Phylotyping and Functional Analysis of Two Ancient Human Microbiomes

Background: The Human Microbiome Project (HMP) is one of the U.S. National Institutes of Health Roadmap for Medical Research. Primary interests of the HMP include the distinctiveness of different gut microbiomes, the factors influencing microbiome diversity, and the functional redundancies of the members of human microbiotas. In this present work, we contribute to these interests by characterizing two extinct human microbiotas. Methodology/Principal Findings: We examine two paleofecal samples originating from cave deposits in Durango Mexico and dating to approximately 1300 years ago. Contamination control is a serious issue in ancient DNA research; we use a novel approach to control contamination. After we determined that each sample originated from a different human, we generated 45 thousand shotgun DNA sequencing reads. The phylotyping and functional analysis of these reads reveals a signature consistent with the modern gut ecology. Interestingly, inter-individual variability for phenotypes but not functional pathways was observed. The two ancient samples have more similar functional profiles to each other than to a recently published profile for modern humans. This similarity could not be explained by a chance sampling of the databases. Conclusions/Significance: We conduct a phylotyping and functional analysis of ancient human microbiomes, while providing novel methods to control for DNA contamination and novel hypotheses about past microbiome biogeography. We postulate that natural selection has more of an influence on microbiome functional profiles than it does on the species represented in the microbial ecology. We propose that human microbiomes were more geographically structured during pre-Columbian times than today

Conserved Orb6 Phosphorylation Sites Are Essential for Polarized Cell Growth in Schizosaccharomyces pombe

The Ndr-related Orb6 kinase is a key regulator of polarized cell growth in fission yeast, however the mechanism of Orb6 activation is unclear. Activation of other Ndr kinases involves both autophosphorylation and phosphorylation by an upstream kinase. Previous reports suggest that the Nak1 kinase functions upstream from Orb6. Supporting this model, we show that HA-Orb6 overexpression partially restored cell polarity in nak1 ts cells. We also demonstrated by coimmunoprecipitation and in vitro binding assays that Nak1 and Orb6 physically interact, and that the Nak1 C-terminal region is required forNak1/Orb6 complex formation in vivo. However, results from in vitro kinase assays did not show phosphorylation of recombinant Orb6 by HA-Nak1, suggesting that Orb6 activation may not involve direct phosphorylation by Nak1. To investigate the role of Orb6 phosphorylation and activity, we substituted Ala at the ATP-binding and conserved phosphorylation sites. Overexpression of kinase-dead HA-Orb6K122A in wild-type cells resulted in a loss of cell polarity, suggesting that it has a dominant-negative effect, and it failed to rescue the polarity defect of nak1 or orb6 ts mutants. Recombinant GST-Orb6S291A did not autophosphorylate in vitro suggesting that Ser291 is the primary autophosphorylation site. HA-Orb6S291A overexpression only partially rescued the orb6 polarity defect and failed to rescue the nak1 defect, suggesting that autophosphorylation is important for Orb6 function. GST-Orb6T456A autophosphorylated in vitro, indicating that the conserved phosphorylation site at Thr456 is not essential for kinase activity. However, HA-Orb6T456A overexpression had similar effects as overexpressing kinase-dead HA-Orb6K122A, suggesting that Thr456 is essential for Orb6 function in vivo. Also, we found that both phosphorylation site mutations impaired the ability of Myc-Nak1 to coimmunoprecipitate with HA-Orb6. Together, our results suggest a model whereby autophosphorylation of Ser291 and phosphorylation of Thr456 by an upstream kinase promote Nak1/Orb6 complex formation and Orb6 activation

Probenecid Blocks Human P2X7 Receptor-Induced Dye Uptake via a Pannexin-1 Independent Mechanism

P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor

TWEAK Appears as a Modulator of Endometrial IL-18 Related Cytotoxic Activity of Uterine Natural Killers

BACKGROUND: TWEAK (Tumor necrosis factor like WEAK inducer of apoptosis) is highly expressed by different immune cells and triggers multiple cellular responses, including control of angiogenesis. Our objective was to investigate its role in the human endometrium during the implantation window, using an ex-vivo endometrial microhistoculture model. Indeed, previous results suggested that basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: Endometrial biopsies were performed 7 to 9 days after the ovulation surge of women in monitored natural cycles. Biopsies were cut in micro-pieces and cultured on collagen sponge with appropriate medium. Morphology, functionality and cell death were analysed at different time of the culture. We used this ex vivo model to study mRNA expressions of NKp46 (a uNK cytotoxic receptor) and TGF-beta1 (protein which regulates uNK cytokine production) after adjunction of excess of recombinant IL-18 and either recombinant TWEAK or its antibody. NKp46 protein expression was also detailed by immunohistochemistry in selected patients with high basic mRNA level of IL-18 and either low or high mRNA level of TWEAK. The NKp46 immunostaining was stronger in patients with an IL-18 over-expression and a low TWEAK expression, when compared with patients with both IL-18 and TWEAK high expressions. We did not observe any difference for TWEAK expression when recombinant protein IL-18 or its antibody was added, or conversely, for IL-18 expression when TWEAK or its antibody was added in the culture medium. In a pro-inflammatory environment (obtained by an excess of IL-18), inhibition of TWEAK was able to increase significantly NKp46 and TGF-beta1 mRNA expressions. CONCLUSIONS/SIGNIFICANCE: TWEAK doesn't act on IL-18 expression but seems to control IL-18 related cytotoxicity on uNK cells when IL-18 is over-expressed. Thus, TWEAK appears as a crucial physiological modulator to prevent endometrial uNK cytotoxicity in human

Fatal Cases of Influenza A(H3N2) in Children: Insights from Whole Genome Sequence Analysis

During the Northern Hemisphere winter of 2003–2004 the emergence of a novel influenza antigenic variant, A/Fujian/411/2002-like(H3N2), was associated with an unusually high number of fatalities in children. Seventeen fatal cases in the UK were laboratory confirmed for Fujian/411-like viruses. To look for phylogenetic patterns and genetic markers that might be associated with increased virulence, sequencing and phylogenetic analysis of the whole genomes of 63 viruses isolated from fatal cases and non fatal “control” cases was undertaken. The analysis revealed the circulation of two main genetic groups, I and II, both of which contained viruses from fatal cases. No associated amino acid substitutions could be linked with an exclusive or higher occurrence in fatal cases. The Fujian/411-like viruses in genetic groups I and II completely displaced other A(H3N2) viruses, but they disappeared after 2004. This study shows that two A(H3N2) virus genotypes circulated exclusively during the winter of 2003–2004 in the UK and caused an unusually high number of deaths in children. Host factors related to immune state and differences in genetic background between patients may also play important roles in determining the outcome of an influenza infection

Association between erythrocyte Na+K+-ATPase activity and some blood lipids in type 1 diabetic patients from Lagos, Nigeria

<p>Abstract</p> <p>Background</p> <p>Altered levels of erythrocyte Na⁺K⁺-ATPase, atherogenic and anti-atherogenic lipid metabolites have been implicated in diabetic complications but their pattern of interactions remains poorly understood.</p> <p>This study evaluated this relationship in Nigerian patients with Type 1 diabetes mellitus.</p> <p>Methods</p> <p>A total of 34 consented Type 1 diabetic patients and age -matched 27 non-diabetic controls were enrolled. Fasting plasma levels of total cholesterol, triglycerides and HDL-cholesterol were determined spectrophotometrically and LDL-cholesterol estimated using Friedewald formula. Total protein content and Na+K+-ATPase activity were also determined spectrophotometrically from ghost erythrocyte membrane prepared by osmotic lysis.</p> <p>Results</p> <p>Results indicate significant (P < 0.05) reduction in Na⁺K⁺-ATPase activity in the Type 1 diabetic patients (0.38 ± 0.08 vs. 0.59 ± 0.07 uM Pi/mgprotein/h) compared to the control but with greater reduction in the diabetic subgroup with poor glycemic control (n = 20) and in whom cases of hypercholesterolemia (8.8%), hypertriglyceridemia (2.9%) and elevated LDL-cholesterol (5.9% each) were found. Correlation analyses further revealed significant (P < 0.05) inverse correlations [r = -(0.708-0.797] between all the atherogenic lipid metabolites measured and Na⁺K⁺-ATPase in this subgroup contrary to group with good glycemic control or non-diabetic subjects in which significant (P < 0.05) Na⁺K⁺-ATPase and HDL-C association were found (r = 0.427 - 0.489). The Na⁺K⁺-ATPase from the diabetic patients also exhibited increased sensitivity to digoxin and alterations in kinetic constants Vmax and Km determined by glycemic status of the patients.</p> <p>Conclusion</p> <p>It can be concluded that poor glycemic control evokes greater reduction in erythrocyte Na⁺K⁺-ATPase activity and promote enzyme-blood atherogenic lipid relationships in Type 1 diabetic Nigerian patients.</p

Expression of TNF-related apoptosis-inducing ligand (TRAIL) in keratinocytes mediates apoptotic cell death in allogenic T cells

The objective of the present study was to evaluate the aptitude of TRAIL gene expression for inducing apoptosis in co-cultivated T-cells. This should allow preparing a strategy for the development of a durable, allogenic skin substitute based on the induction of an immune-privileged transplant. In order to counteract the significant potential of rejection in transplanted allogenic keratinocytes, we created a murine keratinocyte cell line which expressed TRAIL through stable gene transfer. The exogenic protein was localized on the cellular surface and was not found in soluble condition as sTRAIL. Contact to TRAIL expressing cells in co-culture induced cell death in sensitive Jurkat-cells, which was further intensified by lymphocyte activation. This cytotoxic effect is due to the induction of apoptosis. We therefore assume that the de-novo expression of TRAIL in keratinocytes can trigger apoptosis in activated lymphocytes and thus prevent the rejection of keratinocytes in allogenic, immune-privileged transplants

Prion Shedding from Olfactory Neurons into Nasal Secretions

This study investigated the role of prion infection of the olfactory mucosa in the shedding of prion infectivity into nasal secretions. Prion infection with the HY strain of the transmissible mink encephalopathy (TME) agent resulted in a prominent infection of the olfactory bulb and the olfactory sensory epithelium including the olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs), whose axons comprise the two olfactory cranial nerves. A distinct glycoform of the disease-specific isoform of the prion protein, PrPSc, was found in the olfactory mucosa compared to the olfactory bulb, but the total amount of HY TME infectivity in the nasal turbinates was within 100-fold of the titer in the olfactory bulb. PrPSc co-localized with olfactory marker protein in the soma and dendrites of ORNs and VRNs and also with adenylyl cyclase III, which is present in the sensory cilia of ORNs that project into the lumen of the nasal airway. Nasal lavages from HY TME-infected hamsters contained prion titers as high as 103.9 median lethal doses per ml, which would be up to 500-fold more infectious in undiluted nasal fluids. These findings were confirmed using the rapid PrPSc amplification QuIC assay, indicating that nasal swabs have the potential to be used for prion diagnostics. These studies demonstrate that prion infection in the olfactory epithelium is likely due to retrograde spread from the olfactory bulb along the olfactory and vomeronasal axons to the soma, dendrites, and cilia of these peripheral neurons. Since prions can replicate to high levels in neurons, we propose that ORNs can release prion infectivity into nasal fluids. The continual turnover and replacement of mature ORNs throughout the adult lifespan may also contribute to prion shedding from the nasal passage and could play a role in transmission of natural prion diseases in domestic and free-ranging ruminants

Production of Virus-Derived Ping-Pong-Dependent piRNA-like Small RNAs in the Mosquito Soma

The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma. We also present evidence that suggests virus-derived piRNA-like small RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication

Phylogeography of the Microcoleus vaginatus (Cyanobacteria) from Three Continents – A Spatial and Temporal Characterization

It has long been assumed that cyanobacteria have, as with other free-living microorganisms, a ubiquitous occurrence. Neither the geographical dispersal barriers nor allopatric speciation has been taken into account. We endeavoured to examine the spatial and temporal patterns of global distribution within populations of the cyanobacterium Microcoleus vaginatus, originated from three continents, and to evaluate the role of dispersal barriers in the evolution of free-living cyanobacteria. Complex phylogeographical approach was applied to assess the dispersal and evolutionary patterns in the cyanobacterium Microcoleus vaginatus (Oscillatoriales). We compared the 16S rRNA and 16S-23S ITS sequences of strains which had originated from three continents (North America, Europe, and Asia). The spatial distribution was investigated using a phylogenetic tree, network, as well as principal coordinate analysis (PCoA). A temporal characterization was inferred using molecular clocks, calibrated from fossil DNA. Data analysis revealed broad genetic diversity within M. vaginatus. Based on the phylogenetic tree, network, and PCoA analysis, the strains isolated in Europe were spatially separated from those which originated from Asia and North America. A chronogram showed a temporal limitation of dispersal barriers on the continental scale. Dispersal barriers and allopatric speciation had an important role in the evolution of M. vaginatus. However, these dispersal barriers did not have a permanent character; therefore, the genetic flow among populations on a continental scale was only temporarily present. Furthermore, M. vaginatus is a recently evolved species, which has been going through substantial evolutionary changes