Atomistic details of effect of disulfide bond reduction on active site of Phytase B from Aspergillus niger: A MD Study

The molecular integrity of the active site of phytases from fungi is critical for maintaining phytase function as efficient catalytic machines. In this study, the molecular dynamics (MD) of two monomers of phytase B from Aspergillus niger, the disulfide intact monomer (NAP) and a monomer with broken disulfide bonds (RAP), were simulated to explore the conformational basis of the loss of catalytic activity when disulfide bonds are broken. The simulations indicated that the overall secondary and tertiary structures of the two monomers were nearly identical but differed in some crucial secondary–structural elements in the vicinity of the disulfide bonds and catalytic site. Disulfide bonds stabilize the β-sheet that contains residue Arg66 of the active site and destabilize the α-helix that contains the catalytic residue Asp319. This stabilization and destabilization lead to changes in the shape of the active–site pocket. Functionally important hydrogen bonds and atomic fluctuations in the catalytic pocket change during the RAP simulation. None of the disulfide bonds are in or near the catalytic pocket but are most likely essential for maintaining the native conformation of the catalytic site.

Abbreviations

PhyB - 2.5 pH acid phophatese from Aspergillus niger, NAP - disulphide intact monomer of Phytase B, RAP - disulphide reduced monomer of Phytase B, Rg - radius of gyration, RMSD - root mean square deviation, MD - molecular dynamics.     

Context‐specific effects of the identity of detrital mixtures on invertebrate communities

Many aquatic ecosystems are sustained by detrital subsidies of leaf litter derived from exogenous sources. Although numerous studies have examined the effects of litter species richness and identity on decomposition processes, it remains unclear how these effects extend to associated invertebrate communities or how these effects vary spatially according to local environmental context. Using field enrichment experiments, we assessed how the species richness, assemblage composition, and supply of detrital litter resources interact to affect benthic communities of three temperate Australian estuarine mudflats. Our experiments utilized eight litter sources that are presently experiencing human‐mediated changes in their supply to estuarine mudflats. Contrary to predictions, we did not detect effects of the species richness of detrital mixtures on benthic communities. Macroinvertebrate community structure and, in particular, abundance were, instead, influenced by the assemblage composition of detrital mixtures. At two of the three sites, plots receiving the most labile detrital mix, containing the ephemeral algae Chaetomorpha and Ulva, supported the fewest macroinvertebrates of all the experimental enrichments. The large effect of detrital mix identity on macroinvertebrate communities is of concern given present trends of proliferation of macroalgae at the expense of more refractory seagrasses and marsh grasses. As such environmental degradation continues, it will be important to more fully understand under what environmental contexts such compositional changes in detrital resources will have the most detrimental effects on important prey resources for commercially important fish and wading shorebirds.     

Diversity patterns of leaf-associated aquatic hyphomycetes along a broad latitudinal gradient

Information about the global distribution of aquatic hyphomycetes is scarce, despite the primary importance of these fungi in stream ecosystem functioning. In particular, the relationship between their diversity and latitude remains unclear, due to a lack of coordinated surveys across broad latitudinal ranges. This study is a first report on latitudinal patterns of aquatic hyphomycete diversity associated with native leaf-litter species in five streams located along a gradient extending from the subarctic to the tropics. Exposure of leaf litter in mesh bags of three different mesh sizes facilitated assessing the effects of including or excluding different size-classes of litter-consuming invertebrates. Aquatic hyphomycete evenness was notably constant across all sites, whereas species richness and diversity, expressed as the Hill number, reached a maximum at mid-latitudes (Mediterranean and temperate streams). These latitudinal patterns were consistent across litter species, despite a notable influence of litter identity on fungal communities at the local scale. As a result, the bell-shaped distribution of species richness and Hill diversity deviated markedly from the latitudinal patterns of most other groups of organisms. Differences in the body-size distribution of invertebrate communities colonizing the leaves had no effect on aquatic hyphomycete species richness, Hill diversity or evenness, but invertebrates could still influence fungal communities by depleting litter, an effect that was not captured by the design of our experiment.     

Reproduction of Mucohaemorrhagic Diarrhea and Colitis Indistinguishable from Swine Dysentery following Experimental Inoculation with “Brachyspira hampsonii” Strain 30446

Background

Mucohaemorrhagic diarrhea caused by Brachyspira hyodysenteriae, swine dysentery, is a severe production limiting disease of swine. Recently, pigs in western Canada with clinical signs indistinguishable from swine dysentery were observed. Despite the presence of spirochetes on fecal smears, recognized Brachyspira spp. including B. hyodysenteriae could not be identified. A phylogenetically distinct Brachyspira, called “B. hampsonii” strain 30446, however was isolated. The purpose of this study was to experimentally reproduce mucohaemorrhagic colitis and characterize strain 30446 shedding following inoculation.

Methods and Findings

Eighteen 13-week-old pigs were randomly assigned to inoculation (n = 12) or control (n = 6) groups in each of two trials. In trial 1, pigs were inoculated with a tissue homogenate collected from clinically affected field cases. In trial 2, pigs were inoculated with a pure broth culture of strain 30446. In both trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls, all of which remained healthy. In animals with mucohaemorrhagic diarrhea, significantly more spirochetes were observed on Gram stained fecal smears, and higher numbers of strain 30446 genome equivalents were detected by quantitative PCR (qPCR). Strain 30446 was cultured from colon and/or feces of all affected but no control animals at necropsy.

Conclusions

“Brachyspira hampsonii” strain 30446 causes mucohaemorrhagic diarrhea in pigs following a 4–9 day incubation period. Fecal shedding was detectable by day 4 post inoculation, and rarely preceded the onset of mucoid or haemorrhagic diarrhea by more than 2 days. Culture and 30446-specific qPCR are reliable methods of detection of this organism in feces and tissues of diarrheic pigs. The emergence of a novel Brachyspira spp., such as “B. hampsonii”, creates diagnostic challenges including higher risk of false negative diagnostic tests. We therefore recommend diagnostic laboratories routinely use Brachyspira culture, nox-based and species-specific PCR, and DNA sequencing to diagnose Brachyspira-associated colitis in pigs.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

Unravelling core microbial metabolisms in the hypersaline microbial mats of Shark Bay using high-throughput metagenomics

Modern microbial mats are potential analogues of some of Earth''s earliest ecosystems. Excellent examples can be found in Shark Bay, Australia, with mats of various morphologies. To further our understanding of the functional genetic potential of these complex microbial ecosystems, we conducted for the first time shotgun metagenomic analyses. We assembled metagenomic next-generation sequencing data to classify the taxonomic and metabolic potential across diverse morphologies of marine mats in Shark Bay. The microbial community across taxonomic classifications using protein-coding and small subunit rRNA genes directly extracted from the metagenomes suggests that three phyla Proteobacteria, Cyanobacteria and Bacteriodetes dominate all marine mats. However, the microbial community structure between Shark Bay and Highbourne Cay (Bahamas) marine systems appears to be distinct from each other. The metabolic potential (based on SEED subsystem classifications) of the Shark Bay and Highbourne Cay microbial communities were also distinct. Shark Bay metagenomes have a metabolic pathway profile consisting of both heterotrophic and photosynthetic pathways, whereas Highbourne Cay appears to be dominated almost exclusively by photosynthetic pathways. Alternative non-rubisco-based carbon metabolism including reductive TCA cycle and 3-hydroxypropionate/4-hydroxybutyrate pathways is highly represented in Shark Bay metagenomes while not represented in Highbourne Cay microbial mats or any other mat forming ecosystems investigated to date. Potentially novel aspects of nitrogen cycling were also observed, as well as putative heavy metal cycling (arsenic, mercury, copper and cadmium). Finally, archaea are highly represented in Shark Bay and may have critical roles in overall ecosystem function in these modern microbial mats.     

Opt-Out Panel Testing for HIV,Hepatitis B and Hepatitis C in an Urban Emergency Department: A Pilot Study

Objectives

Studies suggest 2 per 1000 people in Dublin are living with HIV, the level above which universal screening is advised. We aimed to assess the feasibility and acceptability of a universal opt-out HIV, Hepatitis B and Hepatitis C testing programme for Emergency Department patients and to describe the incidence and prevalence of blood-borne viruses in this population.

Methods

An opt-out ED blood borne virus screening programme was piloted from March 2014 to January 2015. Patients undergoing blood sampling during routine clinical care were offered HIV 1&2 antibody/antigen assay, HBV surface antigen and HCV antibody tests. Linkage to care where necessary was co-ordinated by the study team. New diagnosis and prevalence rates were defined as the new cases per 1000 tested and number of positive tests per 1000 tested respectively.

Results

Over 45 weeks of testing, of 10,000 patient visits, 8,839 individual patient samples were available for analysis following removal of duplicates. A sustained target uptake of >50% was obtained after week 3. 97(1.09%), 44(0.49%) and 447(5.05%) HIV, Hepatitis B and Hepatitis C tests were positive respectively. Of these, 7(0.08%), 20(0.22%) and 58(0.66%) were new diagnoses of HIV, Hepatitis B and Hepatitis C respectively. The new diagnosis rate for HIV, Hepatitis B and Hepatitis C was 0.8, 2.26 and 6.5 per 1000 and study prevalence for HIV, Hepatitis B and Hepatitis C was 11.0, 5.0 and 50.5 per 1000 respectively.

Conclusions

Opt-out blood borne viral screening was feasible and acceptable in an inner-city ED. Blood borne viral infections were prevalent in this population and newly diagnosed cases were diagnosed and linked to care. These results suggest widespread blood borne viral testing in differing clinical locations with differing population demographic risks may be warranted.     

Abundance of RNase4 and RNase5 mRNA and protein in host defence related tissues and secretions in cattle

Members of the RNaseA family are present in various tissues and secretions but their function is not well understood. Some of the RNases are proposed to participate in host defence. RNase4 and RNase5 are present in cows' milk and have antimicrobial activity. However, their presence in many tissues and secretions has not been characterised. We hypothesised that these two RNases are present in a range of tissues and secretions where they could contribute to host defence. We therefore, determined the relative abundance of RNase4 and RNase5 mRNA as well as protein levels in a range of host defence related and other tissues as well as a range of secretions in cattle, using real time PCR and western blotting. The two RNases were found to be expressed in liver, lung, pancreas, mammary gland, placenta, endometrium, small intestine, seminal vesicle, salivary gland, kidney, spleen, lymph node, skin as well as testes. Corresponding proteins were also detected in many of the above tissues, as well as in seminal fluid, mammary secretions and saliva. This study provides evidence for the presence of RNase4 and RNase5 in a range of tissues and secretions, as well as some major organs in cattle. The data are consistent with the idea that these proteins could contribute to host defence in these locations. This work contributes to growing body of data suggesting that these proteins contribute to the physiology of the organism in a more complex way than acting merely as digestive enzymes.     

Dieback of riparian alder caused by the Phytophthora alni complex: projected consequences for stream ecosystems

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