Functional characterization of two M42 aminopeptidases erroneously annotated as cellulases

Several aminopeptidases of the M42 family have been described as tetrahedral-shaped dodecameric (TET) aminopeptidases. A current hypothesis suggests that these enzymes are involved, along with the tricorn peptidase, in degrading peptides produced by the proteasome. Yet the M42 family remains ill defined, as some members have been annotated as cellulases because of their homology with CelM, formerly described as an endoglucanase of Clostridium thermocellum. Here we describe the catalytic functions and substrate profiles CelM and of TmPep1050, the latter having been annotated as an endoglucanase of Thermotoga maritima. Both enzymes were shown to catalyze hydrolysis of nonpolar aliphatic L-amino acid-pNA substrates, the L-leucine derivative appearing as the best substrate. No significant endoglucanase activity was measured, either for TmPep1050 or CelM. Addition of cobalt ions enhanced the activity of both enzymes significantly, while both the chelating agent EDTA and bestatin, a specific inhibitor of metalloaminopeptidases, proved inhibitory. Our results strongly suggest that one should avoid annotating members of the M42 aminopeptidase family as cellulases. In an updated assessment of the distribution of M42 aminopeptidases, we found TET aminopeptidases to be distributed widely amongst archaea and bacteria. We additionally observed that several phyla lack both TET and tricorn. This suggests that other complexes may act downstream from the proteasome

How a haemosporidian parasite of bats gets around: the genetic structure of a parasite, vector and host compared.

Parasite population structure is often thought to be largely shaped by that of its host. In the case of a parasite with a complex life cycle, two host species, each with their own patterns of demography and migration, spread the parasite. However, the population structure of the parasite is predicted to resemble only that of the most vagile host species. In this study, we tested this prediction in the context of a vector-transmitted parasite. We sampled the haemosporidian parasite Polychromophilus melanipherus across its European range, together with its bat fly vector Nycteribia schmidlii and its host, the bent-winged bat Miniopterus schreibersii. Based on microsatellite analyses, the wingless vector, and not the bat host, was identified as the least structured population and should therefore be considered the most vagile host. Genetic distance matrices were compared for all three species based on a mitochondrial DNA fragment. Both host and vector populations followed an isolation-by-distance pattern across the Mediterranean, but not the parasite. Mantel tests found no correlation between the parasite and either the host or vector populations. We therefore found no support for our hypothesis; the parasite population structure matched neither vector nor host. Instead, we propose a model where the parasite's gene flow is represented by the added effects of host and vector dispersal patterns

Constant regulation for stable CD8 T-cell functional avidity and its possible implications for cancer immunotherapy.

The functional avidity (FA) of cytotoxic CD8 T cells impacts strongly on their functional capabilities and correlates with protection from infection and cancer. FA depends on TCR affinity, downstream signaling strength, and TCR affinity-independent parameters of the immune synapse, such as costimulatory and inhibitory receptors. The functional impact of coreceptors on FA remains to be fully elucidated. Despite its importance, FA is infrequently assessed and incompletely understood. There is currently no consensus as to whether FA can be enhanced by optimized vaccine dose or boosting schedule. Recent findings suggest that FA is remarkably stable in vivo, possibly due to continued signaling modulation of critical receptors in the immune synapse. In this review, we provide an overview of the current knowledge and hypothesize that in vivo, codominant T cells constantly "equalize" their FA for similar function. We present a new model of constant FA regulation, and discuss practical implications for T-cell-based cancer immunotherapy

MAGE-A protein and MAGE-A10 gene expressions in liver metastasis in patients with stomach cancer

Tumour samples from 71 patients with stomach cancer, 41 patients with liver metastasis (group A) and 15 patients each in stages II–IV (group B) and stage I (group C) without liver metastasis were analysed. MAGE-A protein expression was evaluated by immunohistochemistry using a 6C1 monoclonal antibody and MAGE-A10 mRNA expression was detected by highly sensitive in situ hybridisation using a cRNA probe. Expressions of MAGE-A protein and MAGE-A10 mRNA in group A were detected in 65.9 and 80.5%, respectively. Both protein and gene showed significantly higher expression in group A than those in groups B (6.7, 26.7%) and C (0, 0%) (P=0.0003, P=<0.0001, respectively). MAGE-A10 mRNA expression in liver metastasis was found in eight (88.9%) out of nine patients. The concordant rate between MAGE-A family protein expression and MAGE-A10 mRNA expression in the primary sites was 81.7% (P<0.0001). MAGE-A10 gene expression was associated with reduced survival duration. The results of this study suggest that MAGE-A10 is a possible target in active immunotherapy for advanced stomach cancer

Analytical variables influencing the performance of a miRNA based laboratory assay for prediction of relapse in stage I non-small cell lung cancer (NSCLC)

<p>Abstract</p> <p>Background</p> <p>Laboratory assays are needed for early stage non-small lung cancer (NSCLC) that can link molecular and clinical heterogeneity to predict relapse after surgical resection. We technically validated two miRNA assays for prediction of relapse in NSCLC. Total RNA from seventy-five formalin-fixed and paraffin-embedded (FFPE) specimens was extracted, labeled and hybridized to Affymetrix miRNA arrays using different RNA input amounts, ATP-mix dilutions, array lots and RNA extraction- and labeling methods in a total of 166 hybridizations. Two combinations of RNA extraction- and labeling methods (assays I and II) were applied to a cohort of 68 early stage NSCLC patients.</p> <p>Results</p> <p>RNA input amount and RNA extraction- and labeling methods affected signal intensity and the number of detected probes and probe sets, and caused large variation, whereas different ATP-mix dilutions and array lots did not. Leave-one-out accuracies for prediction of relapse were 63% and 73% for the two assays. Prognosticator calls ("no recurrence" or "recurrence") were consistent, independent on RNA amount, ATP-mix dilution, array lots and RNA extraction method. The calls were not robust to changes in labeling method.</p> <p>Conclusions</p> <p>In this study, we demonstrate that some analytical conditions such as RNA extraction- and labeling methods are important for the variation in assay performance whereas others are not. Thus, careful optimization that address all analytical steps and variables can improve the accuracy of prediction and facilitate the introduction of microRNA arrays in the clinic for prediction of relapse in stage I non-small cell lung cancer (NSCLC).</p

Cytolytic T-cell response against Epstein-Barr virus in lung cancer patients and healthy subjects

<p>Abstract</p> <p>Background</p> <p>This study aimed to examine whether EBV seropositive patients with lung cancer have an altered virus-specific CTL response, as compared to age-matched healthy controls and whether any variation in this response could be attributed to senescence.</p> <p>Methods</p> <p>Peripheral blood mononuclear cells from lung cancer patients, age-matched and younger healthy individuals were used to measure EBV-specific CTLs after in vitro amplification with the GLCTLVAML and RYSIFFDYM peptides followed by HLA-multimer staining.</p> <p>Results</p> <p>Lung cancer patients and aged-matched controls had significantly lesser EBV-specific CTL than younger healthy individuals. Multimer positive populations from either group did not differ with respect to the percentage of multimer positive CTLs and the intensity of multimer binding.</p> <p>Conclusions</p> <p>This study provides evidence that patients with lung cancer exhibit an EBV-specific CTL response equivalent to that of age-matched healthy counterparts. These data warrant the examination of whether young individuals have a more robust anti-tumor response, as is the case with the anti-EBV response.</p

[multi’vocal]: reflections on engaging everyday people in the development of a collective non-binary synthesized voice

The growing field of Human-Computer Interaction (HCI) takes a step out from conventional screenbased interactions, creating new scenarios, in which voice synthesis and voice recognition become important elements. Such voices are commonly created through concatenative or parametric synthesis methods, which access large voice corpora, pre-recorded by a single professional voice actor. These designed voices arguably propagate representations of gender binary identities. In this paper we present our project, [multi’vocal], which aims to challenge the current gender binary representations in synthesized voices. More specifically we explore if it is possible to create a non-binary synthesized voice through engaging everyday people of diverse backgrounds in giving voice to a collective synthesized voice of all genders, ages and accents

HIV-Specific T-Cells Accumulate in the Liver in HCV/HIV Co-Infection

BACKGROUND AND AIMS: Hepatitis C Virus (HCV)-related liver disease progresses more rapidly in individuals co-infected with Human Immunodeficiency Virus-1 (HIV), although the underlying immunologic mechanisms are unknown. We examined whether HIV-specific T-cells are identified in the liver of HCV/HIV co-infected individuals and promote liver inflammation through bystander immune responses. METHODS: Ex-vivo intra-hepatic lymphocytes from HCV mono-infected and HCV/HIV co-infected individuals were assessed for immune responses to HIV and HCV antigens by polychromatic flow cytometry. RESULTS: HCV/HIV liver biopsies had similar frequencies of lymphocytes but lower percentages of CD4+ T-cells compared to HCV biopsies. In co-infection, intra-hepatic HIV-specific CD8+ and CD4+ T-cells producing IFN-gamma and TNF-alpha were detected and were comparable in frequency to those that were HCV-specific. In co-infected individuals, viral-specific CD8+ T-cells produced more of the fibrogenic cytokine, TNF-alpha. In both mono- and co-infected individuals, intra-hepatic HCV-specific T-cells were poorly functional compared to HIV-specific T-cells. In co-infection, HAART was not associated with a reconstitution of intra-hepatic CD4+ T-cells and was associated with reduction in both HIV and HCV-specific intra-hepatic cytokine responses. CONCLUSION: The accumulation of functional HIV-specific T-cells in the liver during HCV/HIV co-infection may represent a bystander role for HIV in inducing faster progression of liver disease