Background: Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein–DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid, and simplified Bind-n-seq protocol coupled with a user-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well-known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc). Results: The improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes. Informatics analysis revealed that many of these genes regulate functions associated with virulence, motility, and biofilm formation and included genes previously found involved in virulence. Additionally, electromobility shift assays show that YipR binds to the promoter region of XC_2633 in a CCCTCTC motif-dependent manner. Conclusion: We present a new and rapid Bind-n-seq protocol that should be useful to investigate DNA-binding proteins in bacteria. The analysis of YipR DNA binding using this protocol identifies a novel DNA sequence motif in the promoter regions of target genes that define the YipR regulon.

Introduction: Pandemic A/H1N1/2009 influenza causes severe lower respiratory complications in rare cases. The association between host immune responses and clinical outcome in severe cases is unknown. Methods: We utilized gene expression, cytokine profiles and generation of antibody responses following hospitalization in 19 critically ill patients with primary pandemic A/H1N1/2009 influenza pneumonia for identifying host immune responses associated with clinical outcome. Ingenuity pathway analysis 8.5 (IPA) (Ingenuity Systems, Redwood City, CA) was used to select, annotate and visualize genes by function and pathway (gene ontology). IPA analysis identified those canonical pathways differentially expressed (P < 0.05) between comparison groups. Hierarchical clustering of those genes differentially expressed between groups by IPA analysis was performed using BRB-Array Tools v.3.8.1. Results: The majority of patients were characterized by the presence of comorbidities and the absence of immunosuppressive conditions. pH1N1 specific antibody production was observed around day 9 from disease onset and defined an early period of innate immune response and a late period of adaptive immune response to the virus. The most severe patients (n = 12) showed persistence of viral secretion. Seven of the most severe patients died. During the late phase, the most severe patient group had impaired expression of a number of genes participating in adaptive immune responses when compared to less severe patients. These genes were involved in antigen presentation, B-cell development, T-helper cell differentiation, CD28, granzyme B signaling, apoptosis and protein ubiquitination. Patients with the poorest outcomes were characterized by proinflammatory hypercytokinemia, along with elevated levels of immunosuppressory cytokines (interleukin (IL)-10 and IL-1ra) in serum. Conclusions: Our findings suggest an impaired development of adaptive immunity in the most severe cases of pandemic influenza, leading to an unremitting cycle of viral replication and innate cytokine-chemokine release. Interruption of this deleterious cycle may improve disease outcome.

Background: Colon cancer patients with the same stage show diverse clinical behavior dueto tumor heterogeneity. We aimed to discover distinct classes of tumorsbased on microarray expression patterns, to analyze whether the molecularclassification correlated with the histopathological stages or otherclinical parameters and to study differences in the survival.Methods: Hierarchical clustering was performed for class discovery in 88 colon tumors(stages I to IV). Pathways analysis and correlations between clinicalparameters and our classification were analyzed. Tumor subtypes werevalidated using an external set of 78 patients. A 167 gene signatureassociated to the main subtype was generated using the 3-Nearest-Neighbormethod. Coincidences with other prognostic predictors were assesed.Results: Hierarchical clustering identified four robust tumor subtypes withbiologically and clinically distinct behavior. Stromal components(p < 0.001), nuclear β-catenin (p = 0.021),mucinous histology (p = 0.001), microsatellite-instability(p = 0.039) and BRAF mutations (p < 0.001) wereassociated to this classification but it was independent of Dukes stages(p = 0.646). Molecular subtypes were established from stage I.High-stroma-subtype showed increased levels of genes and altered pathwaysdistinctive of tumour-associated-stroma and components of the extracellularmatrix in contrast to Low-stroma-subtype. Mucinous-subtype was reflected bythe increased expression of trefoil factors and mucins as well as by ahigher proportion of MSI andBRAFmutations. Tumor subtypes werevalidated using an external set of 78 patients. A 167 gene signatureassociated to the Low-stroma-subtype distinguished low risk patients fromhigh risk patients in the external cohort (Dukes B andC:HR = 8.56(2.53-29.01); Dukes B,C andD:HR = 1.87(1.07-3.25)). Eight different reported survival genesignatures segregated our tumors into two groups the Low-stroma-subtype andthe other tumor subtypes.Conclusions: We have identified novel molecular subtypes in colon cancer with distinctbiological and clinical behavior that are established from the initiation ofthe tumor. Tumor microenvironment is important for the classification andfor the malignant power of the tumor. Differential gene sets and biologicalpathways characterize each tumor subtype reflecting underlying mechanisms ofcarcinogenesis that may be used for the selection of targeted therapeuticprocedures. This classification may contribute to an improvement in themanagement of the patients with CRC and to a more comprehensiveprognosis.

Journal of Medical Internet Research - International Scientific Journal for Medical Research, Information and Communication on the Internet

The environment's contribution to health has been conceptualized as the exposome. Biomedical research interest in environmental exposures as a determi

HIV-1 replication is efficiently controlled by the regulator protein Tat (101 amino acids) and codified by two exons, although the first exon (1-72 amino acids) is sufficient for this process. Tat can be released to the extracellular medium, acting as a soluble pro-apoptotic factor in neighboring cells. However, HIV-1-infected CD4(+) T lymphocytes show a higher resistance to apoptosis. We observed that the intracellular expression of Tat delayed FasL-mediated apoptosis in both peripheral blood lymphocytes and Jurkat cells, as it is an essential pathway to control T cell homeostasis during immune activation. Jurkat-Tat cells showed impairment in the activation of caspase-8, deficient release of mitochondrial cytochrome c, and delayed activation of both caspase-9 and -3. This protection was due to a profound deregulation of proteins that stabilized the mitochondrial membrane integrity, such as heat shock proteins, prohibitin, or nucleophosmin, as well as to the up-regulation of NF-κB-dependent anti-apoptotic proteins, such as BCL2, c-FLIPS, XIAP, and C-IAP2. These effects were observed in Jurkat expressing full-length Tat (Jurkat-Tat101) but not in Jurkat expressing the first exon of Tat (Jurkat-Tat72), proving that the second exon, and particularly the NF-κB-related motif ESKKKVE, was necessary for Tat-mediated protection against FasL apoptosis. Accordingly, the protection exerted by Tat was independent of its function as a regulator of both viral transcription and elongation. Moreover, these data proved that HIV-1 could have developed strategies to delay FasL-mediated apoptosis in infected CD4(+) T lymphocytes through the expression of Tat, thus favoring the persistent replication of HIV-1 in infected T cells.