Species-level functional profiling of metagenomes and metatranscriptomes.

Functional profiles of microbial communities are typically generated using comprehensive metagenomic or metatranscriptomic sequence read searches, which are time-consuming, prone to spurious mapping, and often limited to community-level quantification. We developed HUMAnN2, a tiered search strategy that enables fast, accurate, and species-resolved functional profiling of host-associated and environmental communities. HUMAnN2 identifies a community's known species, aligns reads to their pangenomes, performs translated search on unclassified reads, and finally quantifies gene families and pathways. Relative to pure translated search, HUMAnN2 is faster and produces more accurate gene family profiles. We applied HUMAnN2 to study clinal variation in marine metabolism, ecological contribution patterns among human microbiome pathways, variation in species' genomic versus transcriptional contributions, and strain profiling. Further, we introduce 'contributional diversity' to explain patterns of ecological assembly across different microbial community types

Genetic Loci Involved in Antibody Response to Mycobacterium avium ssp. paratuberculosis in Cattle

Background: Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic enteritis in a wide range of animal species. In cattle, MAP causes a chronic disease called Johne's disease, or paratuberculosis, that is not treatable and the efficacy of vaccine control is controversial. The clinical phase of the disease is characterised by diarrhoea, weight loss, drop in milk production and eventually death. Susceptibility to MAP infection is heritable with heritability estimates ranging from 0.06 to 0.10. There have been several studies over the last few years that have identified genetic loci putatively associated with MAP susceptibility, however, with the availability of genome-wide high density SNP maker panels it is now possible to carry out association studies that have higher precision. Methodology/Principal Findings: The objective of the current study was to localize genes having an impact on Johne's disease susceptibility using the latest bovine genome information and a high density SNP panel (Illumina BovineSNP50 BeadChip) to perform a case/control, genome-wide association analysis. Samples from MAP case and negative controls were selected from field samples collected in 2007 and 2008 in the province of Lombardy, Italy. Cases were defined as animals serologically positive for MAP by ELISA. In total 966 samples were genotyped: 483 MAP ELISA positive and 483 ELISA negative. Samples were selected randomly among those collected from 119 farms which had at least one positive animal. Conclusion/Significance: The analysis of the genotype data identified several chromosomal regions associated with disease status: a region on chromosome 12 with high significance (P<5 710-6), while regions on chromosome 9, 11, and 12 had moderate significance (P<5 710-5). These results provide evidence for genetic loci involved in the humoral response to MAP. Knowledge of genetic variations related to susceptibility will facilitate the incorporation of this information into breeding programmes for the improvement of health status

Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle

Background Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne’s disease. This is mainly due to the time it takes for Mycobacterium avium subsp. paratuberculosis (MAP) to grow and lack of sensitivity of culture. Viable MAP cells can be detected in the blood of cattle suffering from Johne’s disease within 48 h using peptide-mediated magnetic separation (PMMS) followed by bacteriophage amplification. The aim of this study was to demonstrate the first detection of MAP in the blood of experimentally exposed cattle using the PMMS-bacteriophage assay and to compare these results with the immune response of the animal based on serum ELISA and shedding of MAP by faecal culture. Results Using the PMMS-phage assay, seven out of the 19 (37 %) MAP-exposed animals that were tested were positive for viable MAP cells although very low numbers of MAP were detected. Two of these animals were positive by faecal culture and one was positive by serum ELISA. There was no correlation between PMMS-phage assay results and the faecal and serum ELISA results. None of the control animals (10) were positive for MAP using any of the four detection methods. Investigations carried out into the efficiency of the assay; found that the PMMS step was the limiting factor reducing the sensitivity of the phage assay. A modified method using the phage assay directly on isolated peripheral blood mononuclear cells (without PMMS) was found to be superior to the PMMS isolation step. Conclusions This proof of concept study has shown that viable MAP cells are present in the blood of MAP-exposed cattle prior to the onset of clinical signs. Although only one time point was tested, the ability to detect viable MAP in the blood of subclinically infected animals by the rapid phage-based method has the potential to increase the understanding of the pathogenesis of Johne’s disease progression by warranting further research on the presence of MAP in blood

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death’ (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death’ (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death

Secreted and tumour targeted human carboxylesterase for activation of irinotecan

Irinotecan (CPT-11) is an anticancer agent for the treatment of colon cancer. CPT-11 can be considered as a prodrug, since it needs to be activated into the toxic drug SN-38 by the enzyme carboxylesterase. An approach to achieve tumour specific activation of CPT-11 is to transduce the cDNA encoding carboxylesterase into tumour cells. A secreted form of carboxylesterase may diffuse through a tumour mass and may activate CPT-11 extracellularly. This could enhance the antitumour efficacy by exerting a bystander effect on untransduced cells. In addition a secreted tumour-targeted form of carboxylesterase should prevent leakage of the enzyme from the site of the tumour into the circulation. We have constructed a secreted form of human liver carboxylesterase-2 by deletion of the cellular retention signal and by cloning the cDNA downstream of an Ig kappa leader sequence. The protein was secreted by transfected cells and showed both enzyme activity and efficient CPT-11 activation. To obtain a secreted, tumour-targeted form of carboxylesterase-2 the cDNA encoding the human scFv antibody C28 directed against the epithelial cell adhesion molecule EpCAM, was inserted between the leader sequence and carboxylesterase-2. This fusion protein showed CPT-11 activation and specific binding to EpCAM expressing cells. Importantly, in combination with CPT-11 both recombinant carboxylesterase proteins exerted strong antiproliferative effects on human colon cancer cells. They are, therefore, promising new tools for gene directed enzyme prodrug therapy approaches for the treatment of colon carcinoma with CPT-11

Physics of Neutron Star Crusts

The physics of neutron star crusts is vast, involving many different research fields, from nuclear and condensed matter physics to general relativity. This review summarizes the progress, which has been achieved over the last few years, in modeling neutron star crusts, both at the microscopic and macroscopic levels. The confrontation of these theoretical models with observations is also briefly discussed.Comment: 182 pages, published version available at <http://www.livingreviews.org/lrr-2008-10

Thymidine phosphorylase expression in normal, hyperplastic and neoplastic prostates: correlation with tumour associated macrophages, infiltrating lymphocytes, and angiogenesis

Thymidine phosphorylase is an angiogenic factor primarily expressed by cancer cells, stromal cells and tumour-associated macrophages in many human malignancies. These different types of thymidine phosphorylase-expressing cells, however, may have a distinct place in the angiogenic process, and this question was addressed in the present study. A series of 20 normal/hyperplastic prostate glands and 60 prostate carcinomas was investigated by immunohistochemistry, using specific antibodies for thymidine phosphorylase (P-GF.44C), tumour-associated macrophages (CD68), endothelium (CD31) and prostate specific antigen (ER-PR8). Thymidine phosphorylase expression by normal and hyperplastic epithelial or stromal cells occurred almost exclusively in the context of an intense lymphocytic infiltrate. High thymidine phosphorylase cancer cells and thymidine phosphorylase stromal cells expression was associated with high angiogenesis in prostate carcinomas, and this significant association was extended to include both tumour-associated macrophages and tumour-infiltrating lymphocytes. Thymidine phosphorylase expression and tumour-infiltrating lymphocytes were related inversely with prostate specific antigen reactivity. In conclusion, thymidine phosphorylase is a major angiogenic factor in prostate carcinomas and its up-regulation is likely to occur in the context of a host immune response

Whole-cell cancer vaccination: from autologous to allogeneic tumor- and dendritic cell-based vaccines

The field of tumor vaccination is currently undergoing a shift in focus, from individualized tailor-made vaccines to more generally applicable vaccine formulations. Although primarily predicated by financial and logistic considerations, stemming from a growing awareness that clinical development for wide-scale application can only be achieved through backing from major pharmaceutical companies, these new approaches are also supported by a growing knowledge of the intricacies and minutiae of antigen presentation and effector T-cell activation. Here, the development of whole-cell tumor and dendritic cell (DC)-based vaccines from an individualized autologous set-up to a more widely applicable allogeneic approach will be discussed as reflected by translational studies carried out over the past two decades at our laboratories and clinics in the vrije universiteit medical center (VUmc) in Amsterdam, The Netherlands

Sea Urchins Predation Facilitates Coral Invasion in a Marine Reserve

Macroalgae is the dominant trophic group on Mediterranean infralittoral rocky bottoms, whereas zooxanthellate corals are extremely rare. However, in recent years, the invasive coral Oculina patagonica appears to be increasing its abundance through unknown means. Here we examine the pattern of variation of this species at a marine reserve between 2002 and 2010 and contribute to the understanding of the mechanisms that allow its current increase. Because indirect interactions between species can play a relevant role in the establishment of species, a parallel assessment of the sea urchin Paracentrotus lividus, the main herbivorous invertebrate in this habitat and thus a key species, was conducted. O. patagonica has shown a 3-fold increase in abundance over the last 8 years and has become the most abundant invertebrate in the shallow waters of the marine reserve, matching some dominant erect macroalgae in abundance. High recruitment played an important role in this increasing coral abundance. The results from this study provide compelling evidence that the increase in sea urchin abundance may be one of the main drivers of the observed increase in coral abundance. Sea urchins overgraze macroalgae and create barren patches in the space-limited macroalgal community that subsequently facilitate coral recruitment. This study indicates that trophic interactions contributed to the success of an invasive coral in the Mediterranean because sea urchins grazing activity indirectly facilitated expansion of the coral. Current coral abundance at the marine reserve has ended the monopolization of algae in rocky infralittoral assemblages, an event that could greatly modify both the underwater seascape and the sources of primary production in the ecosystem