contrast, only 25 average-agers (48%) and 14 sub-agers (30%) had AA/AG alleles. Chi square analysis of AA/AG allelic frequency was significant  with greater frequency of AA/AG alleles in the resilient-agers than sub-agers. Descriptive analysis of the 17 resilient-agers revealed that 7 (41%) were carriers of both CR1 AA/AG alleles and the APOE e4 allele.Analysis of p < 0.05). There was no difference in CC volume between resilient-agers and average-agers.Resilient-agers had larger CC volume at baseline than sub-agers (p < 0.05). There was no difference in IL-6 levels between resilient-agers and average-agers.Resilient-agers exhibited lower levels of IL-6 at baseline than sub-agers (p < 0.05). There was no difference in insulin level between resilient-agers and average-agers.No differences between groups were observed on baseline levels of BMI, HDL, LDL, or MAP. Resilient-agers exhibited lower levels of fasting insulin than sub-agers (p < 0.01).Resilient-agers reported more activity than both average- and sub-agers, endorsing more physical activity on the PASE at baseline . Likelihood ratio tests indicated that IL-6  and PASE  were significant predictors. Parameter estimates with resilient-agers as the reference group indicated PASE as a significant predictor with respect to average-agers . Parameter estimates with resilient-agers as the reference group indicated PASE  and IL-6  as significant predictors with respect to sub-agers.To further explore the contributions of these factors we constructed a multinomial logistic forced entry regression with the three CPS groups as the dependent variable and all significant continuous outcome variables as predictors . We also included age as a covariate. Due to availability of data for the significant outcome variables, the sample size for this model included seven resilient-agers, 27 average-agers, 22 sub-agers. The multinomial logistic regression indicated that the overall model was significant . Physical activity is another factor that has been studied in healthy older adult APOE e4 carriers. APOE e4 carriers reporting high amounts of leisure time physical activity demonstrated greater white matter integrity  than APOE e4 carriers reporting low physical activity. These results suggest that physical activity among APOE e4 carriers may offer unique protective effects against white matter demyelination, which in turn could contribute to the maintenance of CPS. While the finding in this study needs replication, it opens exciting lines of inquiry to further examine whether the factors identified in this study or other factors may modify the effects of these genetic risk factors, providing protective effects in the presence of genetic predisposition toward neuropathology.One such potential factor is completed years of education. APOE, CR1) represent upstream causal mechanisms with downstream effects on vascular health (insulin resistance) and inflammatory measures (IL-6), which in turn have downstream effects on neurodegeneration (CC volume) and cognitive function (CPS). A strength of the current study is the robust measure of CPS, incorporating 14 task conditions. This method represents a more valid measure of CPS than most studies investigating CPS, which make use of a single measure of CPS (Results from this study underscore the heterogeneity of cognitive aging even among typically aging older adults. Recent advances in biological aging models posit a multi-determined, mechanistic account of biological aging, with the causal factors of environment, lifestyle factors, genetics, and disease interacting with redox balance, inflammatory status, and vascular health to affect aging, cognitive function, and neurodegeneration . While oe of CPS . The comWe acknowledge several limitations of this study. The sample size was relatively small, particularly the cohort of resilient-agers. While by definition this group will likely be smaller, future studies should seek to identify a larger number of these resilient-agers. The majority of our findings dissociated resilient-agers and sub-agers, and these results may reflect differences between normal and abnormal cognitive decline. On the other hand, results of the CC volume, IL-6, and insulin data demonstrated numeric differences and medium effect sizes between the resilient-ager and average-ager groups, so the lack of statistical significance may be an issue of power. We did not incorporate multimodal imaging such as amyloid and tau imaging, or DTI, which would have provided more robust measures of white matter integrity. Our measures of cardiovascular health and inflammation were also limited and the overall impact of these factors on CPS may be even greater. The secondary regression analysis included a reduced sample, which could affect the stability of the model. This initial study included measurement of CPS over time, but we did not investigate longitudinal change in other variables of interest. While the findings in this small group of resilient-agers offers validity to the characterization of unusually successful domain-specific cognitive aging, future studies including more time points and investigating additional cognitive domains will allow for distinct longitudinal aging trajectories to be plotted and more robustly characterized.The current study adds to a growing body of literature identifying subgroups of older adults that demonstrate uniquely strong domain-specific cognitive abilities that resist age-related cognitive decline. “Super-agers” have previously been characterized in individuals over age 80 with episodic memory performance at least as good as normative values for 50- to 65-year-olds . This isNB made a substantial contribution to the design of the work, the acquisition and interpretation of the work, drafted the work, approved the final version to be published and agreed to be accountable for all aspects of the work. BB, JK made a substantial contribution to the acquisition and interpretation of the work, revised the work critically for important intellectual content, approved the final version to be published and agreed to be accountable for all aspects of the work. JY, DF, and KY made a substantial contribution to the interpretation of the work, revised the work for important intellectual content, approved the final version to be published and agreed to be accountable for all aspects of the work. MW, AK made a substantial contribution to the acquisition of the work, approved the final version to be published and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer ER and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Background and Aim. MicroRNAs (miRNAs) have been implicated in the pathophysiology of numerous human diseases including gastroesophageal reflux disease (GERD). The objective of this study was to investigate the miRNA expression of exfoliated cells of the tongue in patients with GERD versus healthy controls (Ctrls). Methods. Using quantitative reverse-transcription PCR (qRT-PCR), expression levels of six candidate miRNAs  were examined across a discovery cohort of patients with GERD (n = 24) versus Ctrls (n = 24). These findings were confirmed across a validation cohort . Differences in miRNA expression levels were evaluated using the Mann-Whitney U test while the specificity and sensitivity were obtained using receiver-operator characteristic (ROC) curves. Results. miR-203 was significantly downregulated in GERD patients as compared to Ctrls (P < 0.0001) with ROC curve of 0.94 (95% CI: 0.90–0.97). The sensitivity and the specificity of miR-203 were 91.7% and 87.3%, respectively, in the GERD and Ctrls. These results suggest that miR-203 may be a useful diagnostic marker for discriminating GERD from Ctrls. Conclusions. miR-203 testing may assist in the diagnosis of patients with symptoms suggestive of GERD. Gastroesophageal reflux disease (GERD) is a chronic disease of mucosal damage caused by the backflow of stomach acid into the esophagus. GERD is a condition that knows no boundaries, affecting 5–7% of the population worldwide. It has been shown to have a significant negative impact on the quality of life of those affected, disrupting their daily activities . DiagnosThe tongue is increasingly considered, an extension of the upper gastrointestinal tract that can provide important clues to the body's current condition and health status . The furMicroRNAs (miRNAs) are evolutionarily conserved, small  RNA molecules that modulate the expression levels of target genes and are thus actively involved in a wide range of physiologic and pathologic processes. As such, numerous studies have highlighted the use of miRNAs as highly sensitive and specific biomarkers for the diagnosis of several diseases –9. In prThe objective of this study was to investigate the differences of miRNAs expression in exfoliated cells of the tongue coating between patients with GERD and healthy controls (Ctrls). We evaluated the expression levels of miR-143, miR-145, miR-192, miR-194, miR-203, and miR-205 in exfoliated cells of the tongue coating in patients with GERD and Ctrls across a discovery cohort of 48 specimens. We validated our findings across an independent cohort, thereby confirming that miR-203 was significantly downregulated in GERD as compared to Ctrl levels. Our study demonstrated that miR-203 in exfoliated cells of the tongue coating can be used as a potential noninvasive biomarker for the diagnosis of GERD.n = 72) healthy normal volunteers who had a physical examination  and health history including question about drug use were recruited and used to represent the normal control group. Only those evaluations  needed to determine eligibility for a particular study will be done. The clinical parameters of these patients were shown in From January 2014 to March 2015, 166 patients with GERD were recruited from Shanghai Yueyang Hospital of Integrated Traditional Chinese and Western Medicine. All patients underwent an open-access transoral upper gastrointestinal endoscopy, and those who had typical symptoms (heartburn and regurgitation) or abnormal acid contact time detected at ambulatory esophageal pH testing were considered for inclusion in the study. Exclusion criteria were as follows: (1) GERD combined with other structural gastrointestinal disorders, such as peptic ulcer disease or esophageal or gastric malignancy; (2) prior gastric surgery; and (3) pregnancy. Seventy-two  according to the manufacturer's protocol followed by the treatment of RNase-free DNase I  to eliminate DNA contamination.ΔCt, where ΔCt = Ct of internal reference −Ct of target miRNA. U6 RNA was used as a miRNA internal control. Data obtained by real-time PCR were translated in log 10 (relative level).Quantification of miRNAs was based on the stem-loop RT-PCR method. First, a miRNA-specific stem-loop RT primer is hybridized to the miRNA and then reverse transcribed. Next, the RT product is amplified and monitored in real-time using a miRNA-specific forward primer and the universal reverse primer . PCR was U test. Receiver-operator characteristic (ROC) curves were established to evaluate the difference in the levels of miRNAs among GERD patients and Ctrls. All tests were two-tailed and P < 0.05 was considered statistically significant.Comparisons between groups were analyzed using Mann-Whitneyn = 42, 25%), grade C , and grade D . Only 4 study subjects were diagnosed with Barrett esophagus.A total of 166 GERD patients and 72 healthy controls were included in this study. Patient demographics are shown in n = 24) and Ctrls (n = 24), 6 candidate miRNAs  were selected. In this manner, we found that miRNAs can be found in exfoliated cells of the tongue coating in Ctrls and patients with GERD. Meanwhile, we observed a statistically significant downregulation of miR-203 in GERD as compared to Ctrls (P < 0.0001)  .P < 0.0001)  .Comparing GERD subjects with Ctrls, ROC curve areas of miR-203 were found to be 0.94 (95% CI: 0.90–0.97) . The senThe tongue is part of digestive system and acts as a very good mirror. The tongue has a coating, which represents the state of digestive function in our body. The tongue coating has been shown to play an important role in reflecting the occurrence, development, and prognosis of numerous diseases –4. The nInterestingly, the stability of miRNAs has been readily described not only in tissues or cultured cell lines, but also in human body fluid . Moreover, the expression of miRNA species has been recently shown to correlate with different physiological stages and pathological conditions. As such, some disease-specific profiles can provide crucial diagnostic and prognostic information, highlighting the necessity to exploit miRNAs for possible use as biomarkers –9.Several recent studies have documented the aberrant expression of miRNAs in the pathogenesis of GERD. The expressions of miR-143, miR-145, and miR-205 were significantly higher in oesophageal squamous mucosa from subjects with GERD compared to squamous mucosa from subjects without pathological reflux . SubsequP < 0.0001) with ROC curve areas of 0.94 (95% CI: 0.90–0.97) and a sensitivity and specificity of 91.7% and 87.3%, respectively, in the GERD subjects and Ctrls. These results suggest that the expression level of miR-203 may be useful a marker for discriminating GERD from Ctrls, with the advantage that this technique is noninvasive and inexpensive.To the best of our knowledge, tongue coating miRNAs have not been previously compared between GERD patients and Ctrls. In this study, we hypothesised that miR-143, miR-145, miR-192, miR-194, miR-203, and miR-205 expression might be altered in tongue coating in response to chronic gastroesophageal reflux. Therefore, we evaluated these six miRNAs expression levels from the tongue coating in patients with GERD and Ctrls. qRT-PCR results demonstrated that six miRNAs can be found in exfoliated cells of the tongue coating in patients with GERD and Ctrls. miR-203 in exfoliated cells of the tongue coating was significantly downregulated in GERD patients as compared with Ctrls (Studies interrogating the expression of miR-203 have suggested that high-level expression is observed in skin and in the esophagus, an organ sharing anatomical similarities with skin . SimilarRecently, miR-203 has been indicated to play a key role in the regulation of normal immunity or inflammation response , 24. HowOur study has some limitations. Most of the GERD patients had a grade of A and B, 62%, 25%, respectively. No difference was found in miR-203 expression between the patients with grades A and B. Another limitation was the failure to recruit patients with non-GERD-related disease. Moreover, as global miRNA expression changes were not assessed it is possible that the expression of other miRNAs may be altered in response to chronic reflux.In summary, we demonstrated that miR-203 was significantly downregulated in exfoliated cells of the tongue coating in patients with GERD. We suggest that miR-203 testing may assist in the diagnosis of patients with symptoms suggestive of GERD. This may lessen the use of unnecessary antireflux therapy and the need for further invasive and expensive diagnostic methods. Nevertheless, this approach should be further validated in larger cohorts of patients, especially those with non-GERD-related disease, to assess their efficacy and potential applicability in a screening setting.


Apis mellifera) has changed dramatically since the emergence of the parasitic mite Varroa destructor, which increased the spread of virulent variants of viruses such as deformed wing virus. Previous genomic studies have focused on colonies suffering from infections by Varroa and virulent viruses, which could mask other viral species present in honey bees, resulting in a distorted view of viral diversity. To capture the viral diversity within colonies that are exposed to mites but do not suffer the ultimate consequences of the infestation, we examined populations of honey bees that have evolved naturally or have been selected for resistance to Varroa. This analysis revealed seven novel viruses isolated from honey bees sampled globally, including the first identification of negative-sense RNA viruses in honey bees. Notably, two rhabdoviruses were present in three geographically diverse locations and were also present in Varroa mites parasitizing the bees. To characterize the antiviral response, we performed deep sequencing of small RNA populations in honey bees and mites. This provided evidence of a Dicer-mediated immune response in honey bees, while the viral small RNA profile in Varroa mites was novel and distinct from the response observed in bees. Overall, we show that viral diversity in honey bee colonies is greater than previously thought, which encourages additional studies of the bee virome on a global scale and which may ultimately improve disease management.Understanding the diversity and consequences of viruses present in honey bees is critical for maintaining pollinator health and managing the spread of disease. The viral landscape of honey bees (IMPORTANCE Honey bee populations have become increasingly susceptible to colony losses due to pathogenic viruses spread by parasitic Varroa mites. To date, 24 viruses have been described in honey bees, with most belonging to the order Picornavirales. Collapsing Varroa-infected colonies are often overwhelmed with high levels of picornaviruses. To examine the underlying viral diversity in honey bees, we employed viral metatranscriptomics analyses on three geographically diverse Varroa-resistant populations from Europe, Africa, and the Pacific. We describe seven novel viruses from a range of diverse viral families, including two viruses that are present in all three locations. In honey bees, small RNA sequences indicate that these viruses are processed by Dicer and the RNA interference pathway, whereas Varroa mites produce strikingly novel small RNA patterns. This work increases the number and diversity of known honey bee viruses and will ultimately contribute to improved disease management in our most important agricultural pollinator. Apis mellifera  and Deformed wing virus (DWV) (Black queen cell virus (BQCV) and Acute bee paralysis virus (ABPV)  , 23, ands (ABPV) . More res (ABPV) , which ls (ABPV) . LSVs ars (ABPV) .Varroa destructor  pathway , 37, 38.Varroa parasites but do not appear to suffer any negative consequences , Stellenbosch and Robben Island (South Africa), and Vava'u and Tongatapu islands (Kingdom of Tonga)  . The colof mites . Colonieto mites . The honmain low . In the f Vava'u . We syntf Vava'u . We asse Trinity  and compVarroa-resistant populations. Contigs for DWV, BQCV, and sacbrood virus (SBV) were present in all three locations. ABPV was present in one colony from Robben Island, and LSV was present in one colony each from The Netherlands and Tonga and in two colonies from South Africa  found in Drosophila  (Apis mellifera), followed by the name or category of virus and number if more than 1 .Next, we examined contigs that showed similarity to previously characterized virus sequences from positive-sense, negative-sense, and double-stranded RNA genomes available in GenBank. We found genomic evidence of seven previously undescribed viruses in the honey bee transcriptomes, including four negative-sense and three positive-sense RNA viruses  and 3. Oavirales , and twoaviridae . The throsophila , and oneus (DCV)  to E. FoOrthomyxoviridae family (22% identity), which suggests that this glycoprotein gene may be acquired through an inter-virus-family horizontal transfer event  has a 14,613-nt genome with a prototypic rhabdovirus structure  correspoer event . Intereser event  and 3, wer event . ARV-2 wer event  and 3. Ter event . The RdRBunyaviridae are negative-sense RNA viruses with three genome segments , each containing a separate ORF . Ph. PhBunyatebrates  and placFlaviviridae (Flaviviridae placed AFV in a clade of other recently discovered flavi-like viruses with large genomes (Drosophila species (Takaungu virus and Hermitage virus) (Apis mellifera flavivirus (AFV) was identified in one colony from Robben Island. The 20,414-nt positive-sense RNA genome contains a single ORF of 6,615 aa . BLAST siviridae . The phy genomes   .Cricket paralysis virus (CrPV), Drosophila C virus, and Anopheles C virus (We identified a novel dicistrovirus from one colony at the Amsterdam Water Dunes (Apis mellifera dicistrovirus [ADV]). The 9,126-nt genome contains two ORFs encoding the replication enzyme polypeptide and the capsid proteins, respectively, which is typical of dicistroviruses . The pol C virus .Drosophila species  and Sanger sequencing using primers spanning the neighboring contigs. The resulting Apis mellifera Nora virus (ANV) partial genome sequence is 10,091 nt long and covers the entire replicase, although it is missing the first ORF at the 5′ end of a typical Nora virus genome  53). In. In53).  species .One way of confirming that a putative virus genuinely infects the host from which it is sampled is the presence of an antiviral immune response. In insects, likely candidates are the small RNA pathways that are utilized in viral defense , 40, 59.A. mellifera samples: two from the Amsterdam Water Dunes and two from Robben Island. For each of these geographic locations, we used PCR to screen for individuals that were positive for ARV-1 and used one individual that tested positive for ARV-1 (Amsterdam Water Dunes positive [AWD+] and Robben Island positive [RI+]) and one individual that tested negative (Amsterdam Water Dunes negative [AWD−] and Robben Island negative [RI−]). The RI+ sample also tested positive for ARV-2. The four libraries were subjected to 50-bp single-end sequencing, resulting in between 10 million and 18 million reads per sample.We generated small RNA libraries from the abdomens of four Apis mellifera genome and then aligned the unmapped reads to the ARV-1 and ARV-2 genomes  starting at the dsRNA termini and fading with increasing distance from the termini, with a characteristic 2- to 3-nt overhang . We lookP < 0.01 for both by a chi-squared test)   and H.Taken together, our data suggest that the ARV-1 and -2 small RNAs have been generated by Dicer acting on a double-stranded RNA replication intermediate and that the small RNAs are subsequently bound by Argonaute proteins, indicating that the bees have an active antiviral immune response against ARV-1 and ARV-2.V. destructor mites collected from A. mellifera individuals from Robben Island and performed Illumina 50-bp single-end sequencing, resulting in 18 million to 20 million reads per sample , no 5′-nucleotide bias for antisense reads for ARV-1 (not significant [NS] by a chi-squared test), and no 5′-nucleotide bias for sense or antisense reads mapping to ARV-2 (NS by a chi-squared test)   and J. TAedes aegypti  ping-pong amplification pathway, as this pathway has been implicated in viral defense in  aegypti , 63. The aegypti . We geneThe small RNA profiles in mites do not mimic those in honey bees, indicating that the mite reads do not come from ingested honey bee material. Thus, the high abundance of reads in the mites  and the Varroa-resistant or Varroa-free honey bee populations from three locations in Europe, Africa, and the Pacific. We present genomic evidence of seven new RNA viruses, including three novel positive-sense and four novel negative-sense viruses. Our study therefore increases the number of known honey bee viruses from 24 to 31. ARV-1 and -2 were found in three geographically distinct populations. Using small RNA sequencing, we show that honey bees exhibit classic Dicer-mediated siRNA profiles, suggesting an active bee immune response. We also report the first analysis of small RNAs in Varroa destructor mites and show that ARV-1 and -2 are present in these mites, although the small RNA profile is distinctly different from that for honey bees.We describe a diverse set of new viruses in Osmia cornuta, including one virus from the order Mononegavirales and one from the family Bunyaviridae, indicating that viral diversity in other hymenopteran species extends to negative-sense viruses  are related to viruses known to be present in insects . Our fin viruses . One of pmorLBV1 . LepmorLra flies . Interesa passim . We therThe small RNA patterns of the rhabdoviruses in honey bees show classical Dicer-mediated degradation profiles, providing strong evidence that ARV-1 and -2 enter the cells of bees and begin to replicate. During viral replication, a double-stranded RNA replication intermediate is formed, which can be recognized by the RNAi machinery and chopped by Dicer. The resulting small RNAs become part of an antiviral immune response . This stVarroa, the strong bias for 24-nt antisense small RNAs and the lack of phasing suggest that replicating dsRNA is not a template for Dicer. Internal secondary structures within a negative-sense virus genome can provide dsRNA templates for Dicer . In July 2014, 10 colonies of A. mellifera bees from an apiary at the Amsterdam Water Dunes, The Netherlands, where Varroa has been present since the 1980s and where natural selection for Varroa-resistant colonies has been ongoing since 2008, were sampled  and transported at room temperature prior to storage at −80°C until processing. In Africa and The Netherlands, a minimum of 10 adult worker honey bees were sampled per colony, frozen immediately on dry ice, and stored at −80°C until processing.Honey bee colonies and mites from Europe, Africa, and the Pacific were sampled in 2013 to 2015. Seven  5 years . On Robb sampled . In OctoThorax and abdomen tissues of five adult bees from each colony (thorax only from Tonga) were homogenized in TRIzol reagent (Thermo Fisher), and total RNA was extracted according to the manufacturer's protocol. RNA from each individual was diluted to 200 ng/μl, and 2.65 ng RNA from each of the five individuals from each colony was pooled prior to DNase treatment (Ambion), followed by column purification . Total RNA was transported to the Australian Genome Research Facility (AGRF) on dry ice. Sample RNA integrity was confirmed by using a Bioanalyzer (Agilent). RNA was subjected to a ribosome depletion step (Ribo-Zero-Gold Human/Mouse/Rat) prior to the preparation of Illumina TruSeq Stranded Total RNA paired-end libraries according to the manufacturer's instructions. Libraries were run on an Illumina HiSeq2000 100-bp paired-end sequencing system for a total data yield of 4 to 9 Gb per sample.de novo by using Trinity . We also confirmed the arrangement of the ARV-2 G and L proteins and the putative inter-virus-family horizontal transfer event, using PCR for amplification across the two ORFs and Sanger sequencing for confirmation.We used Bowtie2  to map rVarroa mites was used to generate a small RNA library by using a TrueSeq small RNA kit (Illumina) according to the manufacturer's protocol. Samples were barcoded appropriately for pooling on an Illumina HiSeq2000 instrument, with 50-bp single-end sequencing.One microgram of total RNA (prepared as described above) from individual bees and A. mellifera genome, allowing for up to two mismatches (length fraction of 1 and similarity fraction of 0.9) due to the divergence between honey bee strains and the reference genome. The unmapped reads were subsequently mapped to ARV-1 and ARV-2 with the same stringency settings. The Amsterdam Water Dunes and Robben Island samples were mapped to the consensus viral genome generated by RNA sequencing (see above) from their geographic location. For further small RNA analysis , the mapped reads were exported as BAM files, indexed by using Samtools . The reads were first mapped to the Samtools , and theSamtools  and custx + 22 nucleotides along the sequence (iP)  using custom R scripts and then divided this value by the number of remaining out-of-phase reads (U). Phasing scores were calculated for each phase cycle position in the 22-nt window over 8 cycles, as the signal is expected to degrade over time due to imprecision in the cut length. The relative accumulation of reads at a particular phase cycle position will give a high phasing score. For the honey bee data, we specifically looked for a phase cycle of 22, since this was the predominant size of the small RNAs. For the Varroa data, we analyzed four different phase cycle lengths  using the following equation:i is the phasing cycle, iP is the number of small RNA reads at a given phase cycle position, U is the number of small RNA reads within the phase cycle not at the phase cycle position (out of phase), and n is the number of phase cycle positions occupied by at least one small RNA within the 8 cycles.To determine whether the small RNAs produced from the viral genomes showed evidence of Dicer phasing, we adapted an algorithm designed for detecting phased RNAs in plants . The logKY354230 to KY354234 and KY354236 to KY354244. Annotated NCBI protein sequence accession numbers are ARO50020 to ARO50067. Raw sequence data have been deposited in the Sequence Read Archive (SRA) (accession number SRP095071) (BioProject accession number PRJNA357165 and BioSample accession numbers SAMN06140203 to SAMN06140219).All virus genome sequences generated in this study have been deposited in the GenBank database under accession numbers


NRAS-mutations - most commonly in codon 61 (Q61R or Q61K) - can be detected in around 20% of cutaneous melanomas and have been found more frequently in nodular primary melanomas and primary tumors from skin sites with chronic UV-exposure , a substLancet Oncology [Recently, the results of this randomized multicenter trial were reported in Oncology . The triTaken together, binimetinib is the first kinase inhibitor to show clinical activity in a phase III trial in NRAS-mutated melanoma. However, despite statistical significance, the PFS improvement compared to chemotheraphy is modest, which has led to withdrawal of the new drug application for binimetinib at the FDA by the drug’s manufacturer in early 2017.n = 57) showed an improved median PFS of 5.5 months. Furthermore, treatment responses in this subgroup seemed to be more durable, lasting for a median of 11 months. The response rate in immunotherapy treated patients, in turn, remained unchanged (15.8% versus 15.2%). These observations point to a certain interaction between MEK-inhibitors and immunotherapy, with a positive outcome concerning MEK-inhibitor efficacy. We have recently summarized the various effects that MEK-inhibitors can have on the interaction between tumors and the immune system and, in particular, how they can positively influence the T-cell repertoire in the tumor microenvironment [Yet, a certain subgroup of patients with NRAS-mutated melanoma may achieve increased benefit from binimetinib treatment, which is worth clinical consideration. The pre-specified subgroup of patients with prior immunotherapy treated with binimetinib in the NEMO trial n = 5 showed aMeanwhile, before the advent and availability of further clinical studies combining MEK-inhibitors and immunotherapy, we believe that in patients with NRAS-mutated melanoma, binimetinib represents a new and valuable treatment option after failure of immunotherapy which is worth to be considered alternative to palliative chemotherapy or merely best supportive care, regardless of its current regulatory approval status. The efficacy of binimetinib treatment may also be improved through combination with other molecules such as CDK4/6, MDM2 or ERK inhibitors in the future.


Besides causing neuronal damage, traumatic brain injury (TBI) is involved in memory reduction, which can be a result of alterations in vasculo-neuronal interactions. Inflammation following TBI is involved in elevation of blood content of fibrinogen (Fg), which is known to enhance cerebrovascular permeability, and thus, enhance its deposition in extravascular space. However, the localization of Fg in the extravascular space and its possible interaction with nonvascular cells are not clear. The localization of Fg deposition in the extravascular space was defined in brain samples of mice after cortical contusion injury (CCI) and sham-operation (control) using immunohistochemistry and laser-scanning confocal microscopy. Memory changes were assessed with new object recognition and Y-maze tests. Data showed a greater deposition of Fg in the vascular and astrocyte endfeet interface in mice with CCI than in control animals. This effect was accompanied by enhanced neuronal degeneration and reduction in short-term memory in mice with CCI. Thus, our results suggest that CCI induces increased deposition of Fg in the vasculo-astrocyte interface, and is accompanied by neuronal degeneration, which may result in reduction of short-term memory. Traumatic brain injury (TBI) is one of the most common neurological disorders leading to memory impairment, causing reduction in short-term memory, in particular . The harC) in memory reduction has been shown [Sc [C complex and its association with reduction of short-term memory after CCI [It is known that increased presence of Fg-containing plaques leads to severe impairment in memory in diseases such as vascular dementia  and Alzhen shown ,10. It whown [Sc . Our prefter CCI . Howeverfter CCI . Whilst fter CCI .Since astrocytes are the main connective links between the cerebral vessels and neurons, we hypothesized that during inflammatory pathology , after crossing the blood vessel wall, Fg deposits at the interface between the blood vessel and the astrocyte endfeet. The place of Fg deposition in the extravascular space is important, as it may cause activation of existing astrocytes and thus result in vasculo-neuronal physical, and possibly functional, uncoupling, leading to neuronal dysfunction and thus reduction in memory. Therefore, in the present study, we investigated the location of Fg deposition 14 days after CCI when Fg can appear in the extravascular space only via crossing intact vascular walls. All animal procedures for the study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Louisville in accordance with National Institute of Health Guidelines for animal research.Polyclonal rabbit anti-human Fg antibody was obtained from Dako . Anti-NeuN monoclonal antibody (cloneA60), Anti-glial fibrillary acidic protein (GFAP) cocktail (SMI-22), and Fluoro-Jade C were purchased from EMD Millipore/Life Science . Secondary antibodies conjugated with Alexa-Fluor 488, Alexa-Fluor 594, or Alexa-Fluor 647 were from Invitrogen . 4,6-diamidino-2-phenyl-indole HCl (DAPI) was from Santa Cruz Biotechnology . Normal Donkey Serum (NDS) was purchased from Jackson ImmunoResearch . Texas Red-conjugated Lycopersicon Esculentum agglutinin (TXR-LEA) tomato lectin was from Vector laboratories . Male C57BL/6J wild-type (WT) mice were purchased from the Jackson Laboratory  and bred in-house. Twelve-week old, 26–30 g mice were anesthetized with 2.5% isoflurane and placed in a stereotaxic frame . The shaved skull was exposed, and a 4 mm diameter craniotomy was made at −2.5 mm bregma and 2.75 mm lateral to the midline over the left hemisphere without disturbing the dura mater . The impIt has been shown that after an inflammatory insult, the blood level of Fg increases starting from day 4 and reaches its maximal levels in 12–16 days . TherefoFourteen days after CCI and sham-operation, anesthetized mice were infused with TXR-LEA via carotid cannulation to fluorescently label the intravascular endothelial surface ,13. AnimFor further processing, samples were warmed and the mounting matrix was removed . The secIn another series of experiments, for Fluoro-Jade C staining, the mouse brain tissue preparation was done according to the manufacturer’s  recommendation. Briefly, slides were incubated in 80% ethanol containing 1% sodium hydroxide, then rinsed for 2 min in distilled water after rinsing in 70% ethanol followed by incubation in 0.06% potassium permanganate for 10 min. After rinsing in distilled water, slides were transferred to 0.0001% Fluoro-Jade C solution for 10 min and then thoroughly washed in distilled water. The slides were air dried at 50 °C for 5 min, cleared in xylene for 1 min and covered with glass coverslips.Brain samples were observed with a laser-scanning confocal microscope . Off-line image analysis software  was used to assess expression of Fluoro-Jade C, NeuN, GFAP, and deposition of Fg. For each experimental group, 3–4 brain slices were analyzed. Fluorescence intensities of Fluoro-Jade C and NeuN were measured in 5 randomly placed constant size areas of interest (AOI). The results were averaged for each experimental group and values were presented as fluorescence intensity units (FIU).To define the level of Fg deposition and its location in the extravascular space a total of 4 focal planes of the tissue samples were analyzed. Distance between the analyzed focal planes was set at 0.5 µm. Fluorescence intensity of Fg was measured in each image and was normalized by the length of the respective vascular segment. Data were presented as FIU. Co-localization of GFAP and Fg in brain tissue was assessed by measuring the number of spots generated in result of co-localization of respective colors in images formed after deconvolution of an original image as described earlier . ObtaineA novel object recognition test (NORT) was used to assess visual short-term memory , and wasUsing a Y-maze, a spontaneous alternation test was performed to assess spatial working short-term memory  as descrp < 0.05.All data are expressed as mean ± SEM. The experimental groups were compared by one-way ANOVA. Differences were considered significant if n = 4) in the extravascular space after CCI compared to that  in brains of sham-operated mice. We found that deposition of Fg occurred mainly in the vasculo-astrocyte endfeet interface (Immunohistochemistry analysis showed that Fg deposition was increased (61.1 ± 13.5 FIU; nterface A. Moreovnterface A. Signifnterface A. It is Expression of NeuN was decreased A,B, whilDR that was defined by NORT was lower in mice with CCI than in the control group A. DI andTo our knowledge, this is the first study to specifically show the location of Fg that appears in the perivascular space from non-ruptured microvessels after CCI. While previous studies found the presence of Fg/fibrin in amyloid plaques ,24 and FThe present data confirm our previous findings that deposition of Fg in the extravascular space is increased after CCI . HoweverHere we showed that after crossing the wall of non-damaged cerebral vessels 14 days after mild brain injury when vascular ruptures are minimal, Fg deposits in the existing vasculo-astrocyte endfeet interface. Being in close proximity to astrocytes, Fg may activate them and result in further mobilization of astrocytes to the location of Fg deposition, as shown by Schachtrup et al. .C [In conclusion, we present data that support the new hypothesis that after CCI, inflammation-induced increases in the blood level of Fg  enhancesC ,20 and/oC ,20, formC ,30, and


This gene-environment interaction (GxE) has been previously tied to the processing of emotional stimuli, which is increasingly recognized to be a key factor in ADHD-related impairment. The executive control and default mode brain networks play an important role in the regulation of emotion processing, and altered connectivity of these networks has also been associated with ADHD. We therefore investigated whether resting-state connectivity of either of these networks mediates the relation of 5-HTTLPR and stress exposure with ADHD severity. Resting-state functional magnetic resonance imaging, genetic, and stress exposure questionnaire data was available for 425 adolescents and young adults (average age 17.2 years). We found that 5-HTTLPR S-allele carriers showed a more negative relation between stress exposure and connectivity of the executive control network than L-allele homozygotes, specifically in the pre/postcentral gyrus, striatum, and frontal pole. In the default mode network, we found a positive association between the GxE and supramarginal gyrus connectivity. Connectivity of either network did not significantly mediate the effect of this GxE on ADHD. Opposite effects of stress exposure on connectivity in the executive and default mode networks may contribute to findings that stress exposure is associated with lowered cognitive control and heightened levels of rumination and worrying, for S-allele carriers but not L-allele homozygotes. When combined, these effects on connectivity of both networks may relate to the emotional problems seen in individuals with ADHD.We recently reported that the The online version of this article (doi:10.1007/s11682-016-9633-3) contains supplementary material, which is available to authorized users. Numerous papers have reported that people carrying the short variant  of 5-HTTLPR have a stronger association between stress exposure and psychiatric disorders than people homozygous for the long variant of this gene  consisting of individuals with and without ADHD, thus enabling analysis within a wide range of ADHD severity, in accordance with the continuous distribution of ADHD within the population  study  and the medical ethical committee of the VU University Medical Center. All participants signed informed consent .After selection of those who met the inclusion criteria and had complete phenotypic and good quality rs-fMRI data available, our sample consisted of 425 participants from 255 families. This sample contained 161 participants with a full diagnosis of ADHD, 53 participants with subthreshold ADHD .Genotyping was performed as described in Brookes et al.  and in tAs a measure of socio-economic status, the highest successfully completed education level of the parents was recoded into a measure reflecting years of education. This scale contained nine levels, ranging from 0  to 17 (university) years of education Buis . The aveAll subjects were scanned with either a Siemens MAGNETOM Sonata 1.5 Tesla (at VU UMC in Amsterdam) or a Siemens MAGNETOM Avanto 1.5 Tesla (at Donders Centre for Cognitive Neuroimaging in Nijmegen) MRI scanner , using identical protocols. Functional images during rest were obtained using a gradient echo echo-planar imaging (GE-EPI) sequence . Participants were instructed to relax with their eyes open during the rs-fMRI scan, which lasted 8 minutes.Preprocessing consisted of removal of the first five volumes, primary head movement correction by realignment to the middle volume, global 4D mean intensity normalization and spatial smoothing with a 6 mm Gaussian kernel. We corrected for secondary effects of head motion by applying ICA-AROMA, a robust ICA-based strategy for automatic detection and removal of motion-related artifacts , as calculated by the root mean squared of the frame-wise displacement time series. Subsequently, 19 individuals were removed based on scan quality , and 18 due to incidental findings after visual inspection (e.g. enlarged ventricles or unexpected hypo-intensities), for our final sample size of n = 425.Starting out with 486 participants who had behavioral and rs-fMRI data available, we first excluded the top 5 % of participants with highest motion score , as previously reported in a sample from which the current sample is a subset , but not in L-allele homozygotes .The executive control network covers large portions of the medial frontal lobe, frontal pole, and basal ganglia. The corresponding brain map is displayed in the 5-HTTLPR genotype bilaterally in the precentral gyrus extending into the postcentral gyrus, right frontal pole, left thalamus and caudate nucleus


Recently, numerous compounds have been studied in order to develop antibacterial agents, which can prevent colonized wounds from infection, and assist the wound healing. For this purpose, novel silver chloride nanoparticles stabilized with chitosan oligomer (CHI-AgCl NPs) were synthesized to investigate the influence of antibacterial chitosan oligomer (CHI) exerted by the silver chloride nanoparticles (AgCl NPs) on burn wound healing in a rat model. The CHI-AgCl NPs had a spherical morphology with a mean diameter of 42 ± 15 nm. The burn wound healing of CHI-AgCl NPs ointment was compared with untreated group, Vaseline ointment, and chitosan ointment group. The burn wound treated with CHI-AgCl NPs ointment was completely healed by 14 treatment days, and was similar to normal skin. Particularly, the regenerated collagen density became the highest in the CHI-AgCl NPs ointment group. The CHI-AgCl NPs ointment is considered a suitable healing agent for burn wounds, due to dual antibacterial activity of the AgCl NPs and CHI. Among various materials known to be effective against a variety of bacteria, silver (Ag) compounds  are unique materials due to their powerful antibacterial activities against nearly 650 bacteria strains. In addition, Ag compounds are well-known and have already been used in many biological and medical fields, such as biosensors, wound healing materials, dental resin composites, and cancer therapeutics [Antibacterial materials could be used in food packaging or handling, medical tools, hospitals where people are more vulnerable to infections, possible areas like bathrooms where personal hygiene is important, air and water filtration, apeutics ,2.+ or Ag0. In addition, this antibacterial effect accelerates wound healing [Traumatic wounds including burn wound occur frequently in skin loss, and bacterial infections often result from heavy contamination. There are many dressings and creams available to clinicians for use in treating burn wounds. Numerous agents are valued for their antibacterial activity, which can prevent colonized wounds from becoming infected, thereby assisting the wound to heal. Wound infection is one of the main problems and serious consequence in severe burns or extensive skin loss since bacteria produce leukocidin and tissue-destroying enzymes that further impair healing. As a result, wound infections alter and delay normal the wound healing mechanism. One of the approaches for treating a wound infection is the use of wound dressings containing antibacterial materials with a broad-spectrum of activity. Ag compounds have been widely found in wound dressings in various forms, such as elemental Ag , inorganic compounds , and organic complexes  ,4,5. Rec healing ,7. The s healing ,9. Indee healing ,11,12.Chitosan, a β-1,4-linked polysaccharide of glucosamine (2-amino-2-deoxy-β-D-glucose) with lesser amounts of N-acetylglucosamine, is a natural non-toxic biopolymer derived by deacetylation of chitin . Chitosain vivo wound healing by antibacterial CHI-AgCl NPs have not been studied.In previous study , AgCl NPThe present study was carried out to give an example of an effective burn wound healing agent based on synergistic effect from using both AgCl and CHI. We synthesized CHI-AgCl NPs using water-soluble chitosan oligomer by an environment-friendly method. Furthermore, the CHI-AgCl NPs ointment was prepared to evaluate the healing effect for burn wound using rat model with the expectation of applications in pharmaceutical and biomedical fields.-) remained in CHI molecule because CHI was made by acidic hydrolysis using chitosan in hydrochloric acid. CHI was used as a resource of Cl ions and stabilizing agent for preparing AgCl NPs. Distilled water was used as solvent in all the syntheses to provide benign environmental conditions in this system. In a typical preparation, 0.5 mL of 0.1 M AgNO3 solution was added to 60 mL of 5% (w/v) CHI solution. After complete dissolution, the mixture was reacted in a three-necked glass-stopper flask fitted with a double-walled spiral condenser to prevent evaporation and heated to 70 °C for 300 min. All solution components were purged with nitrogen gas to eliminate oxygen. After the formation of CHI-AgCl NPs, the suspension was freeze-dried immediately at –85 °C to produce the powder CHI-AgCl NPs. A series of experiments were performed to obtain transparent CHI-AgCl NPs by varying the order of reactants and the reaction temperature.Oligomeric chitosan  was supplied by Hyosung Co. (Korea), and its composition is as follows: dimer 2.31, trimer 12.53, tetramer 15.11, pentamer 13.59, hexamer 8.86, heptamer 6.46, octamer 8.87, and nonamer or higher 32.27 mol %. Cl ions  was used to record absorption spectra in the suspension of CHI-AgCl NPs using a cell with path length of 1.0 cm. The morphology of CHI-AgCl NPs was observed using a transmission electron microscope (TEM) . The samples for TEM observation were prepared by spotting a few microliters of the suspension of synthesized CHI-AgCl NPs onto a holey carbon TEM grid followed by drying before putting them into the TEM sample chamber.in-vivo burn wound healing were prepared from Vaseline, CHI, and CHI-AgCl NPs powder. The burn wound healing effect of CHI-AgCl NPs against CHI alone was examined, and Vaseline was used as a base component of the ointment. The composition of each ointment for burn wound healing is described in Three ointments for ad libitum under temperature-, humidity-, and lighting-controlled conditions . Procedures involving animals and their care were conducted in accordance with our institutional guidelines, which comply with international laws and policies [2 (AD = 6 mm) was made. Before creation of the burn wound, rats were anaesthetized by Tiletamine plus Zolazepam, (30 mg/kg + 10 mg/kg) according to body weight. Hair on the dorsal side of the rats was removed, and a burn wound was inflicted by placing the circular iron disc (heated to 95 °C) over the dorsal side for 20 s. Second-degree burns without cellular and tissue structure in the dermis were observed by sections stained with hematoxylin and eosin (H&E). Animals were divided after 7 days of acclimation in the cage, and then assigned equally (n = 30) to one of the following groups: Group 1-untreated controls dressed with gauze; Group 2-treated with the Vaseline ointment with gauze; Group 3-treated with CHI ointment with gauze; and group 4-treated with CHI-AgCl NPs ointment with gauze. Small amounts of ointment were applied everyday for 21 days. The sample tissue was enucleated after treatment 1, 3, 7, 14, and 21 days before sacrifice. The skin tissue for histopathological analysis was fixed in 10% buffered formalin, subsequently dehydrated, and embedded in paraffin. The tissue paraffin was cut into 5 µm sections. Fixed sections were then stained with H&E and Masson’s trichrome staining (MT).The experiment was approved by the institutional committee for animal care in laboratory research. For all experiments, four-week-old Sprague-Dawley rats were housed and bred at the experimental animal center of Chungnam National University. The animals were provided with a commercial diet and water policies . To induThe biochemical analysis of the whole blood was performed to confirm the healing process. The blood sample was obtained from the abdominal aorta of animals before sacrifice. After being placed in a serum tube containing the EDTA, and the tube was shaken with an orbital shaker. Complete blood counts were measured using an automatic blood chemical analyzer .The formation of CHI-AgCl NPs was confirmed easily by both the color change from yellow to yellowish brown with increasing reaction time and the strong surface plasmon resonance (SPR) peak around 400 nm due to the formation of spherical AgCl NPs . Figure in-vivo burn wound treatment to determine the effects of burn trauma on skin that was infected and dehydrated after sustaining injury. The survival curves were found to be significantly different between the ointment-treated groups (Groups 2–4) and untreated group (Group 1). The survival rates of the CHI-AgCl NPs ointment group were 90%, but those of the untreated, Vaseline, and CHI groups were 80% at 21 days (n = 30). In all groups of animals, most of the fatalities occurred between Day 1 and 6 because of the post-burning infection (3, the amount of WBC was somewhat increased in all of the untreated and treated groups after sustaining injury [A clinical pathology study was conducted during nfection a. In parg injury . It was The healing pattern of the burn wounds was studied to examine the histology for the untreated, Vaseline, CHI, and CHI-AgCl NPs groups on Days 1, 3, 7, 14, and 21 using H&E staining method . Generalp < 0.05 compared with the untreated, Vaseline, and CHI groups.Collagen is the main structural protein component of connective tissue, and is mainly found in skin. Therefore, it is desirable to observe the regenerated collagen in damaged skin during the healing process. To observe the collagenous components, the wounds with or without treated ointment were stained using a Masson’s trichrome staining method. In view of the rapid progress of application of nanomaterials in bioengineering fields, environmentally friendly methods should be required since the common methods generate toxic and biological hazards. The CHI-AgCl NPs was successfully obtained by a simple and environmentally benign method that used water and CHI as a biomaterial. The CHI is fundamental in the formation and stabilization of well-dispersed AgCl NPs with a mean diameter of 42 ± 15 nm. The burn wound treated with CHI-AgCl NPs ointment showed the highest percent of survival, and a reasonable number of white blood cells because of the prevention of infection in wound healing. The collagenous components were more regenerated in the CHI-AgCl NPs ointment group than the other treated groups. It was demonstrated that the CHI-AgCl NPs were more effective as a wound-healing accelerator because of improved antibacterial activity of CHI exerted by the AgCl NPs. Consequently, CHI-AgCl NPs ointment is considered a promising material for burn wound healing.