The hypothesized effect of vitamin D on C-reactive protein (CRP) has received substantial attention as a potential means to alleviate the risk for cardiovascular disease. However, observational studies have been inconsistent in their reporting of associations between serum 25-hydroxyvitamin D (25(OH)D) and CRP concentrations, and trials and meta analyses have been inconsistent in their conclusions regarding the effect of vitamin D supplementation on CRP concentrations. These supplementation trials were mostly conducted among patients with more or less inflammatory complications and did not consider potential distinctive effects by weight status. To further our understanding of the potential influences of vitamin D on CRP, we analyzed longitudinal observations of 6755 participants of a preventative health program. On average, serum 25(OH)D concentrations increased from 88.3 to 121.0 nmol/L and those of CRP decreased from 1.7 to 1.6 mg/L between baseline and follow up. Relative to obese participants without temporal increases in 25(OH)D, those who showed improvements of <25, 25–50, 50–75, and more than 75 nmol/L at follow up were 0.57 , 0.54 (0.34–0.85), 0.49 (0.30–0.80), and 0.48 (0.29–0.78) times as likely to have elevated CRP concentrations (≥1 mg/L), respectively. These associations were less pronounced and not statistically significant for normal weight and overweight participants. Herewith, the findings suggest that promotion of adequate serum 25(OH)D concentrations among obese individuals along with healthy lifestyles may alleviate the public health burden associated with cardiovascular disease. C-reactive protein (CRP) is a novel risk factor for cardiovascular disease (CVD) ,2,3. It Vitamin D is synthesized in the skin when exposed to sunlight, and can be ingested through the diet and supplementation . Where thttp://purenorth.ca/pure-north-program/). In brief, established in 2007, this program employs health professional to offer lifestyle counseling to volunteer participants. At enrollment, all participants complete a demographic and lifestyle questionnaire, and provide information regarding their medical history and medication and supplement use. Blood pressure and anthropometric measurements are taken by trained professionals, and blood is drawn for the assessment of several biomarkers including serum 25(OH)D and high sensitive CRP (hs-CRP). The information collected is used to guide lifestyle counseling. Vitamin D supplementation is encouraged given Canada’s Northern latitude, limited sunlight and inadequate cutaneous synthesis of vitamin D. Follow-up visits, which include both assessments and counseling, are scheduled annually. Data collected from October 2007 through April 2014 were used for the current analyses. Participants signed and granted written informed consent for the use of their relevant information for research purposes. All data were anonymized by PN prior to it being transferred to the University of Alberta for data analyses. Ethical approval was obtained from the Human Research Ethics Board at the University of Alberta (Pro00028578).The data for the present study were obtained from the Pure North S’Energy Foundation (PN), a not-for-profit organization, in Calgary, Alberta, Canada that offers a preventative health program to community volunteers. Details of the program have been previous publish elsewhere ,26 and fn = 606) due to the possibility that such a high concentration was caused by acute infections or inflammations rather than chronic inflammation [n = 139) due to the possibility of an acute infection. Observations were also excluded if information on weight or height was missing (n = 79). The final analysis included data from 6755 participants contributing to 6755 baseline visits and 10,383 follow-up visits  , was det® Liason chemiluminescent immunoassay with an inter-assay coefficient of variation (CV) of 11%. Baseline 25(OH)D were categorized into five groups  based on their distribution against serum CRP. The change in 25(OH)D was calculated by subtracting the baseline concentration from the follow-up concentration. Changes were categorized into five groups .Serum 25(OH)D concentrations were determined using DiaSorin2 (kg/m2); BMI was then categorized as “underweight (<18.5 kg/m2)”, “normal weight (18.5 to <25 kg/m2)”, “overweight (25 to <30 kg/m2)”, and “obesity (≥30 kg/m2)” [To determine body weight status, body mass index (BMI) was calculated for each participant as weight in kg/heightt 25 to < kg/m2”, ® 8000 Modular Analyzer Series was used to measure concentrations of serum triglycerides (inter-assay CV = 2%), total cholesterol (inter-assay CV = 1.5%), and high-density lipoprotein . LDL concentrations were calculated using the formula, “LDL = Total Cholesterol − HDL − (Triglycerides/2.2)”, and categorized as “Normal (<2.6 mmol/L)” and “Elevated (≥2.6 mmol/L)”. Blood pressure status was defined as “Normal” if systolic and diastolic pressures were <140/90 mmHg, and as “Elevated” if they were ≥140/90 mmHg or if the participant was using an anti-hypertensive medication. Information on age, gender, smoking status , alcohol consumption and physical activities for a typical week  was ascertained from the self-reported lifestyle questionnaire. The alcohol consumption status was defined as “non-drinker” for those who reported drinking <2 glasses per week and “drinker” for those who drank ≥2 glasses per week. Physical activity levels were determined by estimating the metabolic equivalent of task (MET) per week for each physical activity recorded by participants on their questionnaires. MET values were then multiplied by the time participants spent performing those activities per week (MET * hours per week), and the total for each week was categorized as “low (<10 MET hours/week)”, “moderate (10 to <20 MET hours/week)” and “high (≥20 MET hours/week)”. MET hours per week at baseline were subtracted from the MET hours per week at the follow-up visit to determine change in physical activity. Change in physical activity was categorized as “Negative change” if MET hours per week were greater at baseline than follow-up, “No change” if MET hours per week remained constant and “Positive change” MET hours per week were greater at follow-up than baseline.Low-density lipoprotein (LDL) cholesterol, age, gender, blood pressure, smoking, alcohol consumption, and physical activity have all been associated with CVD and were considered as potential confounding variables in the present study. Automated CobasDescriptive statistics were presented for both baseline visits and the last follow-up visits. Serum CRP concentrations of <1 mg/L representing “low CVD risk” were contrasted with serum CRP concentration of ≥1 mg/L representing “elevated CRP” that combines the categories “average CVD-risk” and “high CVD-risk” associated with elevated CRP . MultiplParticipant characteristics at baseline and at the last follow-up visit are presented in In the above analyses, we considered serum CRP concentration of ≥1 mg/L as “elevated CRP” to capture both average CVD-risk (≥1 mg/L and <3 mg/L) and high CVD-risk (≥3 mg/L). A comparison considering serum CRP concentration of ≥3 mg/L as “elevated CRP” and <3 mg/L as “not elevated” revealed similar findings in that the associations of baseline 25(OH)D concentrations and temporal changes in 25(OH)D concentrations with the risk for elevated CRP concentrations at follow-up were statistically significant for obese participants, but not for underweight and normal weight participants. These results are included in We considered various confounders in the analyses presented in We revealed that higher serum 25(OH)D concentrations at baseline were associated with a reduced risk for elevated CRP concentrations at follow-up among overweight and obese participants, and that temporal increases in serum 25(OH)D concentrations were paralleled with a reduction in the risk for elevated CRP concentrations among obese participants. No statistically significant associations of 25(OH)D with risk for elevated CRP were observed for underweight and normal weight participants. To the best of our knowledge, this is the first study that shows that changes in the risk for elevated CRP in response to temporal changes in 25(OH)D concentration differ by body weight.Several observational studies, though not consistently, reported inverse associations of serum 25(OH)D concentrations with CRP concentrations ,15,31,32Heaney et al.  showed tIn addition to excess body weight, the present study confirmed that many of the conventional CVD risk factors including elevated LDL-cholesterol , increasThe longitudinal design, large sample size and the wide range of serum 25(OH)D concentrations are strengths of the present study. As limitations we acknowledge that this study was conducted among volunteer participants of a preventive health program. These participants may not be representative of the general population. The preventive health program not only encourages supplementation with vitamin D, but rather healthy lifestyles in general. In this regard, we did report that physical active levels improved during follow-up. However, other lifestyle changes may also have occurred, though not recorded, and thus not allowing us to adjust for this in the analyses. Other limitations relate to inaccuracies of the vitamin D assay and the fact that vitamin D was routinely collected and subjected to the assay rather than batch-wise. The absence of information on serum parathyroid hormone concentrations as well as missing values for the confounding variables may have contributed to a partial adjustment for confounding. These limitations underline that caution is warranted in the interpretation and generalization of the present findings.In conclusion, we revealed high serum 25(OH)D concentrations at baseline and prospective improvement in 25(OH)D concentrations decrease the risk for elevated serum CRP concentration in individuals with excess body weight but not in those with normal weight. In Canada, where 20% of adults are obese  and 39%


Accumulated lines of evidence suggests that Na+ transport is most important in the ES, and that mitochondria-rich cells play crucial roles in Na+ transport in the ES. Several lines of evidence support the hypothesis that aldosterone may regulate Na+ transport in ES, resulting in endolymph volume regulation. The presence of molecules related to acid/base transport, such as H+-ATPase, Na+–H+ exchanger (NHE), pendrin (SLC26A4), Cl−–HCO3− exchanger (SLC4A2), and carbonic anhydrase in ES epithelial cells, suggests that acid/base transport is another important one in the ES. Recent basic and clinical studies suggest that aldosterone may be involved in the effect of salt-reduced diet treatment in Meniere’s disease.Ion transport and its regulation in the endolymphatic sac (ES) are reviewed on the basis of recent lines of evidence. The morphological and physiological findings demonstrate that epithelial cells in the intermediate portion of the ES are more functional in ion transport than those in the other portions. Several ion channels, ion transporters, ion exchangers, and so on have been reported to be present in epithelial cells of ES intermediate portion. An imaging study has shown that mitochondria-rich cells in the ES intermediate portion have a higher activity of Na The endolymphatic system homeostasis is crucial to maintain the normal function in the inner ear . The strOne of the pathological findings caused by the disturbance of homeostasis in the endolymphatic system is endolymphatic hydrops, which is known to be the typical pathological finding of Menière’s disease , 5. The The ES is divided into the following three parts on the basis of morphological features: proximal, intermediate, and distal portions , 7. The + and lower K+ and Cl− concentrations and lower pH than cochlear and vestibular endolymph.Endolymph in the ES is quite different in resting potential, ion concentration and pH from that in the other parts of the inner ear, such as the cochlea or vestibular organ Table  16–27].–27.16–27ESP has no negative potential induced by anoxia , 31.ESP shows different responses to several diuretics from resting potentials in cochlear and vestibular endolymph , 31–34. 2 adrenergic action [Catecholamines produce a reversible depression in ESP by βc action .ESP is mainly composed of an acetazolamide-sensitive part and an isoproterenol-sensitive part .Resting potential in the ES named endolymphatic sac potential (ESP) , 29 was +-ATPase [+ transport from the ES lumen to the outside although its physiological roles remain to be clarified.ESP is assumed to have plural origins, one of which may be H+-ATPase . The pre+–K+–2Cl− cotransporter 2 (NKCC2) [+–Cl− cotransporter (NCC) [Molecules related to ion transport in epithelial cells in the intermediate portion of the ES are shown in Table  (NKCC2) , 59, 60 er (NCC) , 58, whiSeveral isoforms of aquaporin (AQP) as molecules related to water transport in ES epithelial cells have been reported, as shown in Table ES epithelial cells have resting membrane potential of approximately −60 mV .ES endolymph has resting potential of approximately +15 mV , 37, 64.+ concentration and lower K+ and Cl− concentrations. There are active Na+ and Cl− outflows from the ES lumen into the outside [ES endolymph has a higher Na outside , 24.+ and Na+ are permeable ions, but Cl− is a negligible ion in the ES isolated epithelial cells [Kal cells .+, K+-ATPase and a higher Na+ permeability [Mitochondria-rich cells in the ES have a higher activity of Naeability .+-ATPase may be involved [ES endolymph has a weak acidity , in whicinvolved .+-ATPase [+–H+ exchanger (NHE) [−–HCO3− exchanger (SLC4A2) [ES epithelial cells have molecules related to acid/base transport, such as H+-ATPase , 50, Na+er (NHE) , 55, pener (NHE) , 50, 55,(SLC4A2) , and car(SLC4A2) .Recent lines of evidence on ion transport in the ES  is as follows:+ imaging study [+, K+-ATPase and a higher Na+ permeability, strongly suggesting that molecules related to Na+ transport may be densely located in mitochondria-rich cells. Mitochondria-rich cells in ES have characteristic qualities of Na+ absorption , which is involved in the maintenance of negative intracellular potential. The model is similar to that found classically in several other Na+-absorbing epithelia [+ transport is a major part of ion transport system in ES.Our Nang study  demonstrion Fig. . Na+ entpithelia . Na+ tra+-ATPase, Na+–H+ exchanger (NHE) and pendrin in the apical membrane, Cl−–HCO3− exchanger (SLC4A2) in the basolateral membrane, and intracellular and membrane-bound carbonic anhydrase [+-ATPase, pendrin, and carbonic anhydrase have been shown to be localized in the same type of ES epithelial cells [+ into the lumen is necessary to be larger than the inflow of HCO3−. NHE in the apical membrane, which is presumed to be active due to a large Na+ inflow into the cell, besides H+-ATPase may be largely involved in the acidity of ES endolymph. Acid–base transport is another important part of ion transport system in ES.Molecules related to acid/base transport are Hnhydrase . H+-ATPaal cells . Pendrinal cells . For the− transport have been reported to be an ion channel  in the apical membrane [−–HCO3− exchanger (SLC4A2) in the basolateral membrane [− concentration of ES epithelial cells. When intracellular Cl− concentration of ES epithelial cells is assumed to be similar to 4 mM in other tissues [− into the cell is calculated to be around 0 mV. Since the results using whole cell patch clamp showed that Cl− current was negligible in the ES isolated epithelial cells [− is a leading ion in ES ion transport system. Cl− is assumed to be transported following Na+ transport and acid/base transport.Molecules related to Clmembrane , ion cotmembrane , 58, andmembrane , and Cl−membrane ). CFTR hmembrane . There h tissues , the eleal cells , it is u+ transport in other tissues such as the kidney [+ transport in ES has been accumulated in comparison with other candidates as follows:The presence of mineralocorticoid receptors (MRs) in ES epithelial cells has been shown .11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which enables aldosterone selectively to bind to MRs by converting cortisol (corticosterone) into inactive metabolites, has been detected in ES epithelial cells . The preThe presence of ENaC in ES epithelial cell has been shown .The presence of NCC in ES epithelial cells has been shown . NCC hadThe antagonist of aldosterone, canrenoate, intravenously produced a decreased ESP change with no change in the endocochlear potential, suggesting that aldosterone could act more sensitively on the ES , 31.Several agents, such as vasopressin , 69, 70,e kidney , 80, 81.+ transport from endolymph into ES epithelial cells, mainly mitochondria-rich cells in a similar manner to epithelial cells in other aldosterone-target tissues (Fig. + absorption increases water absorption, resulting in increased endolymph absorption.Aldosterone activates Naues Fig. . Activat+ transport, resulting in efficient endolymph absortption in the ES.Several AQP isoforms including AQP2 activated by vasopressin have been detected in the ES epithelial cells, as shown in Table + transport in the ES give an experimental support to empirical salt-reduced diet treatment in Meniere’s disease. Our recent study [Plasma aldosterone concentration has been reported to be within the normal range in patients with Meniere’s disease , 87. Thent study . The prent study , 58 may The presence of vasopressin-AQP-2 system in ES epithelia may play important roles in endolymph volume regulation , 61, sugThe findings that catecholamines increased the hydrostatic pressure of cochlear and vestibular endolymph , 77 prob


The Random Forest (RF) algorithm for supervised machine learning is an ensemble learning method widely used in science and many other fields. Its popularity has been increasing, but relatively few studies address the parameter selection process: a critical step in model fitting. Due to numerous assertions regarding the performance reliability of the default parameters, many RF models are fit using these values. However there has not yet been a thorough examination of the parameter-sensitivity of RFs in computational genomic studies. We address this gap here.p/n ratios: sequencing summary statistics (low p/n) and microarray-derived data (high p/n). Here, p, refers to the number of variables and, n, the number of samples. Our findings demonstrate that parameterization is highly correlated with prediction accuracy and variable importance measures (VIMs). Further, we demonstrate that different parameters are critical in tuning different datasets, and that parameter-optimization significantly enhances upon the default parameters.We examined the effects of parameter selection on classification performance using the RF machine learning algorithm on two biological datasets with distinct p/n data. Therefore, there is significant benefit to be gained by model tuning RFs away from their default parameter settings.Parameter performance demonstrated wide variability on both low and high The online version of this article (doi:10.1186/s12859-016-1228-x) contains supplementary material, which is available to authorized users. Machine learning (ML) techniques are widely used in the analysis of high-throughput data to answer a broad range of biological questions. Applications in the field of medicine have transformed our understanding of complex genomic interactions and measurements . ML has p) than samples ; potentially involving highly noisy and significantly correlated variables [Recently, the random forest (RF) algorithm  for ML hariables , 20. Dueariables , 22. Conariables .sampsize [sampsize setting, after each sample is drawn, a decision tree is created. In the most commonly-used implementation, fully-grown or unpruned decision trees are created [ntree [mtry. Of this subset of randomly selected variables, the one that forms the best split is selected [To train a random forest model, a bootstrap  sample isampsize . By defasampsize . Indepen created . The numd [ntree . This cod [ntree  and is cd [ntree , 29. Sind [ntree , 29; howselected , 30. Theselected . Severalselected , 31–34.Machine learning algorithms frequently require estimation of model parameters and hyper-parameters, commonly through grid-searching . Surprisp/n ratio studies. Each sample was classified as “good library” or “bad library” based on information external to the 15 features, and our models aimed to predict this binary response variable.To evaluate the sensitivity of RF models to parameterization, we selected two datasets representative of those commonly used in computational biology. The first studies quality-control metrics in next-generation sequencing  and compp/n studies and comprises three categorical clinical variables and 12,135 continuous mRNA abundances for Non-Small Cell Lung Cancer (NSCLC) patients [The second dataset reflected high ρ) and Lin's Concordance Correlation Coefficient (ρc).For both datasets, we performed two model-fitting steps Fig. . First, p/n dataset. We created 1,500 different sets of parameters and evaluated the performance of each. Most models succeeded at this task  performed well, with an AUC of 0.9726 and ranked in the top 12 % of all models . Interestingly though, the global error rate was not dramatically different between these two groups (20 % for “good library” vs. 14 % for “bad library” samples).We asked if models were consistently struggling with the same samples. We looked for samples in the validation dataset where at least 50 % of models trained with different parameter sets made incorrect predictions. In total 73/576 (12.7 %) of validation samples were difficult to classify. These were strongly asymmetrically distributed between the classes with 72/432 (17 %) “good library” validation samples difficult to classify relative to only 1/144 (1 %) “bad library” validation samples (mtry) was strongly negatively correlated with AUC (ρmtry = -0.895) and mtry ≤ 3 resulted in higher classification accuracy . In contrast, models were relatively robust to changes in the ntree and sampsize parameters .Parameterization was strongly correlated to AUC score Fig.  in this sampsize values did not differ significantly from each other, however, ntree of 10 had significantly lower AUCs (q < 0.05) than other setting . All validation folds were pooled to evaluate AUC and cross-validated models were compared to non-cross-validated models using Spearman's ρ and Lin's ρc  had a RMSE of 0.00203, whereas the default model (rank = 579) had a RMSE of 0.0273. The optimal model in stratified 10-fold cross-validation  had a RMSE of 0.0119, whereas the default model (rank = 319) had a RMSE of 0.0229. Overall, we found that 39 % (578/1500) and 21 % (318/1500) of models outperformed the untuned model , respectively. Twenty one percent (310/1500) of these models shared the same parameter values and were found to perform better than the default settings in both cross-validated and non-cross-validated results. We found the addition of a second metric, RMSE useful in breaking ties and assessing model performance for low p/n data.We found that cross-validation and stratified cross-validation resulted in 97 % of models having an AUC of 1, including the defaults. We used an additional metric, root mean squared error (RMSE) to break ties. The optimal model in 10-fold cross-validation  performed well relative to other models, with an AUC of 0.6098 and ranked 10th. This demonstrates the near optimal performance of the default settings.To contrast these data, we examined the effects of parameterization on prediction accuracy for high p/n data  . The error rate was significantly different between these two groups .We asked if models were consistently struggling with the same samples. We looked for samples in the validation dataset where at least 50 % of models trained with different parameter sets made incorrect predictions. In total 89/186 (48 %) of validation samples were difficult to classify. These were symmetrically distributed between the classes with 37/74 (50 %) “death” events difficult to classify compared to 52/112 (46 %) “no death” samples  was the most correlated, followed by ntree  and sampsize .Parameterization was strongly correlated to AUC in this dataset, with contribution from all parameters. We observed that ntree values resulted in higher prediction accuracy and reduced performance variability compared to lower values (q < 10−8), with no significant difference observed between values ntree ≥ 10,000 . We observed that mtry demonstrated the highest Gini VIM for low p/n data . Interestingly, the parameters demonstrated relatively balanced importance measures with sampsize demonstrating the highest Gini VIM and ntree with the lowest  , 36. We mtry. On the other hand, “% bases ≥ 50 %” was found to have higher VIMs with lower mtry values.Variables differed in their sensitivity to parameter changes when evaluating variable importance Fig. . The varntree values (n = 10) while holding mtry and sampsize constant . These findings challenge the assumption that RFs are relatively robust. Parameters that did not a play key role independently had an observable and significant synergism when constructing RF regression models with interaction terms (from section Parameters can be used to predict performance).To our knowledge, this is the first computational genomic study that addresses parameter sensitivities using a comprehensive range of values for two unique biological data types. In particular, we observed that the low We also noted that our variable importance ranks did not coincide with . This wap > > n and minor class imbalances were observed. The numerous variables in the high p/n data constrained the selection range of mtry parameters, potentially confounding the results. In such samples, mtry ≠ p. This was not the sample for the low p/n data, where we were able to test all possible values of mtry. This limitation may also be viewed as beneficial since the number of randomly selected variables at each split is constrained and therefore, limits tree correlation within a forest.Moreover, characteristics of the data, such as, p/n data and high p/n data respectively, had a higher classification accuracy suggesting, in some instances, the heterogeneity of a sample is more influential on classification accuracy. We also aimed to mitigate class imbalance effects through stratified sampling and by using the AUC performance metric. Alternate methods such as, cost sensitive learning [An additional data characteristic limiting the classification accuracy in RF could be class imbalance , 46. Thelearning  and artilearning , over salearning , or bothlearning  have beep/n and high p/n data. The model fitting process is a fundamental step in machine learning and careless parameter selection can lead to sub-optimal models and potentially missed findings.We analysed the effects of parameterization using exhaustive selection methods and showed that tuning can be successfully applied to a non-parametric machine learning algorithm to improve prediction accuracy. Although we only examined two different genomic datasets, we observed that parameter sensitivities are data-specific, necessitating per-dataset tuning. Our findings illustrate this through discordant correlations between parameters and performance scores for low p/n data) [p/n data) [p/n data, respectively.We explored parameterization of RFs on two datasets. The first was a sequencing-derived dataset (low /n data)  and the /n data) , reflectp/n data  contained 15 quality metrics describing overall coverage, coverage distribution, basewise coverage and basewise quality of 53 whole genomes. The data was derived for the International Cancer Genome Consortium (ICGC) project to predict the amount of sequencing that is required to reach a given coverage depth for 1/8 lane samples [p/n paper [The low p/n data contained gene expression data for 442 lung adenocarcinomas and basic clinical covariates  to predict lung cancer patient outcome (0 for “no death” or 1 for “death”). The data were collected from six contributing institutions and grouped into four subsets based on the laboratory where processed (University of Michigan Cancer Center (UM), Moffitt Cancer Center (HLM), Memorial Sloan-Kettering Cancer Center (MSKCC), and Dana-Farber Cancer Institute (DFCI)). All facilities processed the data using the same robust and reproducible protocol.The high The first two datasets, UM and HLM, were grouped together to form the training set , while the MSKCC data (104 samples) and DFCI data (82 samples) formed the validation set (186 samples).mtry parameter values were selected using factor levels of the default value. Since the nature of this supervised learning problem is that of classification and not regression, the default value of mtry is the square root of the number of variables or features in the data 18 √p, whereas, in regression the default is p/3. The study by [mtry as the most sensitive parameter with values of mtry factor = 1/2 (1/2• 18 √p), mtry factor = 1 (18 √p) and mtry factor = 2 (2•18√p) showing good performance. Given this information and the number of variables in the data, one to all variables were selected as mtry values for the SeqControl dataset (p = 1-15), the mtry values 1, 5, 11, 22, 55, 110, 220, 550, 1100, 2200 were selected for the NSCLC data . The NSCLC values were obtained by selecting factor levels , multiplying them with p and taking the largest integer preceding a specified number i.e., for a value of 3.4, 3 was used.The study by  reportedntree were selected similarly to those for mtry. We imposed factor levels to the default value and took the product to create the ntree values. The factor levels were 1/50, 1/10, 1/5, 4/10, 1, 2, 20, 100, 200 and 1000. The final ntree values were 10, 50, 100, 200, 500, 1000, 1e4, 5e4, 1e5, 5e5. The selected ntree values were the same for both datasets.The values for sampsize, had the same factor levels for both datasets and was a sequence of values from 0.1–1, increasing by increments of 0.1. To obtain the final sampsize values, we multiplied the total number of samples in training by the sampsize factor levels and took the smallest integer proceeding a number i.e., for a value of 3.4, 4 was used.The final parameter Selected parameters were used to train models with the function “randomForest” using sampling with replacement. The data was partitioned according to the original papers, as described above. In the SeqControl data experiment, we aimed to predict whether the target of sequencing depth coverage was achieved using 1/8 lane . In the NSCLC data experiment, we aimed to predict patient outcome . A table of complete parameter settings for the SeqControl data and NSCLC data can be found in Additional file ntree, mtry and sampsize. For the SeqControl data, we used 15 mtry values, 10 ntree values, and 10 sampsize values. These values and numbers differed slightly in the NSCLC training: 10 mtry values, 10 ntree values, and 10 sampsize values. A resulting total of 1500 and 1000 unique combination were obtained for model fitting on the SeqControl data and NSCLC data, respectively.The data were trained using the function “randomForest” from the R package “randomForest” (v4.6-10) , 52. A sAfter training, each model was then validated on independent validation data to obtain class probabilities (votes). The votes and true class labels were then used to estimate model performance by calculating the AUC score.ntree = 200, mtry = 2, sampsize = 200) to train a RF model. The response for our model was AUC score and the variables were ntree, mtry and sampsize. The expression for the model formula included the terms in an additive and interaction format i.e., sampsize + mtry + ntree + sampsize*mtry + sampsize*ntree + ntree* mtry + sampsize*ntree*mtry. After training and validating the models, we were able to assess performance using the following metrics, Spearman's ρ, Spearman's p-value (P) and Lin's ρc. Lastly, importance values were found for each variable  in the form of Gini VIM.In order to determine whether model performance could be predicted, we performed regression using RF, on a subset of parameters and their respective AUC scores. AUC scores were calculated by comparing the predicted votes from each model to the true classifications. We initially attempted this from a linear model approach, however, classification accuracy was low due to overfitting. After subsetting 2/3 of the data into training and 1/3 for validation, we performed model tuning and selected the model with the lowest mean squared error. Tuning was conducted using a grid of parameters  for the SeqControl data. This method of cross-validation has been suggested to perform better than the more expensive leave-one-out cross-validation [n = 720).Ten-fold cross-validation was used to estimate the generalization error of each unique RF model   and paraρ and to find the correlation coefficient between the AUC scores and the parameter of interest. Spearman's ρ, Spearman's p-value and the equation for Lin's ρc from the paper [p-values were adjusted using the function “p.adjust” from the base “stats” package (v3.2.0), using the Benjamini-Hochberg procedure.We evaluated the performance of models using several statistical measures in the R statistical environment (v3.1.3) . For clahe paper  were useFigures were generated in the programming language LaTeX and in the R statistical environment (v3.1.3) using custom R scripts for the “lattice” (v0.2-31)  and “lat


By using thefractal theory and the methods of weight function, a Hilbert-type fractal integral inequality and its equivalent form are given. Their constant factors are proved being the best possible, and their applications are discussed briefly. Some researchers have used the fractal theory to discuss and generalize some classical inequalities on fractal sets , 6, but A non-differentiable function α  . If for Suppose thatandThen, forwe have anα-differential formLetIbe an interval, is the interior ofI) such thatThen the following differentiation rules are valid: (i)(ii)(iii)(iv)whereCis a constant;(v)Ify(x)=(f∘g)(x), thenLet Suppose thatthen we haveSuppose thatandthen we haveForwe have the following equations: , 10Ifis a fractal surface, then we have(i)Hölder’s inequality on the fractal set(ii)Hölder’s weighted inequality on the fractal setThe inequality keeps the form of equality, then there exist constantsAandBsuch that they are not all zero anda.e. onSuppose thatand weight functions are defined byThenwhereSet Similarly, we obtain Suppose thatandis small enough, let us define the real functions as follows: then we haveNote the properties of local fractal space , 7: \docLet Further, let Introducing the mark: y)α (see ).Ifandthenwhere the constant factordefined in ,,\documenLetting Under the conditions of Theorem we havewhere the constant factoris the best possible, and inequality )<∞, by  still ke))<∞, by .On the other hand, by Hölder’s inequality on the fractal set and , we findThe above inequality is , therefoIf the constant factor in  is not oηp(α) in  is the bα values in ,,α valuesLetting  formula , we get mentclass2pt{minimLetting  formula , we get Letting  formula , we findIn the paper, based on the local fractional calculus theory, a Hilbert-type fractional integral inequality and its equivalent form are tentatively researched. The results show that some methods and skills of the Hilbert-type integral inequality can be transplanted to the research of Hilbert-type fractional integral inequality, which provides a new direction and field to research Hardy-Hilbert’s integral inequalities.


Accidents or neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) can lead to progressing, extensive, and complete paralysis leaving patients aware but unable to communicate (locked-in state). Brain-computer interfaces (BCI) based on electroencephalography represent an important approach to establish communication with these patients. The most common BCI for communication rely on the P300, a positive deflection arising in response to rare events. To foster broader application of BCIs for restoring lost function, also for end-users with impaired vision, we explored whether there were specific time windows during the day in which a P300 driven BCI should be preferably applied.The present study investigated the influence of time of the day and modality  on P300 amplitude and latency. A sample of 14 patients (end-users) with ALS and 14 healthy age matched volunteers participated in the study and P300 event-related potentials (ERP) were recorded at four different times  during the day.Results indicated no differences in P300 amplitudes or latencies between groups  or time of measurement. In the auditory condition, latencies were shorter and amplitudes smaller as compared to the visual condition.Our findings suggest applicability of EEG/BCI sessions in patients with ALS throughout normal waking hours. Future studies using actual BCI systems are needed to generalize these findings with regard to BCI effectiveness/efficiency and other times of day. For people with advanced neurodegenerative diseases or in states of severely reduced mobility due to accidents or illness, a brain-computer-interface (BCI) might be the only means to communicate with their environment. A BCI uses brain signals most often recorded with electroencephalography (EEG) and converts them into control signals replacing, restoring, or enhancing the natural output of the central nervous system the patient (end-user of BCI technology) is no longer able to perform  .the effect of the daytime is stronger in ALS patients than in healthy controls (group x time interaction).To provide such data, we recorded visual and auditory P300 in patients diagnosed with ALS and age matched healthy participants at four different time points within 1 day in their homes. Specifically, we hypothesized thatData presented in this paper were taken from Ursula Mochty’s dissertation  and reanM =67.93, SD = 10.46), healthy participants were between 38 and 79 years of age . All ALS patients were assessed with the revised ALS functional rating scale , with lower scores indicating more severe physical impairment.A sample of 14 ALS patients and 14 age and sex matched healthy control subjects participated in the study. We only included ALS patients who were still able to communicate a “Yes” or “No” answer in any modality, thus, no ALS patients in the complete locked-in state were included. All participants had normal or corrected to normal vision. ALS patients were between 40 and 73 years old  and auditory oddball (4 min). During oddball paradigms, participants were instructed to silently count the deviant stimuli.Auditory stimuli were 160 standard (1000 Hz) and 40 deviant (2000 Hz) tones (rectangular pulse of 50 ms duration) presented in pseudo-random order with the restriction that any two deviant tones were separated by at least one standard tone. Stimulus onset asynchrony (SOA) was 1200 ms with a random latency jitter of ± 0.15 s to avoid habituation to a certain SOA .Visual stimuli were short (50 ms) presentations of the letters “H”  and “S”  in white font (120 pt) on a black background in the centre of a computer monitor . StimuluEEG was recorded using a g-tec USB amplifier  with a sampling rate of 256 Hz (band-pass filter: 0.01 and 30 Hz) from 16 electrodes placed at Fp1, Fp2, F3, Fz, F4, T7, C3, Cz, C4, T8, CP3, CP4, P3, Pz, P4 and Oz referenced to the right-earlobe (ground at left mastoid). Impedances were kept below 5 kΩ.Offline, EEG was epoched into 900 ms long intervals beginning from −100 ms before the start of a stimulus, corrected for eye movements , and ali2, [Amplitudes and latencies were analyzed at midline electrodes  using repeated measures ANOVAs with within-subject factors time , region , modality  and the between-subjects factor group , and we report Greenhouse-Geisser—corrected p-values (after Mauchly’s Test). Effect sizes are reported as eta-square , and Co2, . Margina2,  with deg2, ) indicatThe correlation between ALSFRS-R and P300 mean amplitude/latency was based on the respective averaged Fisher Z-transformed Spearman correlation coefficients and evaluated using a t-test. EEG curves in Fig. F = 7.23, p = .013, η2 = .07) and region  = 17.62, p < .001, η2 = .06 see Fig. F(6144) = 2.17, p = .049, η2 = .001). Follow-up analysis indicated that amplitudes in the auditory modality were on average smaller than in the visual modality , and amplitudes at Fz were smaller than at Cz  and Pz , but comparable between Cz and Pz . Follow-up analysis  on the interaction of group, time, modality, and region did not reveal significant differences in amplitudes between time of day and group .Analysis of P300 amplitudes indicated significant main effects of modality  = 119.13, p < .001, η2 = 0.38), an interaction of modality and region  = 6.07, p = .011, η2 = 0.02, see Fig. p < .001), but the latency difference between the two modalities was larger at Fz than at Pz  with no significant differences between either Fz and Cz and Cz and Pz .Analysis of P300 latencies revealed a significant main effect of modality  or latency .To test for potential interrelations between severity of ALS (ALSFRS-R score) and P300 amplitude/latency, we calculated correlational analyses in the patient sample. Disease severity did not correlate with P300 peak amplitude  should be considered as factors influencing P300 amplitude and latency when conducting BCI sessions in patients with ALS.Our hypotheses predicted a general decline of P300 amplitudes and increased latencies throughout the day, and that this proposed effect would be even more pronounced in patients with ALS. Our analyses neither revealed significant differences in ERP amplitudes/latencies between patients with ALS and healthy participants, nor changes with time of day. Instead, we only found that in the auditory domain, amplitudes were smaller and latencies shorter than in the visual domain. P300 amplitudes in both modalities were more positive at Cz and Pz than at Fz.These results indicate that in patients with ALS P300 amplitudes comparable to those of healthy participants can be elicited in both modalities, which supports the feasibility of a P300 BCI also in late stage ALS. Although some previous studies obtained similar results , 30, thiIn a previous study, Wesensten and colleagues  found deAlthough auditory stimulation led to overall smaller P300 amplitudes, amplitudes in ALS patients were still comparable to those of healthy controls. Previous studies have already stressed the need to establish auditory BCIs in ALS patients , 54, as Data collection took place at the participants’ homes, and analyses were deliberately restricted to midline positions. This was done bearing in mind real-world application scenarios for BCIs, especially with regard to patient’s desire for limiting the number of electrodes as much as possible . While sTaken together, the data presented in this study suggest that P300-based BCIs could be applicable in patients with ALS throughout normal waking hours (10 am–4 pm). Patients presented with P300 amplitudes and latencies comparable to those of healthy participants in both, the auditory and the visual domain. Furthermore, there was no correlation between the constraints in physical functioning measured by the ALSFRS-R and P300 physiology. This means that reliable P300 potentials can be elicited also in late stages of the disease, thus preserving the basic requirements for BCI use. This is also supported by single-case studies which evaluated the usability of BCIs during long-term independent home use , 64.In sum, the present results show the feasibility of P300 ERP recordings in patients with ALS throughout the day, and, hence, the P300’s potential usefulness as a BCI input signal in long term independent home use scenarios.


Interferon lambdas (IFNλs) are recently discovered cytokines acting not only at the first line of defense against viral infections but also at the mucosal barriers. In fact, a peculiar feature of the IFNλ system is the restricted expression of the functional IFNλR, which is known to be limited to epithelial cells and discrete leukocyte subsets, including the plasmacytoid dendritic cells (pDCs). In the latter case, current data, discussed in this minireview, indicate that IFNλs positively regulate various pDC functions, including pDC expression of interferon-dependent gene (ISG) mRNAs, production of cytokines, survival, and phenotype. Although the knowledge of the effects on pDCs by IFNλs is still incomplete, we speculate that the peculiar pDC responsiveness to IFNλs provide unique advantages for these innate immune cells, not only for viral infections but also during autoimmune disorders and/or tumors, in which pDC involvement and activation variably contribute to their pathogenesis. Humans (pDCs) , but nots (pDCs) , 10. Migs (pDCs) , 12. Oncs (pDCs) , 14.via induction of antiapoptotic genes  . IFNα, iic genes . Activatic genes , 10, 24.Plasmacytoid dendritic cells also produce type III IFNs/IFNλs , for insInterferon lambdas have been described to stimulate the production of cytokines and chemokines in pDCs. We reported that human pDCs incubated for up to 42 h with 30 IU/ml IFNλ1 or IFNλ3 produce variable, but significant, levels of CXCL10, usually  followed by IFNα . Consist+-monocytes in the presence or absence of reagents inhibiting TNFα, namely etanercept (ETA) and adalimumab, revealed that they induced CCL4 and IκBα mRNA expression in a TNFα-dependent manner  and IRF7 mRNAs were found as induced in murine pDCs incubated with 100 ng/ml IFNλ2  of the variable capacity of pDCs to produce IFNα and CXCL10 by the various donor typologies that we described (As mentioned, given the peculiar expression of IFNλR1 in hepatocytes, clinical trials of IFNλ1 therapy for HCV infection have confirmed that this cytokine has antiviral effects equivalent to IFNα without the same level of associated toxicity . Studiesescribed . Regardlescribed . In thesescribed . Moreoveescribed  and in sescribed , 70, thuescribed , 70. In escribed . Similarescribed . Altogetescribed .via IFNα, which mediates NK cell activation. Thus, based on our unpublished observations indicating that IFNλs, in addition to triggering IFNα production, also induce TRAIL mRNA expression in human pDCs, it would be plausible speculating a potential use of IFNλs as adjuvants to chemotherapy regimens (Neoplastic cells frequently express IFNλR1 and, after treatment with IFNλs, stop the cell cycle and/or undergo apoptosis , 73. In regimens . AccordiGF, NT, and MC have contributed by writing the manuscript.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.


There was also a significantly negative association between high level of UCA1 and poor grade cancer  and positive lymphatic metastasis . In conclusion, our study suggested that UCA1 was correlated with more advanced clinicopathological features and poor prognosis as a novel predictive biomarker of patients with various tumors.Urothelial cancer associated 1 (UCA1) as an oncogenic long non-coding RNA (LncRNA) was aberrantly upregulated in various solid tumors. Numerous studies have demonstrated overexpression of UCA1 is an unfavorable prognostic indicator in cancer patients. This study aimed to further explore the prognosis role and clinical significance of UCA1 in cancer. Eligible studies were recruited by a systematic search in PubMed, Embase, Cochrane Library and Web of Science databases. A total of 19/16 studies with 1587/1291 cancer patients were included to evaluate the association between UCA1 expression and overall survival (OS) and clinicopathological factors of malignancies by computing hazard ratio (HR), odds ratios (OR) and confidence interval (CI). The meta-analysis indicated overexpression of UCA1 was significantly correlated with unexpected OS in patients with cancer (pooled HR = 1.85, 95% CI 1.62–2.10,  Non-coding RNAs (ncRNAs) encoded by the eukaryotic genome are considered as a large number of RNAs which are not translated into proteins. Indeed, more than 90% of noncoding RNAs used to be recognized as “biological noises” in transcription progression compared with about 3% protein-coding genes until the development of high-throughput sequencing technology and large-scale mapping of transcriptomes , 2. BaseUrothelial cancer associated 1 also known as UCA1 is located on 19p13.12 encodes 3 isoforms  with ployA tails, in which the 2.2 kb isoform is called cancer upregulated drug resistant (CUDR). Previous study has shown UCA1, a highly conserved nuclear-enriched lncRNA, is ubiquitous in different development stages of urinary system, productive system, digestive system and respiratory system . Cheng eFurthermore, increasing interest has been focused on whether UCA1 acts as a diagnosis and prognosis biomarker in cancer detection and treatment. UCA1 was firstly characterized as an effective diagnosis biomarker in bladder cancer with a high sensitivity and specificity (80.9% and 91.8%) . MeanwhiUpon an electrical search on PubMed, Embase, Cochrane Library and Web of Science databases, a total of 19 eligible OS papers including 1587 tissue specimens from 1774 records published from 2014 to 2017 were enrolled by a cautious searching strategy and full-text screening, which were based on inclusion and exclusion criteria in this meta-analysis Figure . The maip < 0.001) and no obvious heterogeneity existed across 19 studies under a fixed effect model  in Figure To assess the association between the UCA1 expression and OS of all cancer patients, a total of 1587 patients with HRs and 95% CIs were included. A significant association was observed between high UCA1 level and poor OS in patients with all 10 types of cancer , CRC , HCC , and PAC . There showed no statistical significance of heterogeneity test in subgroups of GC , CRC , HCC , and PAC . Then, a significant effect of elevated UCA1 on OS emerged in multivariable analysis subgroup  and univariable analysis subgroup . No statistically significant heterogeneity was in multivariable analysis subgroup  and univariable analysis subgroup . Meanwhile, we also detected a significant correlation between overexpression of UCA1 and poor prognosis of cancer patients in large specimen size subgroup  and small specimen size subgroup  with no evident heterogeneity  respectively. To the quality score subgroups, we obtained similar association of upregulation of UCA1 with OS in high quality score subgroup  and low quality score subgroup . No evidence of significant heterogeneity was found across 2 subgroups . Quantified heterogeneous test by meta-regression indicated that no specific factor accounted for the heterogeneity among studies of interest consistent with the outcomes of subgroup analysis in cancer type, analysis method, tumor sample size and quality score covariates.The subgroup analysis in a fixed effect model, random effect model and meta-regression was conducted by cancer types, analysis methods, sample sizes and quality scores for a subsequent investigation of potential heterogeneity in Table Egger’s = 0.006, Figure Egger’s = 0.001), high quality score (pEgger’s < 0.001) and multivariate analysis subgroup (pEgger’s < 0.001) in  in . Howeverp < 0.001), positive lymphatic metastasis , and distant metastasis  in Table In the clinicopathological studies, 16 researches consisting of 1291 tumor samples with a correlation between clinicopathological features and UCA1 expression were retrieved in OR analysis. Clinical stage, lymph node metastasis, distant metastasis and tumor size data were collected to analyze. UCA1 expression was significantly different in these clinicopathological factors . We foun2 = 56.5%, pH = 0.024) in 8 clinicopathological studies shown in Table 2 = 71.9%, pH = 0.014) subgroup. No evidence of statistical heterogeneity was detected in cancer type and lymphatic metastasis subgroup . However, only CRC showed clinical significance and no obvious heterogeneity  by stratification analysis of cancer type. In subsequent stratification analysis of lymphatic metastasis, there was no evidence of significant heterogeneity in CRC  and EOC  subgroup.The subgroup analysis stratified by cancer type, sample size and quality score uncovered the resource of heterogeneity under the fixed effect model, random effect model and meta-regression analysis Table , 4, 5, 6Egger’s = 0.622 and pEgger’s = 0.653, respectively) and no asymmetry among 4 subgroups in the sensitivity analysis . Meanwhile, the pooled data of eligible studies also indicated that high UCA1 expression was significant correlated with poor grade cancer  and positive lymph node metastasis .Here, we performed this meta-analysis to explore the correlation between high expression of UCA1 and clinicopathological characteristics and evaluate the prognosis role of UCA1 for cancer patients. All of these results above suggest that high UCA1 expression may be regarded as an unfavorable predictor in different cancers  with the following searching strategy: “UCA1” OR “UCA 1” OR “UCA-1” OR “urothelial cancer associated 1” OR “urothelial cancer associated-1” OR “CUDR” OR “LINC00178” OR “NCRNA00178” OR “onco-lncRNA-36” OR “ENSG00000214049”) AND (“cancer” OR “tumor” OR “tumour” OR “carcinoma” OR “neoplasm” OR “adenoma” OR “sarcoma”). We manually searched retrieved references for potentially missing literatures. The cited articles were excluded from duplicated firstly, then titles and abstracts were carefully scanned to eliminate irrelevant studies. Finally, we prudently reviewed full texts of potential retrieved studies. Studies were available upon the eligibility criteria: (1) showed the relationship between the dichotomous UCA1 levels and prognosis and clinicopathological characteristics of patients with any type of cancer; (2) calculated HRs and 95% CIs for overall survival (OS), progression-free survival (PFS), disease-free survival (DFS); (3) were published in English. Duplicated, non-dichotomous expression of UCA1 or non-English articles, absence of key survival outcome such as HRs, 95% CIs or Kaplan-Meier survival curve, reviews, letters, laboratory studies of non-human research or comments were omitted.Eligible data were deliberatively judged and double checked from available studies based on criteria of inclusion and exclusion. To each study, we carefully extracted the following information: first author, journal, year of publication, country, ethnicity of the study population, type of specimen, carcinoma type, number of patients, detection method, cut-off value, follow-up, quality score and HR and 95% CI for OS, PFS, or DFS. Quality assessment of the available studies was performed upon the REMARK guideline . HR, as Q test and Higgins I-squared statistic (PH < 0.1 and I2 > 50%). To further explore the potential heterogeneity factors and outcome stability in studies, a subgroup analysis and a sensitivity analysis were utilized respectively in the meta-analysis. Publication bias was evaluated by a funnel plot and Egger's linear regression test with a significant publication bias by P < 0.05.All analysis was conducted with STATA software version 12.0 . Odds Ratios (ORs) were performed to analyze the association of UCA1 expression with clinical stage, tumor size, lymphatic and distant metastasis and HRs with corresponding 95% CIs were utilized to estimate the relationship strength between UCA1 expression and patients’ prognosis. If the HRs were not directly reported in original articles, we calculated the essential data upon the previously reported methods . The poo


Muscle deoxygenation (HHb) was measured at the vastus lateralis muscle using near-infrared spectroscopy. Economical speed which can minimize the wC in each individual was calculated from a U-shaped relationship. We found a significantly slower economical speed (ES) under severe hypoxia  compared to normoxia [1.334 (0.070) m s−1] and moderate hypoxia  with no differences between normoxia and moderate hypoxia (P>0.05). HHb gradually increased with increasing speed under severe hypoxia, while it did not increase under normoxia and moderate hypoxia. Changes in HHb between standing baseline and the final minute at faster gait speeds were significantly related to individual ES . These results suggested that acute severe hypoxia slowed ES by ∼8%, but moderate hypoxia left ES unchanged.We measured oxygen consumption during walking per unit distance  which can minimize energy cost of walking. Muscle O2 extraction may be one of the determining factors of an individual's ES. As humans moved into high-altitude regions over the past 20,000 years, these populations adapted culturally and physiologically to the reduced availability of oxygen in the atmosphere . CardiovwC; ml kg−1 m−1) and gait speeds    . This ineed (ES) ; howeverAt high altitude, it is known that peak aerobic capacity is reduced with a reduction of inspired oxygen pressure . Under t2 delivery and O2 extraction. O2 extraction occurs at active skeletal muscle, which is defined as an arterial-venous O2 difference (a-v O2 difference). As muscle O2 extraction and skeletal muscle mitochondrial capacity have been related to exercise performance  derived from near infrared spectroscopy (NIRS), because changes in HHb have been considered a surrogate of microvascular O2 extraction , and that possible alterations in ES would account for changes in muscle O2 extraction. To test this hypothesis, in addition to oxygen consumption measurement, muscle deoxygenation profiles  at the vastus lateralis muscle were simultaneously measured by NIRS.In the present study, we sought to investigate the potential impact of hypoxic conditions on an individual's ES during walking. We hypothesized that ES would be slower with a decrease in fractional inspiratory oxygen concentration (FiO2), and energy expenditure (EE) between the different oxygen concentrations. Meanwhile, carbon dioxide output (V̇CO2), pulmonary ventilation (V̇E), respiratory gas exchange ratio (RER) and heart rate (HR) linearly increased as concentration of FiO2 fell (P<0.001). These parameters under severe hypoxia were significantly higher compared to normoxia and moderate hypoxia . In contrast, arterial O2 saturation (SpO2) linearly decreased with decreasing inspired O2 concentrations (P<0.001), and the differences between conditions were statistically significant (P<0.05). During walking, no differences in V̇O2 were observed among conditions at any gait speed, whereas V̇CO2, V̇E, and energy expenditure (EE) at faster gait speeds under severe hypoxia were significantly greater than normoxia and moderate hypoxia  were 0.982 (0.937∼0.999). Two-way repeated measures analysis of variance (ANOVA) revealed no significant main effect of oxygen concentrations (P>0.05), while significant main effects were observed for gait speed and interaction , and faster three gait speeds (between 1.250 and 1.639 m s−1). From ANOVA with linear trend analysis, the averaged wC at faster three gait speeds linearly increased (P<0.05) among three FiO2 with no statistical differences in pairwise comparisons. The averaged wC at the faster gait speeds under severe hypoxia (3.51±0.44 J kg−1 m−1) was ∼6.4% greater than normoxia (3.30±0.24 J kg−1 m−1); meanwhile, almost equivalent values were found for wC during the slower gait speeds in all conditions (P>0.05). 2 fell (P<0.001). Moreover, ES under severe hypoxia was significantly slower than under normoxia and moderate hypoxia , while no differences were observed in ES between normoxia and moderate hypoxia . Yet there were no statistically significant differences in the coefficients a and b of the quadratic equation among conditions . Additionally, significant differences in HHb were observed between moderate and severe hypoxia above 1.250 m s−1 of gait speed (P<0.05).−1) when the data were pooled.wC and ES during walking. The major findings of the present study were threefold. First, averaged wC at faster gait speeds linearly increased as concentration of FiO2 fell, although overall wC under moderate and severe hypoxia was not significantly different from normoxia. Second, ES linearly decreased as concentration of FiO2 fell and a significantly slower ES was observed only under severe hypoxia; there were no differences in ES between normoxia and moderate hypoxia. Finally, relative changes in HHb at the vastus lateralis muscle from standing to faster gait speeds were inversely correlated with decelerated ES. Collectively, these results suggest that the U-shaped relationship between gait speeds and wC showed only a leftward shift under severe hypoxia, resulting in a slower ES. In addition, these changes in ES might be partly accountable for local muscle O2 extraction.To the best of our knowledge, this is the first study to examine whether hypoxic conditions alter wC and ES under hypoxia, hypoxia by itself may reflect relatively higher exercise intensity. However, it is supposed that mechanical energy demands of walking are the same regardless of oxygen concentration, although we did not measure it. Thus, unchanged overall wC, even under severe hypoxia, may not be a surprising finding. Previous studies reported that V̇O2 kinetics slowed at the onset of moderate exercise while breathing hypoxic gas mixture (12∼15% O2); these studies, however, also demonstrated that V̇O2 showed a stable phase 1∼2 min after the onset of exercise with no differences between normoxia and moderate hypoxia  as concentration of FiO2 fell. Specifically, the averaged wC above 1.250 m s−1 under severe hypoxia was 6.4% higher than normoxia despite no significant differences in pairwise comparisons , indicating that a considerable 6.4% higher wC during the faster gait speeds under severe hypoxia could explain the different ES. Otherwise, as the ES was determined by the coefficients a and b [see Eqn (5) in the method section], greater coefficient a and/or lesser coefficient b resulted in slower ES  to exercising muscles, has been suggested to be a contributor for maintaining similar V̇Och speed .2 extraction indirectly. It has been suggested that HHb as an indicator of muscle O2 extraction (2 utilization (V̇O2m) and O2 delivery . Under severe hypoxia, HHb gradually increased with increasing gait speed, showing significantly higher values above 1.250 m s−1 than normoxia and moderate hypoxia. We speculate that an increased leg muscle BF could compensate for a reduction in the arterial O2 content (CaO2) under moderate hypoxia, causing similar results in HHb between normoxia and moderate hypoxia. Conversely, under severe hypoxia, greater reduction in CaO2 could not be compensated by enhanced muscle BF, resulting in greater HHb increase compared with normoxia and moderate hypoxia.We measured muscle deoxygenation profiles using NIRS to assess muscle Otraction  could re2 diffusion capability at high-altitude, which induces a decrease in SpO2 , who engaged in strenuous daily training . Their mean age, height, and body mass were 24±8 years, 1.74±0.06 m, and 70±10 kg, respectively . Researchers explained all procedures, possible risks, and benefits of participation, and obtained written informed consent from each participant. They were asked to refrain from intense physical activity on the 2 days before and from drinking any alcohol and caffeinated beverages the day before testing. This study conformed to the Declaration of Helsinki, and the Mount Fuji Research Institute ethical committee approved all study procedures (No: ECMFRI-03-2014).2; 15%, equivalent to a simulated altitude of 2700 m, at which there is increased risk of acute mountain sickness), and severe hypoxia . Each oxygen concentration was supplied by 200-L Douglas bag with hypoxic gas generator system (see below ‘Measurements’) and performed on different days in random order, and a single blind method was used. Participants began by sitting in a chair for 10 min, and then standing for 5 min on the treadmill while baseline values were measured. They then began to walk on the treadmill. Six gait speeds were set incrementally at 0.667, 0.861, 1.056, 1.250, 1.444, and 1.639 m s−1. In keeping with our recent work . Under all experimental conditions, participants walked on the same treadmill, and they were free to choose their step frequency at each speed. All participants wore underwear, shirts, socks, shorts and lightweight training shoes . They weent work , each gaE and gas-exchange variables were measured by an online computerized breath-by-breath method . Inspired and expired gas volumes were measured using a hot wire respiratory flow system. Flow signals were electrically integrated for the duration of each breath to calculate minute ventilation. The expired fractions of O2 and CO2 were analyzed using a zirconium solid electrolyte oxygen analyzer and an infrared carbon dioxide analyzer, respectively. The standard known gases  and room air were used for the calibration of the gas analyzer. Each gas was supplied via a 200-L Douglas bag with a hypoxic gas generator system . Throughout the study, participants' HR was recorded with a wireless HR monitor .V̇2) and HHb according to the modified Beer-Lambert law , as previously described , 2014b. bert law . NIRS opbert law . The probert law , and to bert law .2 was monitored by pulse oximeter on the left middle finger every 1 min throughout the study .The thigh muscle, with attached optodes and covering, was wrapped with an elastic bandage to minimize movement of the optodes while permitting freedom of movement for treadmill walking. Pen marks were made on the skin to indicate the margins of the holder so that optodes could be positioned in exactly the same place for each test. NIRS signals were measured at 1-s intervals throughout the experiment. SpO2, and NIRS signals) were averages of the last 2 min of standing prior to starting walking. A single sample with an average final 1-min pulmonary V̇O2 value (ml min−1) and V̇CO2 (ml min−1) at each gait speed were used to obtain the energy expenditure (EE) during walking, based on the following equation  of each individual can be described as follows:wC′ (v) equaled zero, that is, the individual ES could be observed using the following equation:2 amounted approximately 50% of the absolute V̇O2 at the level gradient at 0.667 m s−1 under normoxia, indicating that a careful consideration should be necessary to calculate wC and ES by subtracting the standing metabolic rate To cequation :(3)wherlic rate . Indeed,lic rate . With thE, RER, and SpO2 was calculated.A single sample of the mean NIRS signals during the final 1 min at each speed was also analyzed. To compare HHb between participants, the changes in this metric were quantified as percentage changes from the baseline values. Briefly, each resting baseline value, which was represented as an arbitrary unit, was defined as 100%. Thus, changes in NIRS signals at each gait speed were represented as relative changes from baseline values. Similarly, a single sample with an average final 1 min of V̇post hoc tests were used to evaluate the changes in cardiorespiratory variables at rest, the averaged wC during the slower and faster three stages, and the ES among different oxygen concentrations. Moreover, two-way repeated measure ANOVA was used to compare changes in the wC and the NIRS signals between different oxygen concentrations and gait speed within participants. Tukey's post hoc test for two-way ANOVA was employed when interactions were significant. To estimate the relationship between changes in ES and NIRS signals, a Pearson correlation coefficient was conducted. Statistical analysis was performed by commercial software packages: Sigma Stat ver 3.5  and GraphPad Prism 7 . A P-value of <0.05 was considered statistically significant.All data are presented as means±s.d. One-way repeated measures ANOVA with linear trend analysis and pairwise (Tukey)


Retinoic acid receptor-related orphan receptors (RORs) include RORα (NR1F1), RORβ (NR1F2), and RORγ (NR1F3). These receptors are reported to activate transcription through ligand-dependent interactions with co-regulators and are involved in the development of secondary lymphoid tissues, autoimmune diseases, inflammatory diseases, the circadian rhythm, and metabolism homeostasis. Researches on RORs contributing to cancer-related processes have been growing, and they provide evidence that RORs are likely to be considered as potential therapeutic targets in many cancers. RORα has been identified as a potential therapeutic target for breast cancer and has been investigated in melanoma, colorectal colon cancer, and gastric cancer. RORβ is mainly expressed in the central nervous system, but it has also been studied in pharyngeal cancer, uterine leiomyosarcoma, and colorectal cancer, in addition to neuroblastoma, and recent studies suggest that RORγ is involved in various cancers, including lymphoma, melanoma, and lung cancer. Some studies found RORγ to be upregulated in cancer tissues compared with normal tissues, while others indicated the opposite results. With respect to the mechanisms of RORs in cancer, previous studies on the regulatory mechanisms of RORs in cancer were mostly focused on immune cells and cytokines, but lately there have been investigations concentrating on RORs themselves. Thus, this review summarizes reports on the regulation of RORs in cancer and highlights potential therapeutic targets in cancer. Cancer incidence and mortality rates are increasing worldwide with the growing and aging of the population, as well as risk factors such as outdoor pollution, tobacco smoke, and physical inactivity . Due to The nuclear receptors (NRs) have been demonstrated to play essential roles in cancer-related progresses and to be potential therapeutic targets for many malignancies –5. The rMembers of the RORs are typically regarded as noteworthy in inflammation, autoimmune diseases, metabolism disorders, circadian rhythms, development of neuron cells, and immune cell differentiation. Although RORs share some common sequences, the three RORs present a wide assortment of features. RORα and RORγ are important regulators of the immune system. For instance, the development and differentiation of Th17 cells are dependent on these factors . MoreoveRORα, RORβ, and RORγ are all involved in the modulation of circadian rhythms. RORα functions as a positive regulator of the circadian modulator Bmal1 through binding to ROR-responsive elements (ROREs) , 12. RORAccumulating evidence shows that RORα and RORγ are involved in lipid/glucose metabolism, insulin sensitivity, and cardiometabolic control . A reporThe expression sites and producing cells of RORs are also distinct from each other, consistent with their functions in the various diseases mentioned above. RORα and RORγ are expressed in all skin cell types, including epidermal keratinocytes, melanocytes, dermal fibroblasts, and several established lines of malignant melanomas. The expression levels of RORα/γ are dependent on the skin cell type and can be regulated by hydroxy derivatives of vitamin D3 , 21–24. Other expression sites of RORα include the liver, skin, pancreas, brain, adipose tissue, islet cells, and the pineal gland. In addition to its expression and modulation in melanoma described above, RORα has been researched in breast cancer (BC) , melanomThe RORs have been widely investigated in cancer and have shown varying influences in cancer-related processes, these differences may be due to their structures and their tissue-specific expression. Some studies suggest that RORα is a tumor suppressor and a potential therapeutic target for BC; and based on the limited researches on RORβ in cancer, RORβ might be a tumor suppressor as well. Others have proposed that activating RORγ may exert antitumor immunity , while RThe three ROR family members contain sequences similar to the retinoic acid receptor, with certain differences. The three ROR family members contain sequences similar to the retinoic acid receptor, but in minor details, the structures of each are distinct . The RORReceptor-related orphan receptor genes encode proteins of similar amino sequences ranging from 459 to 556 amino acids according to the different isoforms, and they all consist of four domains. These domains include an N-terminal domain, a highly conserved DNA-binding domain, a ligand-binding domain (LBD), and a hinge between the domains. Transcription is regulated by binding to RORE as a monomer .No cognate ligands of RORs had been identified until crystallography studies on the LBD of RORα indicated that cholesterol and cholesterol sulfate function as natural ligands . SeveralExcept for the natural ligands of RORs mentioned above, there are also some synthetic RORγ ligands with therapeutic potential identified in literatures , 44. ForAs illustrated above, RORs have been implicated in autoimmune or immune-mediated disease, the circadian rhythm, and metabolic disorders. RORs are also important regulators in various cancers due to their pivotal roles in immunity, the circadian rhythm, and metabolic homeostasis, which contribute to tumor progression.in vitro and in a xenograft model in vivo  and promoting positive (C/EBPb) regulators of granulopoiesis and RORγ1 promoted expansion of tumor-promoting MDSCs and TAM in fibrocarcinoma mice models . In a stma model .In another study, RORγ-deficient mice showed inhibited melanoma growth, and this effect was identified to be IL-9 dependent . TogetheOur previous study showed that RORγ2 was highly expressed in non-small cell lung cancer (NSCLC) cells and also served as a prognostic factor . The expRORγt mRNA was shown to be increased in HCC compared with a normal control group . By cont+IL-17+ cells in colorectal cancer were found to express RORγt , RORγ was examined as a therapeutic target due to its overexpression and was found to directly drive androgen receptor (AR) hyperactivity through binding to an exonic RORE and partly through the NR coactivators SRC-1 and -3 . TherefoThe three ROR family members are regarded as important regulators of the circadian rhythm, metabolism, and tumorigenesis. As discussed in this review, the protumor or antitumor effects of RORα and RORβ in cancer have not been intensively explored, requiring further study and evidence. However, as the main transcription factor in IL-17-expressing immune cells, RORγ has been investigated in various cancer cells and tumor-infiltrating cells (Figure Moreover, according to this review, we could conclude that the roles that RORs family members play in tumorigenesis vary in different cancers and, to some extent, depend on producing cells in the tumor microenvironment. Further concentration on the relationships between RORs and tumorigenesis should be meticulously organized and should deeply explore the clinical significance and the underlying mechanisms. More importantly, each RORs family members consists of several isoforms, and some previous studies have showed that different RORs isoforms present different biological functions . Thus, pSince RORα and RORγ are dysregulated in multiple cancer types based on published articles, they likely participate in carcinogenesis through modulating molecules such as IL-17, PRMT2, and AR or as receptors for sterols, such as vitamin D3 derivatives. Intriguingly, IL-17, AR, and vitamin D3 are therapeutic targets in rheumatoid arthritis and have potential, as a frontline treatment option for advanced prostate cancer and an adjuvant in melanoma management. Agonists or inverse agonists for RORα and RORγ might be efficiently inhibiting tumor growth and progression through activation or inactivation so that their ligands or targets, such as vitamin D3 derivatives and AR, become valid or invalid. Another promising new strategy for anticancer therapy might involve directly targeting tumor cells with RORα- and RORγ-specific modulators due to the correlations between high or low expression of RORα and RORγ and tumor progression. Third, RORs are sometimes produced by immune cells in tumor microenvironments and then induce antitumor or protumor activity by regulating tumor-related cytokines or chemokines. Accordingly, therapies targeting RORs producing immune cells could be novel treatments for certain cancers.JF, ZL and GY wrote the draft. YJ revised the manuscript. JF, TL, JX, and FW designed the figures. QH, MG, GH, MZ, LD and SL commented and added extra information.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 latent variable “δ” (for “dementia”) uniquely explains dementia severity. Depressive symptoms are independent predictors of δ. We explored 115 serum proteins as potential causal mediators of the effect of depressive symptoms on δ in a large, ethnically diverse, longitudinal cohort. All models were adjusted for age, apolipoprotein E, education, ethnicity, gender, hemoglobin A1c, and homocysteine, and replicated in randomly selected 50% subsets. Alpha1-antitrypsin (A1AT), FAS, Heparin-binding EGF-like Growth Factor (HB-EGF), Insulin-like Growth Factor-1 (IGF-1), Luteinizing Hormone (LH), Macrophage Inflammatory Protein type 1 alpha (MIP-1α), Resitin, S100b, Tissue Inhibitor of Metalloproteinase type 1 (TIMP-1), and Vascular Cell Adhesion Molecule type 1 (VCAM-1) each were partial mediators of depression’s association with δ. These proteins may offer targets for the treatment of depression’s specific effect on dementia severity and Alzheimer’s Disease (AD) conversion risk. Major Depression has been identified as a risk factor for incident clinical “Alzheimer’s disease (AD)” –2. Howevg” . Independently of the covariates , baseline GDS score was significantly directly associated with Wave 2 dEQ , and weakly with the Wave 2 g’ composite . g’ was then dropped from consideration. The baseline GDS score’s significant association with Wave 2 dEQ scores was in a negative direction suggesting an adverse effect on future observed cognitive performance.The Base Model fit well [χ2 = 455.02 (17), p < 0.001; CFI = 0.914; RMSEA = 0.049 , Chromogranin A, and Tumor Necrosis Factor receptor type II (TNF-RII) were associated with the GDS, but unrelated to dEQ by path b. b2M and TNF-RII have been previously recognized as depression-specific serum protein biomarkers. –45 HowevWe have surveyed more than 100 potential mediators of the GDS’s specific and significant association with δ. Our sample size was large, and we were powered to detect even statistically weak effects. All our findings have been replicated in random subsets of TARCC’s data. We have replicated all but one of our previously observed GDS-adjusted biomarker associations with δ, even though 1) our sample size has increased over time, 2) we are using a new δ homolog, 3) the biomarkers are being used to predict future cognitive performance, and 4) the prior associations were obtained using raw biomarker data while these employ normalized variables.We have also independently confirmed 4/9 serum proteins previously recognized as depression-specific serum protein biomarkers by Papakostas et al.  and BileWe could not confirm Papakostas et al.’s association between five serum proteins and depression.  These inHowever, our sample frame is ethnically diverse and has been selected against cases of clinical major depression. The mean GDS score is within the normal range and <20% of cases exhibited clinically significant GDS scores. Papakostas et al.’s  sample win GDS adjusted models [We have identified four classes of proteins: 1) potential mediators of the GDS’s significant direct effect on δ, 2) δ-independent predictors of GDS, 3) GDS-independent predictors of δ, and 4) proteins unrelated to either the GDS or δ. While many proteins were related to the GDS, only a subset were associated with δ. δ in turn, has been associated with atrophy in the Default Mode Network (DMN), as has d models –18. Thisd models .Our observations help clarify depression’s effects on cognitive function. First, although depressive symptoms may have both direct and indirect effects on observed cognitive performance, only indirect effects mediated by δ should be functionally salient, and thus “dementing.” This constrains the “pseudodementia” of depression to an effect on intelligence. This may limit the ability of antidepressant interventions to reverse depression’s more disabling  cognitive sequelae. Successful interventions on the “dementia of depression” (DoD) will have to effect changes in general intelligence, regardless of their effect on self-reported mood. This may explain the relatively slow trajectory of functional recovery after otherwise “successful” antidepressant treatment. –60 UnforOne limitation to our study is that we tested univariate associations. We cannot address whether subsets of these proteins may be acting in concert to effect changes in dementia severity, nor do our results indicate the biological networks or systems through which they act. Such issues are beyond the scope of this data. However, it is unlikely that they contribute to a single dementing process. The wide range of serum proteins in Tables Nevertheless, the mediators identified in Finally, although the GDS was weakly associated with prospective dEQ scores, even small changes in that construct are likely to be clinically meaningful . The GDS


Clostridium difficile diarrhea (CDD) is problematic. Optimal treatment for CDD remains oral vancomycin, but there is little data on oral vancomycin dosing regimens. The objective of this C. difficile diarrhea study was to compare the efficacy of “high dose” vancomycin, 500 mg (PO) q6h, as sole treatment and in those who after 72 h failed to respond to conventional doses of oral vancomycin, 125–250 mg (PO) q6h. Hospitalized adults with CDD were evaluated by various oral vancomycin regimens, i.e., a conventional dose group (125–250 mg (PO) q6h), a “high dose escalation” dose group (250 mg → 500 mg (PO) q6h), and a “high dose” group (500 mg (PO) q6h). Oral vancomycin treatment groups were compared by time to improvement, i.e., decrease in >50% of watery stools/day and duration of therapy. The high dose escalation and high dose oral vancomycin groups showed the most rapid resolution of diarrhea. There was marked decrease in stools/day after “high dose” vancomycin escalation from conventional dosing, i.e., 250 mg (PO) q6h → 500 mg (PO) q6h. This study demonstrated that “high dose” escalation or initial high dose oral vancomycin, i.e., 500 mg (PO) q6h was the most efficacious regimen for CDD.Current therapy of  Clostridium difficile infection (CDI) is an imprecise term that does not describe the severity of infection. Multiple risk factors are now appreciated; among these, advanced age has been found to be important. Patients ≥ 65 years of age represented 92% of C. difficile related hospital stays in the United States. In the United States, 93% of deaths from C. difficile colitis occurred in persons ≥65 years of age [C. difficile diarrhea and/or colitis [C. difficile diarrhea, e.g., clindamycin or β-lactams. Importantly, most antibiotics are rarely associated with C. difficile, e.g., aminoglycosides, aztreonam, colistin, polymyxin B, quinupristin/dalfopristin, tetracyclines, tigecycline, macrolides, sulfamethoxazole-trimethoprim, daptomycin, linezolid, telavancin, dalbavancin, oritavancin, fosfomycin, nitrofurantoin, and chloramphenicol. In our experience, over the last four decades, clindamycin, cephalosporins, and, less commonly, fluoroquinolones  have an increased risk of C. difficile compared to other antimicrobial agents. The risk of C. difficile is greatly increased with concomitant administration of proton pump inhibitors (PPIs). Exposure to clindamycin or β-lactams not only increases C. difficile risk during therapy but also for three months following therapy [C. difficile as 94% of cases were associated with healthcare including recently discharged patients, outpatients, and those in long-term care facilities [C. difficile risk factors include some antipsychotic agents, stool softeners/laxatives, and some chemotherapeutic agents [C. difficile [C. difficile as are some antibiotics, e.g., doxycycline or tigecycline [s of age ,9,10,11. therapy . Data frcilities . Other Cc agents ,15. Someifficile ,17,18,19ecycline ,21,22.C. difficile published by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA) recommend initial treatment of mild to moderate disease with (diarrhea) metronidazole 500 mg (PO) q8h or vancomycin 125 mg (PO) q6h for 10–14 days. For the treatment of severe disease (colitis), a combination of metronidazole (IV/PO) and vancomycin 500 mg (PO) q6h, is recommended [C. difficile (colitis), is considered suboptimal therapy for C. difficile diarrhea . There is no general agreement on optimal treatment for patients who do not respond to guideline recommendations and only limited published data on the treatment of such patients [Current clinical practice guidelines for ommended . Metronipatients ,27,28.C. difficile and the antibiotic standard against which all successive therapies have been compared. Vancomycin is a unique glycopeptide: when administered orally, vancomycin essentially is not absorbed, i.e., ingestion does not result in appreciable serum levels. Vancomycin concentrates intra-luminally in the colon, making it ideal for the treatment of C. difficile diarrhea (CDD), a mucosal process [C. difficile diarrhea. Accordingly, this has resulted in our approach of using 250 mg (PO) q6h as the initial treatment regimen for C. difficile presenting as diarrheal disease (CDD). It also has been our practice to increase vancomycin dosing to 500 mg (PO) q6h if there is lack of clinical improvement (≥50% decrease in the number/volume of watery stools/day) after 72 h following treatment with our standard vancomycin regimen, i.e., 250 mg (PO) q6h. Vancomycin was the first effective antimicrobial for the treatment of  process . This co process . In our Our primary objective was to evaluate the enhanced therapeutic efficacy of oral vancomycin high dose escalation to 500 mg (PO) q6h for those with CDD who failed to improve substantially (≥50% decrease in the number/volume of watery stools/day) after three days of vancomycin 250 mg (PO) q6h therapy. The secondary objective of this retrospective study included assessing the efficacy of “high dose” vancomycin 500 mg (PO) q6h as the primary treatment dose and to examine concomitant metronidazole utilization.Rapid resolution of CDD is best for the patient, decreases potential exposure to other patients, allows earlier discontinuance of CDD precautions, and permits earlier discharge. ®, an online system to identify patients with otherwise unexplained acute watery diarrhea who tested positive for C. difficile toxin via stool polymerase chain reaction (PCR). All data on patients in the study were appropriately de-identified. There is no institutional protocol to treat C. difficile and treatment is at the discretion of the physician. NYU Winthrop Hospital is a 600-bed university teaching hospital located in Long Island, New York. This was a quality assurance retrospective study utilizing electronic medical records (EMR) to review hospitalized adults with CDD from January 2015 to December 2016. Patients were identified utilizing MedMinedC. difficile treated with oral vancomycin were eligible for inclusion. Excluded were patients with fecal collection bags, immunocompromised patients, or those with incomplete documentation regarding diarrhea as we were unable to determine number of stools and days until soft stool to evaluate treatment response.Hospitalized adults with acute onset watery diarrhea who were stool PCR positive for The objective of this retrospective study was to assess the efficacy of “high dose” vancomycin, 500 mg (PO) q6h, in patients with CDD failing to substantially improve (decrease ≥50% of liquid stools/day) after 72 h. Clinical improvement was defined as a ≥50% decrease in the number/volume of watery stools/day. Clinical resolution was defined as days to soft/formed stools.C. difficile toxin by PCR were identified. Of these, 160 were eligible for analysis and were divided into three groups, i.e., conventional dosing group, high dose escalation group, and high dose group as primary treatment. See Over a 24 month period, 203 non-ICU patients with acute watery stools positive for Out of the 160 patients included in the study for analysis, 108 patients were treated with vancomycin 250 mg (PO) q6h for the entire course of therapy and five patients were treated with 125 mg (PO) q6h for the entire course of therapy. The mean age in this group was 69.6 years (range: 20–102) and 57% were female. In this group, the mean number of watery stools upon diagnosis was 5.6/day (range: 2–17/day); after 72 h of therapy, it decreased to 2.68/day (range: 0–9/day). Clinical resolution occurred at 5.08 days (range: 2–15 days). A total of 65 patients in this group (58%) were concurrently treated with metronidazole. In this patient group, the average number of vancomycin doses was 53.4 (range: 12–205) and the average duration of therapy was 14.2 days (range: 4–54 days). Included were two patients who did not achieve clinical resolution including one on concurrent metronidazole.Of the 160 patients included in the study, 33 patients underwent “high dose” escalation after failing to respond to initial conventional dose vancomycin regimens within three days. In this patient population, the mean age was 68.4 (range 20–97) and 48% were female. At presentation, the mean number of watery stools was 6.45/day (range: 4–15/day). After 72 h of initial therapy with 250 mg (PO) q6h, the mean number of watery stools was 5.5/day (range: 3–12 days). As a result of lack of clinical improvement, the dose was increased to 500 mg (PO) q6h and the number of watery stools 72 h after dose escalation was 2.5/day (range: 0–9/day). Clinical resolution occurred at day 10 (range: 4–27) after initiation of vancomycin therapy and 4.2 days (range: 1–6 days) after the first dose of 500 mg was administered. Included were 19 patients also treated with metronidazole at the initiation of therapy (58%) and two patients discontinued metronidazole post-dose escalation. The average number of vancomycin doses was 64.4 (range: 21–180), of which 21.9 (range: 4–78) were 250 mg and 37.3 (range: 13–98) were 500 mg. The average total duration of therapy was 17.97 days (range: 9–53 days). The average length of therapy after transition to “high dose” oral vancomycin was 11.5 days (range: 5–47 days). Only one patient did not achieve clinical resolution after switching to 500 mg PO q6h .Out of the 160 patients included in this study, 14 patients were treated with vancomycin 500 mg (PO) q6h for the entire course of therapy. The mean age was 64.4 (range 54–94) and 50% were female. The mean number of watery stools/day at presentation was 8.8/day (range: 5–17/day) and after 72 h of therapy it was 3.6/day (range: 1–8/day). Clinical resolution was noted at 5.4 days (range: 3–15 days). Included were 11 patients concurrently treated with metronidazole (79%). In this group, the average number of vancomycin doses was 53 (range: 8–114) and the average duration of therapy was 13.1 days (range: 6–31 days). Only two patients did not achieve clinical resolution. A summary of results is provided .The results of this limited retrospective quality assurance study showed that, in patients failing to achieve rapid clinical improvement of CDD after 72 h with conventional vancomycin dosing, i.e., 125–250 mg (PO) q6h, “high dose” vancomycin 500 mg (PO) q6h was a highly effective treatment option. Consistently, “high dose” vancomycin therapy resulted in a rapid decrease in number of watery stools/day. Treatment for CDD failing to respond to standard vancomycin therapy has consistently been a dilemma for clinicians at our institution. For years, we have observed a high degree of efficacy with “high dose” vancomycin 500 mg (PO) q6h, and we were able to demonstrate this via formal review and analysis. We demonstrated a 97% response rate to vancomycin 500 mg PO q6h in patients who failed to response to 250 mg q6h.There are a limited number of studies, all of which involve a small number of patients, specifically analyzing the efficacy of “high dose” vancomycin in the treatment of CDD. All show rapid rates of clinical resolution, ranging from 92% to 100%, with an average time to resolution of three days ,27,28. C. difficile may be discontinued. Compared to hospital costs of lengths of stay (LOS) attributable to C. difficile, the cost of “high dose” oral vancomycin therapy is negligible. The sooner a clinical resolution of diarrhea is achieved, the earlier infection control precautions for An interesting finding of this study was that regarding duration of treatment. Provided the patient’s diarrhea has resolved, national guidelines recommend a 10- to 14-day course of therapy, i.e., 125–250 mg (PO) q6h; however, we found practitioners often continued therapy beyond the recommended duration despite no data to support the continued oral vancomycin beyond clinical resolution. From the perspective of an antimicrobial stewardship program (ASP), it was interesting to note the role of concomitant metronidazole. Metronidazole was frequently used in all patient groups. However, concomitant metronidazole did not offer any additional benefit independent of vancomycin dose. C. difficile. Although this study was intended to limit confounding variables, there were limitations to this study. The main limitations were relatively small sample size and a retrospective study design. Accurate stool documentation relied entirely on the accurate documentation of the patient’s nurse; treatment choice dose and duration was entirely dependent on the patient’s physician. From an ASP perspective, an institutional protocol would provide improved uniformity of outcomes demonstrated by our data as well as the elimination of the use of concomitant metronidazole in these patients with CDD, not C. difficile colitis. This retrospective quality assurance study was conducted to assess the efficacy of “high dose” oral vancomycin as an effective treatment option in patients with CDD (not colitis) who failed to substantially improve with conventionally dosed oral vancomycin regimens after 72 h. Out of 203 patients, 33 underwent “high dose” escalation and 32 demonstrated resolution. The more rapidly a medication is able to provide clinical improvement/resolution, the sooner the patient may be discharged from the hospital, i.e., decreased LOS. Because oral vancomycin is poorly absorbed in the gastrointestinal tract, “high dose” oral vancomycin escalation is safe and highly effective. Large-scale prospective, randomized, control trials are necessary to assess the optimal dose for treating patients who failed to improve on oral vancomycin therapy. Nevertheless, our single center experience provides support for “high dose” vancomycin escalation or as initial therapy . ImportaFrom an infection prevention and ASP perspective, rapid resolution of CDD has several benefits for the patient and hospital. Faster resolution is better for the patient, decreases potential exposure of other patients to CDD, allows earlier discontinuance of precautions, and earlier discharge.