Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community

Impact of historical gold mining activities on marine sediments in Wine Harbour, Nova Scotia, Canada

Past investigations at historical gold (Au) districts in Nova Scotia, Canada have identified elevated concentrations of arsenic (As) and mercury (Hg) in nearby sediments and waters. These metal(loid)s are derived from erosion of mineralized bedrock, and the disposal of mine tailings into the environment during early operations. The Wine Harbour gold district is located along the eastern shore of Nova Scotia, and produced 1329 kg of Au from 75 581 tonnes of crushed rock from 1862 to 1939.The gold occurs in arsenopyrite-bearing quartz-carbonate veins and was extracted using stamp milling and Hg amalgamation. Historical maps document tailings deposits near former stamp mill sites; however, the extent to which these mine wastes influence environmental quality in the adjacent marine environment is uncertain. In this study, we measured metal(loid) concentrations in tailings, marine sediments, and surface waters to assess the lateral and vertical extent of mining-related impacts on Wine Harbour. Chemical analyses of terrestrial and intertidal tailings reveal high concentrations of both As (86–196 000 mg/kg) and Hg (444–320 000 µg/kg). Analyses of marine sediments show a wide range in both As (4–568 mg/kg) and Hg (<5–7430 µg/kg) concentrations. In general, the highest metal(loid) concentrations in sediments were recorded down-gradient of stamp mill sites. Elevated concentrations were also detected in sediments underlying an active mussel aquaculture operation at the western end of the harbour. Results from this study have been used to help assess potential ecosystem and human health risks associated with historical gold mine wastes in the Wine Harbour area

Multisite Evaluation and Validation of a Sensitive Diagnostic and Screening System for Spinal Muscular Atrophy that Reports SMN1 and SMN2 Copy Number, along with Disease Modifier and Gene Duplication Variants

Spinal muscular atrophy is a severe autosomal recessive disease caused by disruptions in the SMN1 gene. The nearly identical SMN2 gene copy number is associated with disease severity. SMN1 duplication markers, such as c.*3+80T>G and c.*211_*212del, can assess residual carrier risk. An SMN2 disease modifier (c.859G>C) can help inform prognostic outcomes. The emergence of multiple precision gene therapies for spinal muscular atrophy requires accurate and rapid detection of SMN1 and SMN2 copy numbers to enable early treatment and optimal patient outcomes. We developed and evaluated a singletube PCR/capillary electrophoresis assay system that quantifies SMN1/2 copy numbers and genotypes three additional clinically relevant variants. Analytical validation was performed with human cell lines and whole blood representing varying SMN1/2 copies on four capillary electrophoresis instrument models. In addition, four independent laboratories used the assay to test 468 residual clinical genomic DNA samples. The results were >98.3% concordant with consensus SMN1/2 exon 7 copy numbers, determined using multiplex ligation-dependent probe amplification and droplet digital PCR, and were 100% concordant with Sanger sequencing for the three variants. Furthermore, copy number values were 98.6% (SMN1) and 97.1% (SMN2) concordant to each laboratory's own reference results. (J Mol Diag

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Identification and Characterization of a Mef2 Transcriptional Activator in Schistosome Parasites

Myocyte enhancer factor 2 protein (Mef2) is an evolutionarily conserved activator of transcription that is critical to induce and control complex processes in myogenesis and neurogenesis in vertebrates and insects, and osteogenesis in vertebrates. In Drosophila, Mef2 null mutants are unable to produce differentiated muscle cells, and in vertebrates, Mef2 mutants are embryonic lethal. Schistosome worms are responsible for over 200 million cases of schistosomiasis globally, but little is known about early development of schistosome parasites after infecting a vertebrate host. Understanding basic schistosome development could be crucial to delineating potential drug targets. Here, we identify and characterize Mef2 from the schistosome worm Schistosoma mansoni (SmMef2). We initially identified SmMef2 as a homolog to the yeast Mef2 homolog, Resistance to Lethality of MKK1P386 overexpression (Rlm1), and we show that SmMef2 is homologous to conserved Mef2 family proteins. Using a genetics approach, we demonstrate that SmMef2 is a transactivator that can induce transcription of four separate heterologous reporter genes by yeast one-hybrid analysis. We also show that Mef2 is expressed during several stages of schistosome development by quantitative PCR and that it can bind to conserved Mef2 DNA consensus binding sequences

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

A novel formulation of inhaled sodium cromoglicate (PA101) in idiopathic pulmonary fibrosis and chronic cough: a randomised, double-blind, proof-of-concept, phase 2 trial

Background Cough can be a debilitating symptom of idiopathic pulmonary fibrosis (IPF) and is difficult to treat. PA101 is a novel formulation of sodium cromoglicate delivered via a high-efficiency eFlow nebuliser that achieves significantly higher drug deposition in the lung compared with the existing formulations. We aimed to test the efficacy and safety of inhaled PA101 in patients with IPF and chronic cough and, to explore the antitussive mechanism of PA101, patients with chronic idiopathic cough (CIC) were also studied. Methods This pilot, proof-of-concept study consisted of a randomised, double-blind, placebo-controlled trial in patients with IPF and chronic cough and a parallel study of similar design in patients with CIC. Participants with IPF and chronic cough recruited from seven centres in the UK and the Netherlands were randomly assigned (1:1, using a computer-generated randomisation schedule) by site staff to receive PA101 (40 mg) or matching placebo three times a day via oral inhalation for 2 weeks, followed by a 2 week washout, and then crossed over to the other arm. Study participants, investigators, study staff, and the sponsor were masked to group assignment until all participants had completed the study. The primary efficacy endpoint was change from baseline in objective daytime cough frequency (from 24 h acoustic recording, Leicester Cough Monitor). The primary efficacy analysis included all participants who received at least one dose of study drug and had at least one post-baseline efficacy measurement. Safety analysis included all those who took at least one dose of study drug. In the second cohort, participants with CIC were randomly assigned in a study across four centres with similar design and endpoints. The study was registered with ClinicalTrials.gov (NCT02412020) and the EU Clinical Trials Register (EudraCT Number 2014-004025-40) and both cohorts are closed to new participants. Findings Between Feb 13, 2015, and Feb 2, 2016, 24 participants with IPF were randomly assigned to treatment groups. 28 participants with CIC were enrolled during the same period and 27 received study treatment. In patients with IPF, PA101 reduced daytime cough frequency by 31·1% at day 14 compared with placebo; daytime cough frequency decreased from a mean 55 (SD 55) coughs per h at baseline to 39 (29) coughs per h at day 14 following treatment with PA101, versus 51 (37) coughs per h at baseline to 52 (40) cough per h following placebo treatment (ratio of least-squares [LS] means 0·67, 95% CI 0·48–0·94, p=0·0241). By contrast, no treatment benefit for PA101 was observed in the CIC cohort; mean reduction of daytime cough frequency at day 14 for PA101 adjusted for placebo was 6·2% (ratio of LS means 1·27, 0·78–2·06, p=0·31). PA101 was well tolerated in both cohorts. The incidence of adverse events was similar between PA101 and placebo treatments, most adverse events were mild in severity, and no severe adverse events or serious adverse events were reported. Interpretation This study suggests that the mechanism of cough in IPF might be disease specific. Inhaled PA101 could be a treatment option for chronic cough in patients with IPF and warrants further investigation

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease