Effects of wood removal on stream habitat and nitrate uptake in two northeastern US headwater streams

Forested headwater streams play an important role in watershed nutrient dynamics, and wood is thought to be a key factor influencing habitat structure and nitrate-nitrogen dynamics in many forested streams. Because wood in streams can promote nitrogen uptake through denitrification, we hypothesized that nitrate uptake velocities would decrease following wood removal. We measured stream characteristics and nitrate uptake velocities before and after wood manipulation experiments conducted at Hubbard Brook Experimental Forest, NH, and the Sleepers River watershed, VT. The mean size of stream substrates and the amount of riffle habitat increased following wood removal. In contrast to our expectations, summer nitrate uptake velocities increased in the wood removal treatments relative to the reference treatments, possibly because wood removal increased the availability of stable substrates for periphyton growth, therefore increasing nitrate demand in these streams. Our results highlight that effects of wood on stream ecosystems occur through multiple pathways and suggest that the relative importance of these pathways may vary seasonally.     

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.     

Conformational restriction in a series of GPR119 agonists: Differences in pharmacology between mouse and human

A series of conformationally restricted GPR119 agonists were prepared based around a 3,8-diazabicyclo[3.2.1]octane scaffold. Examples were found to have markedly different pharmacology in mouse and human despite similar levels of binding to the receptor. This highlights the large effects on GPCR phamacology that can result from small structural changes in the ligand, together with inter-species differences between receptors.     

Impacts of inundation and drought on eukaryote biodiversity in semi‐arid floodplain soils

Floodplain ecosystems are characterized by alternating wet and dry phases and periodic inundation defines their ecological character. Climate change, river regulation and the construction of levees have substantially altered natural flooding and drying regimes worldwide with uncertain effects on key biotic groups. In southern Australia, we hypothesized that soil eukaryotic communities in climate change affected areas of a semi‐arid floodplain would transition towards comprising mainly dry‐soil specialist species with increasing drought severity. Here, we used 18S rRNA amplicon pyrosequencing to measure the eukaryote community composition in soils that had been depleted of water to varying degrees to confirm that reproducible transitional changes occur in eukaryotic biodiversity on this floodplain. Interflood community structures (3 years post‐flood) were dominated by persistent rather than either aquatic or dry‐specialist organisms. Only 2% of taxa were unique to dry locations by 8 years post‐flood, and 10% were restricted to wet locations (inundated a year to 2 weeks post‐flood). Almost half (48%) of the total soil biota were detected in both these environments. The discovery of a large suite of organisms able to survive nearly a decade of drought, and up to a year submerged supports the concept of inherent resilience of Australian semi‐arid floodplain soil communities under increasing pressure from climatic induced changes in water availability.     

Molecular identification of the prey range of the invasive Asian paper wasp

The prey range of the invasive Asian paper wasp, Polistes chinensis antennalis, was studied using molecular diagnostics. Nests of paper wasps were collected from urban residential and salt marsh habitats, larvae were removed and dissected, and DNA in the gut of the paper wasp larvae was amplified and sequenced with cytochrome c oxidase subunit I (COI). Seventy percent of samples (211/299) yielded medium‐to high‐quality sequences, and prey identification was achieved using BLAST searches in BOLD. A total of 42 taxa were identified from 211 samples. Lepidoptera were the majority of prey, with 39 taxa from 91% of samples. Diptera was a relatively small component of prey (three taxa, 19 samples). Conclusive species‐level identification of prey was possible for 67% of samples, and genus‐level identification, for another 12% of samples. The composition of prey taken was different between the two habitats, with 2.5× more native prey species being taken in salt marsh compared with urban habitats. The results greatly extend the prey range of this invasive species. The technique is a more effective and efficient approach than relying on the collection of “prey balls”, or morphological identification of prey, for the study of paper wasps.     

Pseudomonas fluorescens NZI7 repels grazing by C. elegans,a natural predator

The bacteriovorous nematode Caenorhabditis elegans has been used to investigate many aspects of animal biology, including interactions with pathogenic bacteria. However, studies examining C. elegans interactions with bacteria isolated from environments in which it is found naturally are relatively scarce. C. elegans is frequently associated with cultivation of the edible mushroom Agaricus bisporus, and has been reported to increase the severity of bacterial blotch of mushrooms, a disease caused by bacteria from the Pseudomonas fluorescens complex. We observed that pseudomonads isolated from mushroom farms showed differential resistance to nematode predation. Under nutrient poor conditions, in which most pseudomonads were consumed, the mushroom pathogenic isolate P. fluorescens NZI7 was able to repel C. elegans without causing nematode death. A draft genome sequence of NZI7 showed it to be related to the biocontrol strain P. protegens Pf-5. To identify the genetic basis of nematode repellence in NZI7, we developed a grid-based screen for mutants that lacked the ability to repel C. elegans. The mutants isolated in this screen included strains with insertions in the global regulator GacS and in a previously undescribed GacS-regulated gene cluster, ‘EDB'' (‘edible''). Our results suggest that the product of the EDB cluster is a poorly diffusible or cell-associated factor that acts together with other features of NZI7 to provide a novel mechanism to deter nematode grazing. As nematodes interact with NZI7 colonies before being repelled, the EDB factor may enable NZI7 to come into contact with and be disseminated by C. elegans without being subject to intensive predation.     

Akonni TruTip? and Qiagen? Methods for Extraction of Fetal Circulating DNA - Evaluation by Real-Time and Digital PCR

Due to the low percentage of fetal DNA present in maternal plasma (< 10%) during early gestation, efficient extraction processes are required for successful downstream detection applications in non-invasive prenatal diagnostic testing. In this study, two extraction methods using similar chemistries but different workflows were compared for isolation efficiency and percent fetal DNA recovery. The Akonni Biosystems TruTip technology uses a binding matrix embedded in a pipette tip; the Circulating Nucleic Acids Kit from Qiagen employs a spin column approach. The TruTip method adds an extra step to decrease the recovery of DNA fragments larger than 600 bp from the sample to yield an overall higher percentage of smaller molecular weight DNA, effectively enriching for fetal DNA. In this evaluation, three separate extraction comparison studies were performed - a dilution series of fragmented DNA in plasma, a set of clinical maternal samples, and a blood collection tube time point study of maternal samples. Both extraction methods were found to efficiently extract small fragment DNA from large volumes of plasma. In the amended samples, the TruTip extraction method was ~15% less efficient with overall DNA recovery, but yielded an 87% increase in % fetal DNA relative to the Qiagen method. The average percent increase of fetal DNA of TruTip extracted samples compared to the Qiagen method was 55% for all sets of blinded clinical samples. A study comparing extraction efficiencies from whole blood samples incubated up to 48 hours prior to processing into plasma resulted in more consistent % fetal DNA recoveries using TruTip. The extracted products were tested on two detection platforms, quantitative real-time PCR and droplet digital PCR, and yielded similar results for both extraction methods.     

Enhanced Activation of Memory,but Not Na?ve,B Cells in Chronic Hepatitis C Virus-Infected Patients with Cryoglobulinemia and Advanced Liver Fibrosis

Mixed cryoglobulinemia is the most common extrahepatic disease manifestation of chronic hepatitis C virus (HCV) infection, where immunoglobulins precipitate at low temperatures and cause symptoms such as vasculitis, glomerulonephritis and arthralgia. HCV-associated cryoglobulinemia is also strongly linked with the development of B cell non-Hodgkin lymphoma. Abnormal B cell function in HCV infections can lead to the formation of HCV cryoglobulin complexes that usually comprise monoclonal rheumatoid factor and HCV-specific immune complexes. The aim of this study was to characterize the activation phenotype of B cells from patients with chronic HCV infection in comparison to healthy controls using flow cytometry. In addition, we determined how the activation status varies depending on the presence of cryoglobulinemia and advanced liver fibrosis. We found that only memory B cells, not naïve cells, were significantly activated in chronic HCV infection when compared with healthy controls. We also identified markers of memory B cell activation that were specific for HCV patients with cryoglobulinemia (CD86, CD71, HLA-DR) and advanced liver disease (CD86). Our results demonstrate that HCV infection has differential effects on B cells depending on the severity of hepatic and extrahepatic disease.     

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.     

Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial

Background

Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key to the interpretation of epidemiologic studies of these parasites, as well as for monitoring large scale treatment efficacy and effectiveness studies. As morbidity and transmission of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity of intestinal parasite infections, and compare them to standard microscopy.

Methodology/Principal Findings

Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions the first targeting: Necator americanus, Ancylostoma spp., Ascaris spp., and Trichuris trichiura; and the second Entamoeba histolytica, Cryptosporidium spp., Giardia. duodenalis, and Strongyloides stercoralis. Samples were also subject to sodium nitrate flotation for identification and quantification of STH eggs, and zinc sulphate centrifugal flotation for detection of protozoan parasites. Higher parasite prevalence was detected by multiplex PCR (hookworms 2.9 times higher, Ascaris 1.2, Giardia 1.6, along with superior polyparasitism detection with this effect magnified as the number of parasites present increased (one: 40.2% vs. 38.1%, two: 30.9% vs. 12.9%, three: 7.6% vs. 0.4%, four: 0.4% vs. 0%). Although, all STH positive samples were low intensity infections by microscopy as defined by WHO guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections.

Conclusions/Significance

Multiplex PCR, in addition to superior sensitivity, enabled more accurate determination of infection intensity for Ascaris, hookworms and Giardia compared to microscopy, especially in samples exhibiting polyparasitism. The superior performance of multiplex PCR to detect polyparasitism and more accurately determine infection intensity suggests that it is a more appropriate technique for use in epidemiologic studies and for monitoring large-scale intervention trials.