metaBIT,an integrative and automated metagenomic pipeline for analysing microbial profiles from high‐throughput sequencing shotgun data

Micro‐organisms account for most of the Earth's biodiversity and yet remain largely unknown. The complexity and diversity of microbial communities present in clinical and environmental samples can now be robustly investigated in record times and prices thanks to recent advances in high‐throughput DNA sequencing (HTS). Here, we develop metaBIT, an open‐source computational pipeline automatizing routine microbial profiling of shotgun HTS data. Customizable by the user at different stringency levels, it performs robust taxonomy‐based assignment and relative abundance calculation of microbial taxa, as well as cross‐sample statistical analyses of microbial diversity distributions. We demonstrate the versatility of metaBIT within a range of published HTS data sets sampled from the environment (soil and seawater) and the human body (skin and gut), but also from archaeological specimens. We present the diversity of outputs provided by the pipeline for the visualization of microbial profiles (barplots, heatmaps) and for their characterization and comparison (diversity indices, hierarchical clustering and principal coordinates analyses). We show that metaBIT allows an automatic, fast and user‐friendly profiling of the microbial DNA present in HTS shotgun data sets. The applications of metaBIT are vast, from monitoring of laboratory errors and contaminations, to the reconstruction of past and present microbiota, and the detection of candidate species, including pathogens.     

Vitamin D,Fibroblast Growth Factor 23 and Incident Cognitive Impairment: Findings from the REGARDS Study

Vitamin D protects against cognitive decline in animals but evidence in humans has been inconsistent. Fibroblast growth factor 23 (FGF23) is a hormone that inhibits vitamin D activation yet few studies examined whether FGF23 is associated with cognitive impairment. The objective of this study was to examine associations of 25(OH)D and FGF23 with incident cognitive impairment in the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study, a cohort of black and white adults ≥45 years old. FGF23 and 25(OH)D were measured in 474 incident impairment cases and 561 controls. In multivariable-adjusted models, there were no significant associations of FGF23 with incident cognitive impairment. In analyses using clinically-relevant categories of 25(OH)D (< 20 ng/ml, 20–29.9 ng/ml, ≥30 ng/ml), there was no statistically significant association of lower 25(OH)D concentrations with odds of incident cognitive impairment in models adjusted for demographic, clinical, and laboratory variables and season of blood draw (tertile 1 [≥30 ng/ml] reference; tertile 2 [20–29.9 ng/ml], odds ratio [OR] 0.96, 95%CI 0.67, 1.38; tertile 3 [<20 ng/ml] OR 1.26, 95%CI 0.83, 1.91). When 25(OH)D was modeled as race-specific tertiles, there were no significant associations of 25(OH)D with incident cognitive impairment in whites, whereas lower 25(OH)D was associated with higher odds in blacks (tertile 1 [>23 ng/ml] reference; tertile 2 [15–23 ng/ml], OR 2.96, 95%CI 1.48,5.94; tertile 3 [<15 ng/ml] OR 2.40, 95%CI 1.07,5.40) in the fully adjusted model. In this cohort of older adults, lower race-specific tertiles of 25(OH)D were associated with higher incidence of cognitive impairment in black individuals but not white individuals. These data suggest that treating low 25(OH)D may be a novel strategy for addressing racial disparities in neurocognitive outcomes.     

The Honey Bee Pathosphere of Mongolia: European Viruses in Central Asia

Parasites and pathogens are apparent key factors for the detrimental health of managed European honey bee subspecies, Apis mellifera. Apicultural trade is arguably the main factor for the almost global distribution of most honey bee diseases, thereby increasing chances for multiple infestations/infections of regions, apiaries, colonies and even individual bees. This imposes difficulties to evaluate the effects of pathogens in isolation, thereby creating demand to survey remote areas. Here, we conducted the first comprehensive survey for 14 honey bee pathogens in Mongolia (N = 3 regions, N = 9 locations, N = 151 colonies), where honey bee colonies depend on humans to overwinter. In Mongolia, honey bees, Apis spp., are not native and colonies of European A. mellifera subspecies have been introduced ~60 years ago. Despite the high detection power and large sample size across Mongolian regions with beekeeping, the mite Acarapis woodi, the bacteria Melissococcus plutonius and Paenibacillus larvae, the microsporidian Nosema apis, Acute bee paralysis virus, Kashmir bee virus, Israeli acute paralysis virus and Lake Sinai virus strain 2 were not detected, suggesting that they are either very rare or absent. The mite Varroa destructor, Nosema ceranae and four viruses (Sacbrood virus, Black queen cell virus, Deformed wing virus (DWV) and Chronic bee paralysis virus) were found with different prevalence. Despite the positive correlation between the prevalence of V. destructor mites and DWV, some areas had only mites, but not DWV, which is most likely due to the exceptional isolation of apiaries (up to 600 km). Phylogenetic analyses of the detected viruses reveal their clustering and European origin, thereby supporting the role of trade for pathogen spread and the isolation of Mongolia from South-Asian countries. In conclusion, this survey reveals the distinctive honey bee pathosphere of Mongolia, which offers opportunities for exciting future research.     

Effects of the Infusion of 4% or 20% Human Serum Albumin on the Skeletal Muscle Microcirculation in Endotoxemic Rats

Background

Sepsis-induced microcirculatory alterations contribute to tissue hypoxia and organ dysfunction. In addition to its plasma volume expanding activity, human serum albumin (HSA) has anti-oxidant and anti-inflammatory properties and may have a protective role in the microcirculation during sepsis. The concentration of HSA infused may influence these effects. We compared the microcirculatory effects of the infusion of 4% and 20% HSA in an experimental model of sepsis.

Methods

Adult male Wistar rats were equipped with arterial and venous catheters and received an intravenous infusion of lipopolysaccharide (LPS, serotype O127:B8, 10 mg/kg over 30 minutes) or vehicle (SHAM, n = 6). Two hours later, endotoxemic animals were randomized to receive 10 mL/kg of either 4% HSA (LPS+4%HSA, n = 6), 20% HSA (LPS+20%HSA, n = 6) or 0.9% NaCl (LPS+0.9%NaCl, n = 6). No fluids were given to an additional 6 animals (LPS). Vessel density and perfusion were assessed in the skeletal muscle microcirculation with sidestream dark field videomicroscopy at baseline (t0), 2 hours after LPS injection (t1), after HSA infusion (t2) and 1 hour later (t3). The mean arterial pressure (MAP) and heart rate were recorded. Serum endothelin-1 was measured at t2.

Results

MAP was stable over time in all groups. The microcirculatory parameters were significantly altered in endotoxemic animals at t1. The infusion of both 4% and 20% HSA similarly increased the perfused vessel density and blood flow velocity and decreased the flow heterogeneity to control values. Microvascular perfusion was preserved in the LPS+20%HSA group at t3, whereas alterations reappeared in the LPS+4%HSA group.

Conclusions

In a rat model of normotensive endotoxemia, the infusion of 4% or 20% HSA produced a similar acute improvement in the microvascular perfusion in otherwise unresuscitated animals.     

Dynamics of polymorphism of acidocalcisomes in<Emphasis Type="Italic"> Leishmania</Emphasis> parasites

Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes. When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 m diameter distributed throughout the cell. X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc. Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes. When cells were transferred to Warrens medium, the acidocalcisomes transformed from spherical into branched tubular organelles. The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes. X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron. Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture conditions.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Induction of baroresistance by hydrogen peroxide, ethanol and cold-shock in Saccharomyces cerevisiae

The acquisition of tolerance to high hydrostatic pressure of 220 MPa (HHP) in response to a 0.4 mM hydrogen peroxide, 6% ethanol and cold-shock (10 degrees C) pretreatment for different lengths of times was studied in the yeast Saccharomyces cerevisiae. The protection conferred by these different treatments was similar ( approximately 3 log cycles) and time-dependent. Analysis of the induction of the most pressure up-regulated genes under these conditions was investigated by RT-PCR. Our results revealed that the cell stress response to HHP shares common features with hydrogen peroxide and ethanol stresses, but differs in some way to cold-shock.     

3-O-methylfunicone, a secondary metabolite produced by Penicillium pinophilum, induces growth arrest and apoptosis in HeLa cells

3-O-Methylfunicone (OMF) is a secondary metabolite produced by the soil fungus Penicillium pinophilum which has cytostatic properties. The aim of this study was to investigate the mechanisms by which such properties are exerted, with special reference to any anti-proliferative and apoptotic potential, on HeLa cells. OMF treatment caused about 44% inhibition of cell growth after 24 h, and modifications in the tubulin fibre organization. In addition, a significant increase in p21 mRNA expression and a decrease in cyclin D1 and Cdk4 mRNA expression resulted at the same time. Apoptosis induction was demonstrated by the annexin V assay, cytofluorimetric analysis of the DNA content of the sub-G1 fraction and DNA laddering. Taken together, our data showed that the compound inhibits proliferation of HeLa cells by several mechanisms, such as disruption of tubulin fibres, cell cycle arrest and apoptosis induction. The capacity of the compound to affect the cell cycle and to modulate apoptosis is indicative of a potential for the development of a new agent for cancer chemotherapy.     

Identification of 8-iso-prostaglandin F(2alpha) in rat brain neuronal endings: a possible marker of membrane phospholipid peroxidation

Isoprostanes are a family of prostaglandin (PG) F and E isomers generated by free-radical attack from membrane bound arachidonic acid. We measured detectable levels of 8-iso-PGF(2alpha) in the perfusates of synaptosomes obtained from different areas of the rat brain cortex. A small but significant release of this isoprostane was found under basal conditions from all the areas explored; being lower in the dorsal cortex in respect to the frontal, parietal and occipital areas. Exposure of synaptosomes to a phospholipase A(2) activator, i.e. calcium-ionophore A23187, an oxidant agent, such as hydrogen peroxide or amyloid beta-peptide did not modify 8-iso-PGF(2alpha) release when these stimuli were applied separately. However, either hydrogen peroxide or amyloid beta-peptide increased 8-iso-PGF(2alpha) release in a dose-dependent manner, when given in the presence of the calcium-ionophore A23187. Synaptosome treatment with a non-selective cyclooxygenase inhibitor (fenoprofen) did not modify 8-iso-PGF(2alpha) release in any way, but treatment with a water soluble antioxidant (Trolox C) completely suppressed isoprostane release under basal conditions, as well as after the oxidant injury induced either by hydrogen peroxide or amyloid beta-peptide. We conclude that, in neuronal endings, 8-iso-PGF(2alpha) is generated under basal conditions and its formation may be increased in a dose-dependent fashion by oxidant stimuli through a cyclooxygenase-independent mechanism involving free radical-catalyzed oxidation of arachidonic acid on membrane phospholipids.