HIV-infected persons suffering from tuberculosis experience high mortality. No programmatic studies from India have documented the delivery of mortality-reducing interventions, such as cotrimoxazole prophylactic treatment (CPT) and antiretroviral treatment (ART). To guide TB-HIV policy in India we studied the effectiveness of delivering CPT and ART to HIV-infected persons treated for tuberculosis in three districts in Andhra Pradesh, India, and evaluated factors associated with death.We retrospectively abstracted data for all HIV-infected tuberculosis patients diagnosed from March 2007 through August 2007 using standard treatment outcome definitions. 734 HIV-infected tuberculosis patients were identified; 493 (67%) were males and 569 (80%) were between the ages of 24–44 years. 710 (97%) initiated CPT, and 351 (50%) collected >60% of their monthly cotrimoxazole pouches provided throughout TB treatment. Access to ART was documented in 380 (51%) patients. Overall 130 (17%) patients died during TB treatment. Patients receiving ART were less likely to die , while males and those with pulmonary TB were more likely to die .Among HIV-infected TB patients in India death was common despite the availability of free cotrimoxazole locally and ART from referral centres. Death was strongly associated with the absence of ART during TB treatment. To minimize death, programmes should promote high levels of ART uptake and closely monitor progress in implementation. India has the world's highest burden of tuberculosis (TB) with 1.9 million estimated incident cases per year. It also ranks among the world's highest HIV burden with an estimated 2.3 million persons living with HIV/AIDS Pneumocystis pneumonia, toxoplasmosis, and other bacterial opportunistic infections Cotrimoxazole is a broad-spectrum antimicrobial agent that is recommended as primary prevention against opportunistic infections in HIV-infected persons Antiretroviral treatment has been convincingly associated with improved treatment outcomes and reduced mortality To guide TB-HIV policy in India, the Government of India's Revised National TB Control Programme (RNTCP) and NACP implemented a demonstration project in three districts in the state of Andhra Pradesh. The project's goal was to deliver the WHO-recommended interventions of CPT and ART  to all known HIV-infected TB patients. We retrospectively evaluated the efficiency of decentralized CPT delivery from primary health centres, assessed the effectiveness of referral links to ART centres, and studied factors associated with mortality among HIV-infected individuals treated for tuberculosis under programmatic conditions.Three districts in the southern Indian state of Andhra Pradesh—Ananthapur, Vizianagaram, and Vishakapatnam (combined 2001 population 9.7 million)—were intentionally selected to implement these TB-HIV activities on the basis of high HIV prevalence among antenatal clinic attendees during NACP surveillance from 2003–2006. In the three districts there are a total of 154 primary health care facilities which provide general health services including TB diagnostic and treatment services. At the time of implementation of these interventions, there were 47 HIV counseling and testing centres and 3 ART centres for free HIV diagnosis and treatment . HIV treatment and care, including ART, was available free of cost to patients at these ART centres. Patients from Vizianagaram district were able to access ART services in the adjacent district of Vishakapatnam.TB diagnosis under RNTCP is based on initial smear microscopy, but allows for the diagnosis of smear-negative and extrapulmonary TB as well. Pulmonary TB diagnosis is based on a standard diagnostic algorithm Providers were instructed to immediately refer all TB patients with any HIV risk factors to the nearest HIV counseling and testing centre, as per the prevailing national guidelines at the time. HIV test results were communicated to the referring provider either directly by the patient or via direct communication from HIV counselor to the referring provider (with patient assent). An initial 1-month supply of CPT, at the dose of sulfamethoxazole 800 mg/trimethoprim 160 mg daily (one “double strength” cotrimoxazole tablet) was provided to the HIV-infected TB patients from their respective primary health centres, and 1-month refills made available for the duration of their TB treatment at no cost. While being providing CPT, patients were counseled on the benefits of CPT, possible side effects, and the importance of adherence. In an attempt to address concerns about patient privacy, CPT was available at primary health centres only from health care workers, and not through community DOT providers. At the start of the intervention, CPT became available for all HIV-infected TB patients at the same time, regardless of whether a patient was newly diagnosed with TB or already on TB treatment for several months.Providers were asked to refer HIV-infected TB patients to the nearest ART centre as soon as possible, but at least 2 weeks after the initiation of anti-TB treatment (to address the infection control concerns). Patients were asked to continue CPT from ART centres after their TB treatment was completed, or earlier if ART were initiated during TB treatment.The RNTCP individual patient TB treatment cards were modified to enable recording of HIV status, CPT initiation, CPT adherence, and ART information. Trainings were conducted for all district medical officers, programme staff of NACP and RNTCP, and pharmacists at peripheral health facilities. Standard forms were developed to facilitate referrals and feedback on results of HIV testing and ART evaluations. TB programme staff maintained a separate register of HIV-infected TB patients to facilitate supervision and monitoring.Standard definitions were used to categorize patients according to TB type and treatment outcomes We retrospectively abstracted data from programme records for all HIV-infected TB patients notified to the RNTCP between 1 March and 31 August 2007. Treatment cards were collected from all health facilities and data double entered with Microsoft Excel. At the end of 6 months RNTCP and NACP conducted field visits to 30 health service delivery sites in the three districts, and conducted unstructured interviews with 100 randomly selected HIV positive TB patients to identify gaps in the delivery of intervention services.We compared HIV-infected TB patients who died during TB treatment with those who did not die. Risk factors for death analyzed included age, sex, district of residence, type of TB, CPT use, CPT adherence, and ART provision. To assess CPT adherence in the context of a variable duration of TB treatment, we expressed the number of monthly CPT dose pouches picked up—as recorded on TB treatment cards—as a proportion of the number of months which an HIV-infected TB patient was alive and on treatment (rounded down to the nearest month). Data on CD4 cell counts were not available in routine records.For bivariate analysis, we compared proportions using chi-square, and for continuous variables we compared medians using the Wilcoxin Rank-Sum test. The survival distribution of the overall cohort was estimated using the Kaplan-Meier method, and subgroups were compared using the Cox-Mantel (log-rank) test. P-values throughout were 2-sided and we used a 0.05 significance level.For multivariate analysis we calculated hazard ratios for factors associated with death using a Cox proportional-hazards model. The dependent variable was the number of days. Treatment outcomes other than death were censored after the last day of TB treatment recorded. We pre-selected variables for inclusion in the multivariable model based on prior information from studies from other settings on risk factors for mortality in HIV-infected TB patients This retrospective analysis was conducted after review and approval by the National AIDS Control Programme and the Central TB Division, Directorate General of Health Services, Ministry of Health and Family Welfare, Government of India. The activity was determined to be programme evaluation of the implementation of internationally-recommended interventions. Electronic databases created for this analysis were stripped of personal health identifiers and maintained securely.Among the 7,752 patients registered in the three districts from March 1 2007 to August 31 2007, 734 (9.5%) were identified as HIV-infected. The proportion of registered patients reported to be HIV-infected varied by district; 322 (9.9%) out of the 3,239 of TB patients registered from Ananthapur were HIV-infected, as were 317 (11.6%) out of the 2,725 from Vishakapatnam and 95 (5.3%) out of the 1,788 from Vizianagaram. Characteristics of these 734 HIV-infected TB patients are shown in . The exact date of CPT initiation was not available for 26% of patients, as field staff commonly only recorded the month of initiation. Overall 351 (48%) collected ≥60% of the number of monthly cotrimoxazole pouches provided. Anecdotally, we observed that the on-site availability of CPT at primary health centers appeared to motivate providers assess the HIV status of TB patients.710 (97%) of the 734 known HIV-infected TB patients were initiated on CPT during the evaluation period; 22 (3%) of patients were already taking CPT before the start of TB treatment from the ART centre, and no record of CPT initiation was found for two patients. Nearly half  received prophylaxis within 1 week of starting TB treatment Among the 734 HIV-infected TB patients, 133 (18%) were documented as already on ART at the time of TB treatment initiation. Of the 601 HIV-infected TB patients not on ART at the time of TB treatment initiation, 559 (93%) were documented to have been referred to ART centres. Of the 559 referrals to ART centre, 324 (61%) were documented to be subsequently pre-registered at ART centres. Of the 324, 247 (76%) were initiated on ART by the end of TB treatment. In total, 380 of 734 (52%) had access to ART during TB treatment.. Treatment success was reported in 536 (73%) patients; treatment success was highest among new-extrapulmonary patients (89%), and was lowest among re-treatment patients (51%). Treatment default was more common among re-treatment patients (16%) or new smear-negative TB patients (11%), compared to new smear-positive TB patients (4.3%) (p<0.01 for each comparison). The combined treatment success rates in this group of HIV patients was lower than the overall treatment success rate in the three districts (84%) during the same time period.Treatment outcomes were available for all 734 HIV-infected TB patients .Overall 130 (18%) HIV-infected TB patients died during TB treatment. Bivariate risk factors for death are shown in , ART exposure at any time during TB treatment remained independently protective against death . Being a male and having pulmonary TB were also independently associated with a greater risk of death, and there was a trend towards greater risk of death in those with prior anti-TB treatment.In multivariate analysis Limiting the multivariate analysis to those patients who started ART during TB treatment  did not substantially change the association between ART exposure and lower risk of death , nor did limiting the analysis to males or females alone .This evaluation provides the first evidence from India regarding the operational challenges of delivering WHO-recommended mortality-reducing TB-HIV interventions under general field conditions. It also provides information on the daunting problem of the high mortality of HIV-infected TB patients. In this resource-limited setting, nearly 1 in 5 HIV-infected individuals diagnosed with TB died during TB treatment. While previous studies have reported high mortality among HIV-infected persons with tuberculosis With no additional resources or efforts beyond provider trainings and monitoring by regular HIV and TB programme staff, more than 95% of all detected HIV-infected TB patients were initiated on CPT. Mixed adherence may have reduced the effectiveness of CPT in this population, as just half of patients collected >60% of their cotrimoxazole. With increased awareness of CPT, adherence may improve; additional measures the programmes should consider include use of peer-counselor networks and non-governmental organizations to support and encourage patients and providers.We found that patients provided ART during TB treatment had less than half the risk of death as those not provided ART. This finding was independent of sex or previous TB treatment history, and is consistent with findings from several other settings HIV-infected males experienced higher death rates than females; the reasons for this finding are unclear. Overall case-fatality rates among males with tuberculosis in India are modestly higher than in females, and postulated reasons for higher mortality among males include differences in treatment adherence, age distribution, smoking behavior, and comorbidities Our evaluation was subject to several important limitations. This was a retrospective evaluation of a demonstration conducted under routine programme conditions; available information was limited to that recorded on standard patient records which had been modified to include additional HIV-related information. With ascertainment of HIV-infection among TB patients based on selective referral of those persons with HIV risk factors, the patients identified might not be representative of the larger population of HIV-infected TB patients. All 3 districts practiced the same selective referral policy, but in Vizianagaram district a simultaneous population-based survey of HIV infection among TB patients provides insight into the effectiveness of the ascertainment of HIV infection Treatment outcomes were recorded by many providers, and were not independently validated. Information about other biomedical risk factors for death that may have confounded this evaluation, such as severity of TB disease, drug resistance, severity of immune suppression, or comorbidities, were not available. No ‘control’ areas without CPT were assessed because we did not seek to evaluate the efficacy of CPT as an intervention, and observational analysis of the association of CPT and mortality were not possible since almost all patients were exposed to CPT during TB treatment. The efficacy of CPT, however, has been amply demonstrated in multiple settings. The recording of ART information was dependent on feedback from the ART centre. Some patients may not have had ART consumption recorded on their TB treatment card, so we may have misclassified these patients as not provided ART, and thus underestimated the protective effect of ART in this population. We were also unable to collect adequate information about sub-type of extrapulmonary TB, patient CD4 count, or the specific timing of ART initiation. Future studies should seek to collect this information to better understand the finding of lower overall mortality in extrapulmonary TB, and the protective effect of ART against mortality during TB treatment.On the basis of this experience, decentralized CPT delivery for HIV-infected TB patients has been included in national TB-HIV policies, and is being presently implemented in high HIV-prevalence settings throughout India Cotrimoxazole prophylaxis can be delivered to HIV-infected TB patients under programmatic conditions in India. Despite the availability of free cotrimoxazole locally and ART from referral centres, death was common and was strongly associated with the absence of ART during TB treatment. These findings have been translated into national policy and programme implementation through the expansion of cotrimoxazole availability and training on the importance of ART. To minimize death, the TB and HIV programmes should target high levels of ART uptake and closely monitor progress in implementation.


Large-scale compilation of gene expression microarray datasets across diverse biological phenotypes provided a means of gathering a priori knowledge in the form of identification and annotation of bimodal genes in the human and mouse genomes. These switch-like genes consist of 15% of known human genes, and are enriched with genes coding for extracellular and membrane proteins. It is of interest to determine the prediction potential of bimodal genes for class discovery in large-scale datasets.Use of a model-based clustering algorithm accurately classified more than 400 microarray samples into 19 different tissue types on the basis of bimodal gene expression. Bimodal expression patterns were also highly effective in differentiating between infectious diseases in model-based clustering of microarray data. Supervised classification with feature selection restricted to switch-like genes also recognized tissue specific and infectious disease specific signatures in independent test datasets reserved for validation. Determination of "on" and "off" states of switch-like genes in various tissues and diseases allowed for the identification of activated/deactivated pathways. Activated switch-like genes in neural, skeletal muscle and cardiac muscle tissue tend to have tissue-specific roles. A majority of activated genes in infectious disease are involved in processes related to the immune response.Switch-like bimodal gene sets capture genome-wide signatures from microarray data in health and infectious disease. A subset of bimodal genes coding for extracellular and membrane proteins are associated with tissue specificity, indicating a potential role for them as biomarkers provided that expression is altered in the onset of disease. Furthermore, we provide evidence that bimodal genes are involved in temporally and spatially active mechanisms including tissue-specific functions and response of the immune system to invading pathogens. Gene expression is controlled over a wide range at the transcript level through complex interplay between epigenetic modifications, DNA regulatory proteins, and microRNA molecules -3. GenomThe high-dimensionality inherent in genome-wide quantification makes extracting meaningful biological information from gene expression datasets a difficult task. Early attempts at genome-wide expression analysis used unsupervised clustering methods to identify groups of genes or conditions with similar expression profiles -9. BioloMany simple unsupervised learning algorithms rely on distance metrics to either partition profiles into distinct groups ,16 or buMethods such as unsupervised, supervised and model-based classification provide the means to evaluate switch-like gene expression patterns in high-dimensional datasets profiling diverse biological conditions. For this purpose, we compiled two large-scale gene expression microarray datasets from publicly available data repositories. The first dataset included samples spanning nineteen different tissue types from healthy donors. The second dataset included samples from donors with one of a number of infectious diseases including HIV-1 infection, hepatitis C, influenza, and malaria. Our results demonstrate that switch-like genes exhibit tissue and disease-specific expression signatures. Dimension reduction of genome-wide expression data through the identification of switch-like genes enabled highly accurate classification of samples into tissue-specific and disease-specific clusters. Moreover, analysis of activated switch-like genes in various disease and tissue types revealed that these genes participate in specialized or temporally active mechanisms. Further study of genes in the switch-like gene set may provide biologically significant information about the molecular basis of phenotype distinction.A model-based classification algorithm  partitioBinomial tests were used to identify sets of bimodal genes biased toward the "on" mode in the tissues that are well-represented in our microarray dataset (> 25 samples). A gene by sample heat map Figure  shows thFunctional enrichment analysis identified gene sets related to tissue-specific function in sets of bimodal genes expressed in the "on" mode in brain, skeletal muscle, cardiac muscle and lung tissues. The GO categories that are significantly enriched with bimodal genes that are "on" in brain tissue samples included neural tissue-specific processes including neural migration, adhesion, recognition and differentiation, nervous system development, and synaptic transmission Table . SimilarModel-based clustering of bimodal gene expression led to accurate classification of disease phenotypes in an independent dataset of 221 microarray tissue samples profiling infectious diseases. Note that only normal tissue microarray data and not infectious disease data was used in the original annotation of switch-like genes. The posterior pairwise probability matrix derived from model-based clustering partitioned expression profiles of peripheral blood mononuclear cells (PBMC) into disease-specific clusters for HIV-1 infection, hepatitis C, influenza, and malaria Figure . We focuNext, we examined the switch states of bimodal genes in infectious disease associated microarray data. Of the 1295 bimodal genes analyzed, 192, 160, 148 and 117 genes were expressed in the "on" mode in the majority of samples from PBMCs in hepatitis C, influenza A, malaria, and HIV-1 infection, respectively. In liver biopsies from hepatitis C infected individuals, 301 bimodal genes are over-represented in the "on" mode. Biological processes commonly enriched in the set of bimodal genes expressed in the "on" mode in these diseases include B cell receptor signalling and humoral immune response involving circulating immunoglobulins  but had little differentiation potential at small sample sizes. The decrease in classification accuracy observed with the use of distance-based clustering may be due to estimation of the number of clusters via the gap statistic . Incorpoa priori knowledge in the analysis of microarray data produced by different laboratories.A set of 300 bimodal genes expressed on the extracellular matrix or the plasma membrane is sufficient to accurately differentiate between nineteen different tissue types in model-based clustering even at 5 microarray samples for tissue type. This set of genes includes those that code for membrane-bound integrin proteins and ECM proteins belonging to collagen, laminin, and fibronectin families. Genes expressed in the "on" mode in brain tissue and the "off" mode in muscle tissue largely coded for neural-specific cell adhesion molecules. Supervised classification has the potential to further reduce the set of 300 bimodal genes to biomarker sets when considering biomarkers for tissue-specific diseases. Accurate classification with the subset of bimodal genes presented in this article demonstrate the importance of cell/ECM interactions in tissue differentiation  and willOur study showed that the bimodal gene set identified using microarray data associated with healthy tissue is highly effective in differentiating between microarray data from tissues infected by various infectious diseases such as the HIV-1 infection, hepatitis C, influenza and malaria. The classification was unsupervised and the disease signature was conserved across laboratories. Moreover, bimodal gene sets differentiated between liver and blood cell tissues infected with the same hepatitis virus. The identification of bimodal genes expressed in the activated state in various infectious diseases and subsequent enrichment analysis with KEGG pathways provide biological context to the perturbation of various cell signaling networks induced by invading viruses. In the infectious disease states investigated here, bimodal genes expressed in the "on" mode were related to both innate and antigen-mediated immune responses.a priori to reduce the feature space in microarray analysis. The large body of evidence presented in this work points to the success of switch-like gene sets in capturing biologically-relevant gene expression signatures from microarray data.It should be noted that other gene sets determined by feature selection may be even more discriminative of the phenotypes included in this analysis than the switch genes under consideration. Our intent in this study was not to identify discriminative genes but rather to use unsupervised clustering to determine whether switch-like expression patterns are associated with phenotype and whether previously identified switch-like genes could be used Given the demonstrated biological relevance of bimodal expression patterns, it would be worthwhile to determine the clinical relevance of switch-like gene annotation. Identification of bimodal genes expressed in the activated state in complex diseases such as autism, diabetes and cancer may provide a method for dimension reduction in the identification of disease-related single nucleotide polymorphisms (SNPs)  and expra priori knowledge gained from compilation of large-scale microarray datasets from multiple laboratories containing at least 400 samples for each gene in the array could be successfully used in reducing the dimension of features in microarray analysis. We reduced dimensionality by focusing on a set of genes with bimodal expression patterns, i.e. genes that adopt either an "on" or "off" mode of expression and are tightly regulated at the transcript level. Detection of bimodality using expectation maximization revealed a list of 1265 bimodal genes in the human genome. A subset of 300 bimodal genes was sufficient to differentiate between nineteen different tissue signatures even in small sample sizes. These genes code for proteins either on the cell membrane or at the extracellular matrix. Such proteins can be identified in tissue using fluorescence, Q dots and other methods and as such are candidate biomarkers for specific tissues.In this study, we showed that The set of bimodal genes are capable of capturing infectious disease signatures from microarray data corresponding to hepatitis C, influenza, HIV-1 infection and malaria. Disease-specific expression patterns of bimodal genes suggest that infection by different pathogens may initiate different host responses mediated by switch-like gene expression. Determination of "on" and "off" states of switch-like genes in various tissues and diseases allowed for the identification of activated/deactivated pathways that are consistent with existing research data. Classification accuracy was exceptional even with class-specific sample sizes between ten and twenty arrays. The use of a priori knowledge from public microarray datasets in the form of bimodal gene sets has clinical implications in disease subtype classification. Genome-wide association studies for SNP discovery linked to complex diseases such as autism and cancer could potentially benefit from dimension reduction by focusing on regions of DNA that code for switch-like genes and their promoter regions.Microarray datasets used in this study were compiled from the online public repositories Gene Expression Omnibus (GEO)  and ArraDatasets were first filtered such that only the 22,277 probe sets common to both the HGU133A and HGU133plus2 platforms were retained. Reference robust multi-chip averaging (refRMA)  was usedProbe sets were annotated using Entrez Gene ID, Ensembl accession number, gene symbol, Gene Ontology terms  and KEGGλ was used to reject the null hypothesis. As in our previous work [Bimodal genes were identified in expression data of healthy tissues using a statistical method previously applied in the detection of switch-like behavior among mouse  and humaous work ,47, p-vaBimodal gene expression values were binarized by defining a gene-specific threshold at the intersection of the probability density functions of the two-component mixture models . ExpressGene sets characterized by KEGG pathways and GO terms were analyzed to identify functional categories enriched in sets of bimodal genes biased to the "on" or "off" mode in healthy and disease phenotypes. We assessed the enrichment of functional gene sets by comparing the number of "on" or "off" genes observed in a particular functional group to the number expected by chance . The hypTwo distance-based clustering algorithms, Kmeans  and hierk groups  using either Kmeans or hierarchical clustering as described above. A set of B reference datasets were generated by drawing samples from the reference distribution and clustered in the same manner. The gap statistic (Gapk) was calculated as:The optimal number of clusters tatistic . The gapthe data . Expresskb *,  and Wk are within-cluster sums of squares of the reference and observed datasets respectively. The estimated number of clusters k at which:in which Wk is the standard deviation of log(Wkb*).and sdA model-based clustering algorithm , developyi is the expression value in sample i, μ is a vector of mean expression values over all samples, ri ∈ m indicates the relevant genes, δi is a vector of mean shifts and εi is a vector of the variance in expression values. Cluster-specific parameters Θ =  are sampled from a baseline distribution f0 in a Polya urn scheme or Chinese restaurant process as described by Hoff:in which n-1 is the empirical distribution of Θ1,..., Θn and α is a constant. This process potentially results in less than n unique draws from the baseline distribution and therefore naturally leads to clustering. Parameters of the model are fit from the data using a Gibbs sampling algorithm [where flgorithm . We ran lgorithm  in the Rlgorithm ,21.Given a set of clusters obtained from Gibbs sampling, the probability that two observations belong to the same class is approximated by the proportion of clusters in which they are grouped together . For eacci  is a vector indicating which cluster sample i is assigned to. Although the pairwise posterior probability is a useful measure in itself, it does not provide a single cluster partition. For this purpose, a distance metric (Dij) was defined from the pairwise posterior probabilities equal to Dij = 1 - Pij [in which  1 - Pij . A uniqua priori. The Adjusted Rand Index (ARI) was used to measure the amount of agreement between the known and estimated class membership [ij of the contingency table equals the number of observations in cluster i of class j. Row sums of the contingency table are equal to ni. and column sums are equal to n.j. With this notation, the Adjusted Rand Index is calculated by the formula below and takes a value of 1 when the two partitions agree completely and a value of 0 when the index equals its expected value i.e. the partitions are no better than random.In this study, clustering algorithms were applied to data in which the true class membership of all samples was known mbership ,22. Givejth classifier , training samples in class j were assigned to class 1. All other samples were assigned to class 0. Discriminative bimodal genes were identified from the training data according to the ratio of within class to between class sums of squares [i and samples in class 0 (dco) and class 1 (dc1), respectively [dco/(dco+dc1). Values close to 0/1 indicate low/high confidence that test sample i belongs to class j. Confidence measures were compared from each classifier and test sample i was assigned to the class associated with the highest confidence.A multi-class supervised learning scheme was used to classify tissue samples on the basis of bimodal gene expression. In binary classification of microarray data, training data was used to rank features by a two-class test statistic . Discrim squares . Diagonaectively . A confiIn silico expression datasets consisted of 10, 20, 30, 50, or 100 observations/arrays and 1000 features/genes. Initially, a binary vector indicating the class membership of each observation was drawn from a binomial distribution B. A number of 5, 10, 20, 50, or 100 informative gene expression profiles were drawn from a pair of multivariate normal distributions N1 and N2 representing each class of observations. Non-informative expression values representing noise genes were drawn from a mixture of N1 and N2 with mixing probabilities of 1/2 from each distribution. A diagonal covariance matrix (Σ) was used to simulate independent expression values. Effect size was measured by a separation parameter defined for each gene, specifically the distance in class-specific means divided by the pooled variance. Three effect sizes  were investigated. We used logistic regression, implemented in the stats package in the R statistical environment, to generate the response variable that indicates class membership from the expression data. Regression coefficients associated with the informative genes were drawn from a uniform distribution U. By logistic regression, the probability that the ith observation is class 1 is given by πi:Synthetic data was used to determine the effect of sample size, effect size and the number of informative genes on prediction accuracy in binary classification. β1 ... βM are the defined regression coefficients and xi 1, ... xM, i are the expression values of the informative genes in the ith observation [i on the basis of πi (yi = 1 iff πi > 0.5). In this manner, the relationship between the jth gene and the response variable yi can be specified exactly , independent of the sample distribution of gene j.in which ervation . The simARI: Adjusted Rand Index; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; ECM: extracellular matrix; MEM: membrane; PBMC: peripheral blood mononuclear cells; HIV: human immunodeficiency virus; refRMA: reference robust multi-array average; SNP: single nucleotide polymorphism; eQTL: expression quantitative trait loci; AUC: area under the receiver operating characteristic curve.MG and AT conceived and developed the research plan and wrote the manuscript draft. MG implemented classification of expression data and all subsequent analysis. Both authors read and approved the final manuscript.Annotation information for 1265 bimodal genes. For each gene, the Affymetrix probe set ID, full gene name, and mode of expression  in brain, skeletal muscle, cardiac muscle and lung tissue are listed.Click here for filegormley tozeren bmc bioinformatics.Click here for fileAnnotation information for microarrays. For each gene expression array analyzed, the GEO series and sample accession numbers are listed along with phenotype and chip type.Click here for file


Transfusion-associated hepatitis B viral infection (TAHBV) continues to be a major problem in India even after adoption of mandatory screening for HBsAg (hepatitis B surface antigen) detection by ELISA (enzyme-linked immunosorbent assay) method. The high incidence of TAHBV is reported in patients receiving multiple transfusions. It has been demonstrated that some HBsAg-negative and anti HBc antigen positive (anti-hepatitis B core antigen positive) individuals continue to replicate hepatitis B virus (HBV). The negative HBsAg in the blood of apparently healthy individuals may not be sufficient to ensure absence of circulating HBV. Thus the blood containing anti-HBc antigen with or without detectable presence of HBsAg might be infectious.As of today, the blood banks of many countries have adopted anti-HBc testing to decrease HBV transfusion risk, while others have not. Anti-HBc testing is not mandatory in blood banks of many developing countries, and only HBsAg testing by ELISA is used as screening test for HBV infection.et al. reported higher incidence of HBV-carrier state in transfusion recipients as compared to the general population (12.3% vs. 3.6%). These authors found the incidence of TAH-B (transfusion associated hepatitis B) to be 11%.[The prevalence rate of HBsAg-positive adult, healthy donor population in the country is around 4.7%. Data suggests that in the general population about 75% carriers acquire infection by horizontal spread during early childhood, and the remaining acquire it by vertical (mother-to-child) transmission. A study HBV consists of several proteins or antigens to which antibodies are formed. A surface antigen protein HBsAg is present on the outer envelope of the virus, and it can be found floating free in the plasma. Antibodies are also produced against the two proteins present within the core, hepatitis B core antigen (HBcAg) and hepatitis Be antigen (HBeAg).HBV DNA is the first marker to be detected by polymerase chain reaction (PCR) before HBsAg reaches detectable levels. But the PCR or nucleic acid amplification test (NAT) is not performed routinely in blood centers. HBcAg is present in the serum but is undetectable; however, IgM anti-HBc is the first antibody to appear and persists for about six months. IgG anti-HBc is found lifelong in persons who have been infected with HBV and is a marker of past or current viral replication. In the fCarriers with HBsAg below the detection level can transmit HBV by blood transfusion.Subjects infected with HBV may show HBsAg negative result owing to point mutation in the pre-core region of the virus, resulting in inability to synthesize HBsAg. Fulminant hepatitis developed in recipients of HBsAg-negative blood from such donors infected with mutated virus. In all those donors, high levels of anti-HBc were present.In acute infection, there are two periods when HBsAg may be undetectable although the subject can transmit HBV — during early incubation period when both HBsAg and anti-HBc are undetectable; and after clearance of HBsAg but before anti-HBs has become detectable (diagnostic window). In this phase, anti-HBc and anti-HBe can be detected.To encourage voluntary blood donation should be the first step of prevention. In Japan a change from paid donation to voluntary donation decreased the incidence of TAH(transfusion associated hepatitis) from 51% to 16%. Second step is to use sensitive tests for detection of HBV. Anti-HBc testing was introduced in the United States as a surrogate marker to identify units of donated blood that posed increase risk of transmission of HBV infection.5In India, the reported incidence of anti-HBc among blood donors ranges from 17% to 29%. Most of In India seroprevalence of anti-HBc is quite high as compared to western countries; hence screening of donor blood for total anti-HBc may not be practically important and cannot be the criterion to discard blood units, whereas the anti-HBc IgM–reactive samples with negative HBsAg test may identify the potentially infectious blood units.Retrospective studies on regular blood donors and their respective recipients are essential to know the rate of HBV transmissions via anti-HBc–positive and HBsAg-negative donations. Routine anti-HBc IgM screening of blood donors could probably prevent some transfusion-transmitted HBV infections.To reduce the risk of transfusion-associated hepatitis B, test for anti-HBc IgM may be included in routine screening of donors’ blood as it has been proved to be an excellent indicator of occult HBV during window period. However, awareness and education of donors regarding the modes of HBV transmission, a stringent one-to-one donor screening and increasing the voluntary donor base should also be implemented to minimize the rate of transfusion-associated hepatitis B.


Mycoplasma pneumoniae and other infections can be associated with the production of cold agglutinins. In its classic presentation with haemolytic anaemia and Raynaud's syndrome, cold agglutinin disease is usually idiopathic. Several factors play a role in determining the ability of a cold agglutinin to induce a haemolytic anaemia such as antibody concentration and temperature range, in particular the highest temperature at which antibodies interact with red blood cells.Cold agglutinin disease usually develops as a result of the production of a specific immunoglobulin M auto-antibody directed against the I/i and H antigens, precursors of the ABH and Lewis blood group substances, on red blood cells. Autoimmune and lymphoproliferative disorders, A 48-year-old Caucasian man presented to our hospital with symptoms of extreme asthenia caused by severe anaemia. The transfusion of red blood cells (O Rh-positive), started as prescribed by the emergency guidelines in force without pre-transfusion tests, induced fatal haemolysis because of the presence of high levels of anti-H antibodies in his blood, that reacted with the large amount of H antigen in universal (0) red blood cells.Emergency transfusion of universal red blood cells (0 Rh-positive or negative) is usually accepted by the international guidelines in force in emergency departments. In this report we describe a rare complication caused by the very high concentration in the recipient of cold agglutinins and the activation of the complement system, responsible for red blood cell lysis and consequent fatal cardiovascular shock. We conclude that emergency transfusion of universal red blood cells (0 Rh-positive or negative) may be dangerous and its risk should be assessed against the risk of delaying transfusion until the pre-transfusion tests are completed. Cold agglutinins were first described by Landsteiner in 1903 ,3. In 19CAD is characterized by an auto-antibody  which isPatients show haemolytic anaemia of varying degrees of severity, as well as episodes of hemoglobinuria and acrocyanosis, which arise or worsen upon exposure to low temperatures.Mycoplasma pneumoniae or infection by the Epstein-Barr virus, as well as by lymphoproliferative disorders such as Waldenström's macroglobulinemia.Cold agglutinin antibodies are mainly specific for the I/i and H RBCs membrane systems , and theThe auto-antibody involved is usually an IgM, less frequently an IgA or IgG, which is able to agglutinate RBCs at temperatures of between 0 and 5°C. Complement activation generally occurs between 20 and 25°C, but is also possible at normal body temperature. It is also important to note that agglutination is not necessary for complement activation, especially in patients with high levels of auto-antibodies  ,8. This A 48-year-old Caucasian man presented to the Accident and Emergency Department of our hospital with symptoms of extreme asthenia, but showed no evidence of Raynaud's syndrome. In the past few months, he had complained about a productive cough and post-prandial vomiting. At admission, he was evidently dehydrated and undernourished, very pale, dyspnoeic and tachycardiac (110 bpm) at rest. Heart sounds were soft but no other pathologic sign concerning his lungs and abdomen was noted. His blood pressure was 80 over 50 mmHg.A blood cell count showed severe anaemia (haemoglobin = 3.8gr/dl) and the patient was prescribed an emergency transfusion of RBCs (0 Rh-positive), owing to the severe anaemia associated with dyspnoea and tachycardia at rest, and hypotension. Blood samples were also sent to our Blood Transfusion Service at this time. Previous data relating to our patient was not found in our records.After centrifugation, samples showed low hematocrit and normal plasma appearance. The direct blood group test resulted in unequivocally A with Rh phenotype Ccddee, while the indirect test revealed agglutination of B cells and a strong agglutination of 0 cells. Antibody screening also showed strong agglutination (4+) of all panel cells.The above-mentioned Accident and Emergency Department was immediately alerted to our patient's immunohaematological situation, and we advised urgent cessation of the transfusion of RBCs (0 Rh-positive), which the physician had already initiated as prescribed by the emergency guidelines in force. We also recommended our patient's transferral to the Haematology Department in Ferrara City Hospital, where he arrived in a state of severe cardiovascular shock.Blood samples taken from him at this time showed dramatic haemolysis, which led to his death within a few hours.Subsequent blood tests revealed the presence of cold agglutinin syndrome with very high levels of anti-H (1:65.600). He showed positive (3+) results for the direct antiglobulin test for complement fractions, which caused the intravascular haemolysis and consequent cardiovascular shock.His death prevented further attempts to define the aetiology of his condition. However, an emergency ultrasonography had shown several enlarged lymph nodes  along the iliac vessels and the thoracic and abdominal aorta, suggesting a lymphomatous pathology.The dramatic clinical situation of our patient upon presentation led the physician to commence emergency transfusion of universal RBCs (0 Rh-positive) according to the guidelines which were designed to safeguard against major complications such as multi-organ failure due to severe anaemia.However, the transfusion of 0 RBCs, expressing a large amount of H antigens, caused dramatic haemolysis, cardiovascular shock and our patient's death within a few hours.We conclude that emergency transfusion of universal red blood cells (0 Rh-positive/negative) may be dangerous and the risks of the procedure should be assessed against the risks of delaying transfusion until the pre-transfusion tests are completed.The authors declare that they have no competing interests.GL and DR performed pre-transfusion evaluations and made the diagnosis; then they searched the literature for other similar reports. GL and RR drafted the manuscript. All authors read and approved the final manuscript.Written consent was obtained from our patient's next-of-kin for publication of this case study. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Chronic diseases are generating a major health and societal burden worldwide. Healthy lifestyles, including physical activity (PA), have proven efficacy in the prevention and treatment of many chronic conditions. But, so far, national PA surveillance systems, as well as strategies for promotion of PA, have shown low impact. We hypothesize that personalized modular PA services, aligned with healthcare, addressing the needs of a broad spectrum of individual profiles may show cost-effectiveness and sustainability.The current manuscript describes the protocol for regional implementation of collaborative self-management services to promote PA in Catalonia (7.5 M habitants) during the period 2017–2019. The protocols of three implementation studies encompassing a broad spectrum of individual needs are reported. They have a quasi-experimental design. That is, a non-randomized intervention group is compared to a control group  using propensity score methods wherein age, gender and population-based health risk assessment are main matching variables. The principal innovations of the PA program are: i) Implementation of well-structured modular interventions promoting PA; ii) Information and communication technologies (ICT) to facilitate patient accessibility, support collaborative management of individual care plans and reduce costs; and iii) Assessment strategies based on the Triple Aim approach during and beyond the program deployment.The manuscript reports a precise roadmap for large scale deployment of community-based ICT-supported integrated care services to promote healthy lifestyles with high potential for comparability and transferability to other sites.NCT02976064). Registered November 24th, 2016.This study protocol has been registered at ClinicalTrials.org (The online version of this article (10.1186/s12913-018-3363-8) contains supplementary material, which is available to authorized users. Physical inactivity is acknowledged as a significant public health problem worldwide with kernel implications on major chronic conditions , 2. On tThe growing awareness on the health burden generated by insufficient levels of PA has prompted the interest for deploying community-based initiatives aiming at fostering active healthy living . HoweverThe current protocol relies on the hypothesis that properly tailored self-management services, fully integrated in patient’s health action plan with remote off-line professional support, may induce sustained behavioral changes resulting in active lifestyle behavior. A recent study  assessinThe ultimate aim of the novel PA services for chronic patients and citizens at risk is to generate sustained enhancement of PA with health value generation. Hence, is expected a positive impact on multi-morbidities in terms of: i) Preventing occurrence, ii) Reducing exacerbations, and, iii) Modulating disease progress. The main outcome of the current protocol will be a roadmap for large-scale deployment and assessment of novel collaborative self-management PA services in the region of Catalonia (7.5 million citizens). The approach taken for promotion of PA shall have potential for generalization to other interventions aiming at promoting healthy lifestyles, as part of regional integrated care strategies for chronic patients.The protocol has been designed as part of the 2016–2020 Health Plan in Catalonia  that invhttps://lamevasalut.gencat.cat/web/guest/pre-login-cps)  as a self-management tool adopted to facilitate patient empowerment for self-care and enhanced monitoring. Moreover, the PHF is linked to the regional shared electronic health record (HC3) [The technological support used in the studies facilitates collaborative work among different healthcare professionals as well as patient empowerment for self-management. This scenario involves deployment of a Digital Health Framework , 24 bridrd (HC3) , and pro3 and the PHF.Another key ICT-supporting tool for the current PA protocol is an adaptive case management (ACM) system  to suppoEthical Committee for Clinical Research at Hospital Clínic de Barcelona approved the study (HCB/2016/0883) which has been registered at ClinicalTrials.org (NCT02976064). Patients’ acceptance and signature of the informed consent will be required to participate in all the studies.Large scale deployment of the current protocol is supported by convergence of different types of resources from: i) Institutional bodies ; ii) Healthcare providers ; and, iii) EU Grants (see funding section of the manuscript). The The protocol addresses the four aims displayed in Fig. n = 1000) in the current protocol is to assess cost-effectiveness of the deployment of the intervention as mainstream service at Hospital Clinic de Barcelona.A recent randomized controlled trial (RCT) carried out in high-risk candidates to major abdominal surgery has demonstrated high efficacy of prehabilitation reducing postoperative complications in these patients . The main = 800) is to assess both cost-effectiveness and sustainability of a community-based program combining exercise training and promotion of PA in clinically stable chronic patients showing one or more of the following target disorders: i) Chronic obstructive pulmonary disease (COPD) or other chronic respiratory conditions; ii) Cardiovascular disorders (CVD); and/or iii) Type II diabetes mellitus (T2DM).A previous non-randomized pilot study carried out by the research team  identifin = 1800) will assess large-scale deployment and sustainability of a portfolio of services promoting PA and healthy lifestyles for citizens at risk for the targeted chronic conditions and patients showing mild target disease(s). The study will also assess the impact of the intervention on health-related quality of life.Finally, the Lifestyle study  of the corresponding intervention as well as to identify factors modulating the success of large scale deployment. To this end, for each implementation study, an intervention group will be compared to a control group . Briefly, the Prehabilitation study will be deployed at  methods  wherein  methods , 34 willInclusion criteria for the Prehabilitation study are: i) Candidates to major elective abdominal, gynecological, cardiovascular, urological and thoracic surgical procedures; ii) Patients presenting high surgical risk defined by: age above 70 years and/or American Society of Anesthesiologists (ASA) score III/IV ; and, iiThe Rehabilitation study has the following inclusion criteria: i) Patients suffering one or more targeted chronic conditions with moderate-to-severe disease of the main disorder; and, ii) Patients showing history of frequent exacerbations.Finally, the inclusion criteria for the Lifestyle study are as follows: i) Citizens at risk for chronic conditions; and, ii) Patients showing mild target disease(s). All of them recruited through advertisements and through primary care centers.Exclusion criteria for the three implementation studies are as follows: i) Emergency surgery (applicable only for the Prehabilitation study); ii) Unstable cardiac of respiratory disease; iii) Locomotor limitation precluding the practice of exercise; and, iv) Cognitive deterioration impeding the adherence to the program.The entry point to each program, as well as the core management and coordination functions, will be ascribed to the primary care team composed by general practitioner and case manager (physiotherapist). They will be directly in charge of the six common steps of the service workflow, namely: i) inclusion; ii) characterization and re-assessment of patient’s work plan; iii) integration of the intervention into the patient’s action plan; iv) execution of the specific program; v) follow-up/event handling; and, (vi) discharge from the program, as explained in detail in Section 1S.The current protocol aims to rollout the recently reported prehabilitation service  to all hCandidates for the prehabilitation study fulfilling the inclusion criteria will be identified by the anesthesiologist in the pre-anesthesia visit. All candidates will be assessed in order to address a variety of issues: i) Identify the overall needs of the candidate; ii) Perform a baseline evaluation of his/her fitness and physical activity levels; and, iii) Identify his/her adherence profile, as well as factors and circumstances that may modulate practicalities of the intervention. Personalization of the prehabilitation plan involves the following main actions: i) Calendar and planning of face to face visits and remote  contacts with healthcare professionals; ii) Intensity/volume of the supervised endurance training program; iii) Threshold of minimum steps per day to promote physical activity; iv) Specific nutritional counseling and intervention ; v) GroHealth value generation by personalized services including endurance training and promotion of PA will be assessed in chronic patients with target diseases. Briefly, the planned six-month period intervention will have two phases including, as a first step, reassessment of the patient work-plan aiming at optimization of both pharmacological and non-pharmacological therapies. The first phase of the program will start with a motivational interview wherein the intervention will be explained and co-designed with the patient. It will be followed by: i) Supervised endurance training sessions with a flexible duration of 2 to 6 weeks; ii) Promotion of active lifestyle; and, iii) Patient empowerment for self-management of his/her condition aiming at increasing program adherence. The second phase of the intervention will include promotion of PA and self-management using the PHF  with remThe main objective is to assess the impact of a program promoting PA and healthy lifestyles in citizens at risk and patients with mild chronic disease. Briefly, key aspects of the intervention are: i) A motivational interview to personalize the intervention; ii) Familiarization of the candidate with the use of the PHF as a self-management tool; and, iii) Assignment of one case manager for off-line remote surveillance of the service. Moreover, the subject will receive information on a portfolio of optional services offered by the service   methodolThe present protocol aims to evaluate the determinants of large-scale deployment and adoption of three novel collaborative self-management services promoting healthy lifestyles. We believe that the protocol represents a highly relevant step to foster synergies between non-pharmacological and pharmacological care paving the way for enhanced preventive care for chronic patients.The study protocol adopts a population-health approach aiming at articulating a test bed that shall facilitate a successful rollout of the three implementation studies at regional level. Synergies across healthcare tiers will be fostered throughout the program deployment. For example, the current hospital-based prehabilitation study will be expanded and enriched with appropriate health-risk assessment tools aiming at enhancing perioperative risk prediction and care at community level. Thus, it should facilitate productive collaborative work between specialized and primary care professionals. Moreover, the program is expected to be highly transferable to other geographical areas.The current manuscript reports on the protocol for regional deployment of collaborative self-management services promoting healthy lifestyles that will be carried out in Catalonia during the period 2017–2019.Additional file 1:Protocol for regional implementation of collaborative self-management services to promote physical activity. This file includes the specific details of each of the three protocols described in the main manuscript. (DOC 531 kb)


The combined use of immunotherapy and radiation therapy is emerging as a potentially effective treatment for patients with immunogenic tumors such as melanoma; however, evidence for long-term treatment outcomes is lacking. Herein, we summarize our previously described case study of a patient with metastatic melanoma treated with two cycles of ipilimumab, followed by stereotactic body radiotherapy to two of seven liver metastases, with two additional cycles of ipilimumab. In the longest follow-up to date, we report a successful treatment outcome at 6.5 years. Our patient remains in complete remission, with no evidence of disease or recurrence 6.5 years after treatment. He continues to manage chronic hypophysitis developed secondary to immunotherapy and has developed osteopenia from prolonged systemic glucocorticoid use. The use of radiotherapy in combination with targeted immune therapy appears to be an effective treatment strategy, with long-lasting efficacy. Melanoma is a highly immunogenic cancer, and its ability to induce a systemic immune response has made it ideal for immunotherapy treatment. Increasing numbers of reports in the literature have demonstrated the safety and efficacy of combined radiation treatment (RT) with systemic immunotherapy as a means of inducing a clinically meaningful anti-melanoma immune response with an abscopal response at sites of disease distant from those treated with local RT -5.Herein, we present the 6.5 year follow-up of a patient with metastatic (stage IV) melanoma. This case illustrates a promising successful long-term treatment outcome of combined immunotherapy and high-dose stereotactic body radiotherapy in metastatic melanoma. We discuss current findings regarding the abscopal effect as well as current literature exploring the mechanism of action for the abscopal effect.2.In our initial case report , we descHe underwent wide excision of the primary lesion followed by completion lymph node dissection (LND) of the left axillary basin following what was initially deemed a positive sentinel lymph node biopsy, although histology from the LND favored a diagnosis of capsular nevi within several regional lymph nodes (LNs) . The patient remained disease-free for three years, when an in-transit metastasis was detected near the primary site. Following excision of the lymphatic metastasis, he was treated with adjuvant radiotherapy of 50 Gy in 20 fractions to the left posterior arm, followed by one month of adjuvant systemic therapy with high-dose interferon (IFN). One year later, he experienced a second local in-transit recurrence, with positron emission tomography–computed tomography (PET-CT) imaging and tissue confirmation of two to three metastatic melanoma lesions in the liver. BRAF testing on the hepatic metastasis was negative for the V600 mutation.To induce an anti-tumor immune response that could mediate systemic tumor regression , planned treatment for the patient included four doses of ipilimumab (anti-CTLA-4) at 3 mg per kilogram of body weight every three weeks, with radiation to begin after two doses. After two cycles of ipilimumab alone, a PET-CT scan showed progression of liver metastases with enlargement of the two previous liver lesions and development of five new hypermetabolic foci in the liver, the largest measuring 2.3 x 2.5 cm Figure . He thenFollow-upSix and a half years after completing treatment, the patient remains disease-free. He continues to be followed every six months, and as of June 2018, he has no clinical or radiologic evidence of disease recurrence. His most recent PET-CT and maximum intensity projection (MIP) scans were performed in 2016 and showed no evidence of disease Figure . His duaOur patient represents the longest reported follow-up of an abscopal complete response (CR) in metastatic melanoma, remaining without evidence of disease or recurrence for 6.5 years after a combined modality treatment of ipilimumab and radiotherapy to two of his seven metastatic melanoma liver lesions. Reports of this phenomenon in patients with metastatic cancer have increased since Postow et al. first reported it in 2012 [Radiation has long been used as a local therapy for cancer and growing evidence shows that irradiation can cause or contribute to regression of distant tumors at non-irradiated sites. The “abscopal effect” is mediated by the induction of a clinically meaningful anti-tumor immune response. There are multiple factors involved in this process. For example, radiation induces upregulation of the major histocompatibility complex (MHC) I surface expression which has been shown to enhance immune responses . MHC I iThe rarity of cases describing the abscopal effect with RT alone suggest that radiation is not sufficient to induce efficacious systemic antitumor effects in most cases. While radiation promotes immune stimulatory signals, it also has immunosuppressive effects (e.g. stimulating tumor growth factor-b (TGFb) production and upregulating T-regulatory (T-reg) cells) . TherefoWhile the role of ipilimumab in aiding an anti-tumor immune response is promising, it induces immune-related adverse events (irAEs) in up to 64% of patients . CheckpoThe longevity of the remission of this patient demonstrates the potential benefit of combining checkpoint blockade and radiotherapy. Of note, this patient was treated before anti-PD-1 therapy was widely used in melanoma, and he did not receive any additional cancer-directed treatment after completing the four cycles of ipilimumab. Use of a high dose of radiotherapy (54 Gy in three fractions) may have enhanced systemic immune response contributing to effective tumor resolution . The resIn order to optimize this therapy, further investigation into the mechanism of action is needed to understand how best to combine these therapies. Several prospective trials have shown the promise of combining checkpoint blockade with RT to induce a synergistic effect in the treatment of immunogenic cancers ; howeverin vivo tumor vaccine strategy.We report long term successful outcomes 6.5 years following combined radiotherapy and immunotherapy in a patient with metastatic melanoma. This patient has had a durable, complete abscopal response, demonstrating the potential of this combined therapy as a noninvasive


Cortical activity has distinct features across scales, from the spiking statistics of individual cells to global resting-state networks. We here describe the first full-density multi-area spiking network model of cortex, using macaque visual cortex as a test system. The model represents each area by a microcircuit with area-specific architecture and features layer- and population-resolved connectivity between areas. Simulations reveal a structured asynchronous irregular ground state. In a metastable regime, the network reproduces spiking statistics from electrophysiological recordings and cortico-cortical interaction patterns in fMRI functional connectivity under resting-state conditions. Stable inter-area propagation is supported by cortico-cortical synapses that are moderately strong onto excitatory neurons and stronger onto inhibitory neurons. Causal interactions depend on both cortical structure and the dynamical state of populations. Activity propagates mainly in the feedback direction, similar to experimental results associated with visual imagery and sleep. The model unifies local and large-scale accounts of cortex, and clarifies how the detailed connectivity of cortex shapes its dynamics on multiple scales. Based on our simulations, we hypothesize that in the spontaneous condition the brain operates in a metastable regime where cortico-cortical projections target excitatory and inhibitory populations in a balanced manner that produces substantial inter-area interactions while maintaining global stability. The mammalian cortex fulfills its complex tasks by operating on multiple temporal and spatial scales from single cells to entire areas comprising millions of cells. These multi-scale dynamics are supported by specific network structures at all levels of organization. Since models of cortex hitherto tend to concentrate on a single scale, little is known about how cortical structure shapes the multi-scale dynamics of the network. We here present dynamical simulations of a multi-area network model at neuronal and synaptic resolution with population-specific connectivity based on extensive experimental data which accounts for a wide range of dynamical phenomena. Our model elucidates relationships between local and global scales in cortex and provides a platform for future studies of cortical function. Cortical activity has distinct but interdependent features on local and global scales, molded by multi-scale connectivity. Data from multiple species including macaque indicate that the ground state of cortex locally features asynchronous irregular spiking with low pairwise correlations  and low,Most cortical models concentrate on either the local or the global scale, using two basic approaches. The first approach represents each neuron explicitly in networks ranging from local microcircuits to small numbers of areas , 15. TheCortical processing is not restricted to one or few areas, but results from complex multi-area interactions , 22. SimTwo main obstacles of multi-scale simulations are gradually being overcome. First, recent progress in simulation technology enables the efficient use of supercomputers . Second,2 microcircuit with the full density of neurons and synapses. A mean-field method ∀A.To investigate inter-area propagation, we determine the temporal order of spiking based on the location of the extremum of the correlation function for each pair of areas. This measure is chosen to characterize the relative timing of activity fluctuations across areas, as opposed to measures of causal interactions like Granger causality and the directed transfer function . In an a library  with pea library  with ranPSCj(t) = exp[−t/τs], * indicating convolution, synaptic time constant τs, the population firing rate νj of source population j, mean indegree Kij, and mean synaptic weight Jij of the connection from j to target population i containing Ni neurons. The population firing rate νj is defined as the spike histogram with bin width 1 ms averaged over the entire population, thus time t is in discrete increments of 1 ms.In the context of our spiking network model we define functional connectivity (FC) as the zero-time lag cross-correlation coefficient of the area-averaged synaptic inputs, which we approximate asneuRosim , so that the maximal real part of the eigenvalues λ′ of the resulting matrix G′ is max[Re(λ′)] = 1. This scaling modulates the relative strengths of direct and indirect paths: a larger value of max[Re(λ′)] increases the relative weighting of indirect paths. Subsequently we use the same method as ) The model construction starts with the binary connectivity, defines the weighted structural connectivity based on connection densities, and finally yields the functional connectivity from simulations. Comparing with the experimentally observed functional connectivity, we find that each level  adds explanatory power. In relation to fMRI functional connectivity, the role of anesthesia is worth investigating, as anesthetics influence BOLD responses in complex ways, although low doses as used in the experiment described here may have only mild effects, as shown in rat  and marmThe model predicts that population bursts propagate stably across multiple areas, predominantly in the feedback direction, because parietal areas are more unstable than temporal and occipital areas, as revealed by linear stability analysis. This occurs despite higher relative indegrees of feedforward connections, indicating the importance of the dynamical state for the effective influence of the structural connectivity. Specifically, the comparatively high firing rate of the layer 5 excitatory neurons enhances the influence of feedback compared to feedforward connections, which mainly originate in supragranular layers. The systematic activation of parietal before occipital areas in the model parallels human EEG findings on information flow during visual imagery  and top-Granger causality analysis of cortico-cortical interactions reveals layer- and population-specific communication channels that depend on the difference in the architectural types of the areas. Low architectural types correspond to low neuron density and a thin or absent layer 4 (more limbic areas); high architectural types correspond to high neuron density and a pronounced layer 4 (more sensory areas) , 54, 138Comparing these results with the network structure shows that substantial effective communication arises over a range of connection strengths due to the influence of the dynamical state of the populations, but that the weakest connections do not contribute significantly to communication. Our insights open up a new perspective on the significance of weak ties , since oIn the model, cortico-cortical indegrees are higher onto excitatory than onto inhibitory neurons, but stronger synapses onto inhibitory than onto excitatory neurons are required to maintain stability. This stabilization mechanism is similar to stabilization by inhibition locally in V1 , but useRandom connectivity below the population level is unlikely to suffice for the brain to perform its computations. Including higher-order structures such as neuron-level motifs  and patc2 patch of cortex. True area sizes vary from ~3 million cells in TH to ∼300 million cells in V1 for a total of ∼8 ⋅ 108 neurons per hemisphere of macaque visual cortex, a model size that with recent advances in simulation technology [For tractability, the model represents each area as a 1 mmchnology  already chnology  and enabOverall, our model elucidates multi-scale relationships between cortical structure and dynamics, and can serve as a platform for the iterative integration of new experimental data, the creation of hypotheses, and the development of functional models of cortex.


This work investigates the surface plasmon resonance (SPR) response of 50-nm thick nano-laminated gold film using Kretschmann-based biosensing for detection of urea and creatinine in solution of various concentrations (non-enzymatic samples). Comparison was made with the presence of urease and creatininase enzymes in the urea and creatinine solutions (enzymatic samples), respectively. Angular interrogation technique was applied using optical wavelengths of 670 nm and 785 nm. The biosensor detects the presence of urea and creatinine at concentrations ranging from 50–800 mM for urea samples and 10–200 mM for creatinine samples. The purpose of studying the enzymatic sample was mainly to enhance the sensitivity of the sensor towards urea and creatinine in the samples. Upon exposure to 670 nm optical wavelength, the sensitivity of 1.4°/M was detected in non-enzymatic urea samples and 4°/M in non-enzymatic creatinine samples. On the other hand, sensor sensitivity as high as 16.2°/M in urea-urease samples and 10°/M in creatinine-creatininase samples was detected. The enhanced sensitivity possibly attributed to the increase in refractive index of analyte sensing layer due to urea-urease and creatinine-creatininase coupling activity. This work has successfully proved the design and demonstrated a proof-of-concept experiment using a low-cost and easy fabrication of Kretschmann based nano-laminated gold film SPR biosensor for detection of urea and creatinine using urease and creatininase enzymes. End stage renal disease (ESRD) or commonly known as kidney failure is one of the major public health problems affecting approximately 10% of the world population. Imminently, the number of the ESRD patients that require haemodialysis continue to rise mainly due to the increase in cases of hypertension and diabetes and also in the growing elderly population. Kidney failure is often manifested by the unusual albumin secretion and decreased kidney function. During pathophysiological conditions, urea concentration ranges from 30–150 mM while the normal level range is between 2.5–6.7 mM . ConsequHaemodialysis monitoring system may include an optical sensor to detect urea and creatinine as the kidney wastes. The surface-enhance Raman scattering (SERS) which is one of the optical sensing method for urea and creatinine detection have been previously investigated. Leordean and coworkers  reportedAlternatively, the Kretschmann-based surface plasmon resonance (SPR) sensing approach using planar thin metal film offers distinct advantages over other label-free sensing techniques. Firstly, planar layer of high quality and uniformity are readily manufactured. A wide range of commercial SPR sensor slides is available in the market which give remarkably low limit of detection. Secondly, the field confinement resulting from the excitation of propagating surface plasmon polariton is highly uniform over large areas, albeit with weaker field confinement than the localized surface plasmon polariton counterpart using metal nanostructures . Adding The SPR phenomenon, in principle, is due to the confined electromagnetic wave of surface plasmon polariton in the vicinity of dielectric-metal interface. In fact, the Kretschmann configuration of the attenuated total reflection method is frequently used for SPR excitation . It cons-1cm-2 for the detection of urea using multi-wall carbon nanotubes with the presence of Ni-MOF. Finally, Raveendran et al [The urea and creatinine sensing research has been explored in various techniques such as ion sensitive field effect transistors, optical fibre-based SPR, and electrochemical system. Bhatia et al.  reportedan et al  discloseIn this paper, we propose an SPR-based sensor using Kretschmann configuration with urea-urease and creatinine-creatininase enzymatic sample that offers a simple, economical and label-free method of detection with excellent sensitivity. Urea and creatinine solutions of various concentrations in non-enzymatic samples as well as enzymatic samples were analysed by measuring the changes in refractive index of the samples at optical wavelengths of 670 and 785 nm. In brief, the commercially available Lumerical’s finite difference time domain (FDTD) software was used to find the optimum incident wavelength and thickness of the nano-laminated gold film. Afterward, these parameters were used for experimental investigation. This work corresponds to the early stages of designing a Kretschmann-based SPR biosensors for medical applications in artificial kidney. Specifically, the SPR technique can be used as a sensing method for determining the adequacy of dialysis using determination of urea and creatinine concentration in the dialysate effluent from artificial kidney. The knowledge can be subsequently employed in optical fibre-based SPR sensors for portable and cost-effective applications.Sensor slides with 50 nm-thick nano-laminated gold film coating were purchased from BioNavis Ltd. The Urea, creatinine, urease and creatininase powder purchased from Sigma Aldrich were used as received. Ultrapure water was used as a buffer from Milipore® system (resistivity of 18.2 MΩ.cm at room temperature of 25°C) and for dilution of urea, creatinine, urease enzyme and creatininase enzyme.o to 80o), was performed in order to obtain the source angle capable to excite the SPR mode. This happens when the y component of the source wave vector matches the wave vector of the SPR mode. In other words, the SPR occurs when the wave vector components are parallel to the nano-laminated gold film surface. The perfectly matched layer (PML) profile was set to a "steep angle" to better absorb light propagating at large angles away from the normal incidence. A mesh override region was used to force a finer mesh step size in the y-direction which is normal to the metal film. The optical parameter was set as a plane-wave source (Bloch/periodic type) at an optical wavelength of 633 nm. As shown in Initially, nano-laminated gold film thicknesses suitable for Kretschmann SPR-based sensor were simulated for thicknesses ranging from 20 to 80 nm at optical wavelength of 633 nm using angular interrogation technique. Lumerical’s FDTD software was used for the numerical analysis, where a parameter sweep over the incident angle  was used to run the experimental work based on the obtained numerical analysis data. The SPR Navi 200 instrument employed Kretschmann configuration where the incident light is coupled via prism so that the wave number of the incident light is enhanced to match that of the excited surface plasmon. The optical interface between the prism and glass side of the sensor slide contain refractive index matching elastomer to match the refractive index of the prism and the sensor glass slide. Without the use of the refractive index matching elastomer, reflection and refraction might occur at the optical interface and thus the SPR excitation at the gold surface will be affected. As shown in Experiments were conducted with various concentrations of urea and creatinine non-enzymatic samples and also enzymatic samples of urea-urease and creatinine-creatininase composition. The non-enzymatic urea samples were prepared by serial dilution with ultrapure water at 100, 200, 400, and 800 mM concentrations, whereas that of creatinine samples were at 10, 20, 50, 100, and 200 mM concentrations. As for the enzymatic samples, urease and creatininase powder were dissolved in ultrapure water based on their molecular weight calculation. The concentration of urease and creatininase enzymes were fixed at 0.682 μmol and 0.05 mmol, respectively. Ideally, the urease enzyme and creatininase enzyme solution were mixed with urea and creatinine solution right before injection into the SPR instrument as the composition denatured easily at room temperature. Accordingly, urease solution (1 ml) was mixed with urea solution (1 ml) from each concentration . Likewise, creatininase solution (1 ml) was mixed with creatinine solution (1 mm) from each concentration .Prior to the experiment, the peristaltic pump on the SPR instrument was turned on to fill the flow cells with ultrapure water as a buffer at 100 μl/min flow rate. This allows the buffer solution to flow over the nano-laminated gold film. Then, the nano-laminated gold film was exposed to the urea-urease enzymatic sample by injection through inlet 1 while creatinine-creatininase enzymatic sample through inlet 2 using a syringe. The injection valve was kept at ‘inject’ position for two minutes before turning off for another two minutes. The sample solutions flow across the nano-laminated gold film and discharged at outlet. Same procedure was performed on non-enzymatic samples of urea and creatinine solutions at different concentrations.Sn, is defined as the shift in incident angle per unit change in the concentration of urea and creatinine. It was measured by dividing the shifting angle value (from ultrapure water) per molarity of urea and creatinine solutions and express in (°/M). The SPR response curves were also used to obtain refractive index value of each concentration of urea and creatinine solutions using SPR Navi LayerSolver software. Accordingly, the data from SPR response curves obtained from 670 nm wavelength were used to calculate the thickness (d), refractive index (n), and dispersion relation (k) of the Au thin film under air exposure. Afterward, the refractive index of urea, creatinine, urease, and creatininase solutions at 670 nm were constrained from the linear relationship between λ and n (where n = a+bλ).The reflectivity, sensorgram, and sensitivity analysis of this nano-laminated gold sensor for urea and creatinine detection were measured from the SPR response curves obtained. Sensitivity, o. When the thickness is below the optimum value, the SPR response curve becomes broad whereas at above the optimum value, it become shallow indicate low reflectivity of the signal.In order to characterize the behaviour of nano-laminated gold film SPR-based sensor in different thickness, reflectivity was plotted against incident angle in SPR response curves as obtained from the FDTD numerical analysis. o, which is a higher value compared to 594 nm at 46o. Meanwhile, the resonance angle at optical wavelengths of 633, 670 and 785 nm occurred at 45o, 44o and 43o, respectively. The best SPR response curve occurred at 785 nm wavelength with reflectivity of 90.8% (FWHM = 0.55o), followed by 90.2% (FWHM = 0.71o) at 670 nm. At the wavelength of 534 nm, reflectivity and FWHM were not measured due to a very shallow and broad curve as a result of poor SPR excitation and it is therefore not suitable for the sensor design.Further, the numerical analysis conducted in angular interrogation mode with changes of optical parameter for a 50 nm-thick nano-laminated gold film SPR sensor gave plots of reflectivity versus incident angle. Next, a double channel SPR Navi 200 instrument in Kretschmann configuration using attenuated total reflection was used for sensing urea and creatinine in experimental studies. A commercially available 50 nm-thick nano-laminated gold SPR sensor was inserted into the sensor holder of the SPR instrument. Flow cell was set close to the triangular prism so that analyte solutions can easily be introduced to flow across the nano-laminated gold film surface. Analysis was undertaken for sensing of urea and creatinine at different concentrations in enzymatic and non-enzymatic samples using 670 nm and 785 nm optical wavelengths. The enzyme urease and creatininase were mix with urea and creatinine solution and were injected into the SPR instrument within one minute in order to prevent the urease and creatininase enzyme degraded. Afterward, the SPR instrument made the measurement and yield a SPR response curve within 30 seconds after the injection. This assure the materials degradation did not occur in this time scale. In order to observe the SPR response in a clearer manner, a sensorgram of the incident angle shifts versus time for different urea and creatinine concentrations at 670 and 785 nm optical wavelengths were plotted as shown in Results of sensitivity were plotted in sensitivity graph with increment of urea and creatinine concentrations in enzymatic and non-enzymatic samples as shown in It is demonstrated that the enzymatic samples on nano-laminated gold film SPR-based sensor had increased in sensitivity compared to the non-enzymatic samples. The improved sensitivity of the sensor was due to the increased in refractive index of analyte sensing layer as a result of urea-urease and creatinine-creatininase coupling activity. The presence of urease and creatininase enzymes in samples served as biomolecules recognition molecule in the sensor system. This proved that the Kretschmann-based SPR sensor with nano-laminated gold film for urea and creatinine detection using enzymatic solution is indeed a suitable method, especially for artificial kidney applications. It is an economical and label-free detection method with ease of operation, excellent sensitivity, and rapid detection in designing urea and creatinine sensor which will subsequently lead to a portable optical fibre-based SPR sensor applications. This work can be a proof-of-concept experiment and it can be applied to other analyte such as glucose and uric acid. The selectivity study using nano-laminated gold film sensor with enzyme immobilized on the sensing layer will be conducted in our future study with real sample from haemodialysis effluent. Immobilizing the enzyme on the sensor surface allows reusable usage of enzyme to for analyte detection. In this way the cost for operating the Kretschmann based SPR biosensor can be further reduced.We have successfully investigated a nano-laminated gold film SPR-based sensor using Kretschmann configuration for urea and creatinine detection. To the best of our knowledge, this is a novel investigation on the SPR sensitivity of 50 nm-thick nano-laminated gold film towards the different concentration of urea and creatinine in the presence of their respective enzymes using Kretschmann configuration. The sensorgram and sensitivity of the nano-laminated gold film towards the enzymatic and non-enzymatic sample of various concentrations have been successfully investigated and analysed. The effects of couple activity of analyte-enzyme in the enzymatic samples were believed to enhance the sensitivity of the sensor by ~150 to 1057% compared to a non-enzymatic solution (at 670 nm wavelength). Additional advantages of this sensor include low cost, commercial availability of nano-laminated gold film and capability of detecting even a small increase in urea and creatinine concentrations.