Strongyloides questions-a research agenda for the future.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.

Advancing Strongyloides omics data: bridging the gap with Caenorhabditis elegans.

Among nematodes, the free-living model organism Caenorhabditis elegans boasts the most advanced portfolio of high-quality omics data. The resources available for parasitic nematodes, including Strongyloides spp., however, are lagging behind. While C. elegans remains the most tractable nematode and has significantly advanced our understanding of many facets of nematode biology, C. elegans is not suitable as a surrogate system for the study of parasitism and it is important that we improve the omics resources available for parasitic nematode species. Here, we review the omics data available for Strongyloides spp. and compare the available resources to those for C. elegans and other parasitic nematodes. The advancements in C. elegans omics offer a blueprint for improving omics-led research in Strongyloides. We suggest areas of priority for future research that will pave the way for expansions in omics resources and technologies. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.

stepRNA: Identification of Dicer cleavage signatures and passenger strand lengths in small RNA sequences.

The enzyme Dicer is a component of many small RNA (sRNA) pathways involved in RNA processing for post-transcriptional regulation, anti-viral response and control of transposable elements. Cleavage of double-stranded RNA by Dicer produces a signature overhanging sequence at the 3' end of the sRNA sequence relative to a complementary passenger strand in a RNA duplex. There is a need for reliable tools to computationally search for Dicer cleavage signatures to help characterise families of sRNAs. This is increasingly important due to the rising popularity of sRNA sequencing, especially in non-model organisms. Here, we present stepRNA, a fast, local tool that identifies (i) overhang signatures strongly indicative of Dicer cleavage in RNA sequences, and (ii) the length of the passenger strand in sRNAs duplexes. We demonstrate the use of stepRNA with simulated and biological datasets to detect Dicer cleavage signatures in experimentally validated examples. Compared to currently available tools, stepRNA is more accurate, requires only sRNA sequence data rather than a reference genome, and provides information about other important features such as passenger strand length. stepRNA is freely available at https://github.com/Vicky-Hunt-Lab/stepRNA and is easily installable.

piRNA-like small RNAs target transposable elements in a Clade IV parasitic nematode.

The small RNA (sRNA) pathways identified in the model organism Caenorhabditis elegans are not widely conserved across nematodes. For example, the PIWI pathway and PIWI-interacting RNAs (piRNAs) are involved in regulating and silencing transposable elements (TE) in most animals but have been lost in nematodes outside of the C. elegans group (Clade V), and little is known about how nematodes regulate TEs in the absence of the PIWI pathway. Here, we investigated the role of sRNAs in the Clade IV parasitic nematode Strongyloides ratti by comparing two genetically identical adult stages (the parasitic female and free-living female). We identified putative small-interfering RNAs, microRNAs and tRNA-derived sRNA fragments that are differentially expressed between the two adult stages. Two classes of sRNAs were predicted to regulate TE activity including (i) a parasite-associated class of 21-22 nt long sRNAs with a 5' uridine (21-22Us) and a 5' monophosphate, and (ii) 27 nt long sRNAs with a 5' guanine/adenine (27GAs) and a 5' modification. The 21-22Us show striking resemblance to the 21U PIWI-interacting RNAs found in C. elegans, including an AT rich upstream sequence, overlapping loci and physical clustering in the genome. Overall, we have shown that an alternative class of sRNAs compensate for the loss of piRNAs and regulate TE activity in nematodes outside of Clade V.

Genome of the fatal tapeworm Sparganum proliferum uncovers mechanisms for cryptic life cycle and aberrant larval proliferation.

The cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, S. proliferum's phylogeny and life cycle remain enigmatic. To investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the mechanisms underlying pathogenicity, we sequenced the entire genomes of S. proliferum and a closely related non-life-threatening tapeworm Spirometra erinaceieuropaei. Additionally, we performed larvae transcriptome analyses of S. proliferum plerocercoid to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum has experienced a clearly distinct evolutionary history from S. erinaceieuropaei. Moreover, we found that nonordinal extracellular matrix coordination allows asexual reproduction in the host, and loss of sexual maturity in S. proliferum are responsible for its fatal pathogenicity to humans. Our high-quality reference genome sequences should be valuable for future studies of pseudophyllidean tapeworm biology and parasitism.

Sequential Changes in the Host Gut Microbiota During Infection With the Intestinal Parasitic Nematode Strongyloides venezuelensis.

Soil-transmitted helminths (STHs) are medically important parasites that infect 1. 5 billion humans globally, causing a substantial disease burden. These parasites infect the gastrointestinal tract (GIT) of their host where they co-exist and interact with the host gut bacterial flora, leading to the coevolution of the parasites, microbiota, and host organisms. However, little is known about how these interactions change through time with the progression of infection. Strongyloidiasis is a human parasitic disease caused by the nematode Strongyloides stercoralis infecting 30-100 million people. In this study, we used a closely related rodent parasite Strongyloides venezuelensis and mice as a model of gastrointestinal parasite infection. We conducted a time-course experiment to examine changes in the fecal microbiota from the start of infection to parasite clearance. We found that bacterial taxa in the host intestinal microbiota changed significantly as the infection progressed, with an increase in the genera Bacteroides and Candidatus Arthromitus, and a decrease in Prevotella and Rikenellaceae. However, the microbiota recovered to the pre-infective state after parasite clearance from the host, suggesting that these perturbations are reversible. Microarray analysis revealed that this microbiota transition is likely to correspond with the host immune response. These findings give us an insight into the dynamics of parasite-microbiota interactions in the host gut during parasite infection.

Biology and genome of a newly discovered sibling species of Caenorhabditis elegans.

A 'sibling' species of the model organism Caenorhabditis elegans has long been sought for use in comparative analyses that would enable deep evolutionary interpretations of biological phenomena. Here, we describe the first sibling species of C. elegans, C. inopinata n. sp., isolated from fig syconia in Okinawa, Japan. We investigate the morphology, developmental processes and behaviour of C. inopinata, which differ significantly from those of C. elegans. The 123-Mb C. inopinata genome was sequenced and assembled into six nuclear chromosomes, allowing delineation of Caenorhabditis genome evolution and revealing unique characteristics, such as highly expanded transposable elements that might have contributed to the genome evolution of C. inopinata. In addition, C. inopinata exhibits massive gene losses in chemoreceptor gene families, which could be correlated with its limited habitat area. We have developed genetic and molecular techniques for C. inopinata; thus C. inopinata provides an exciting new platform for comparative evolutionary studies.

Comparative transcriptomics gives insights into the evolution of parasitism in Strongyloides nematodes at the genus, subclade and species level.

Strongyloides spp., gastrointestinal nematode parasites of humans and other animals, have genetically identical parasitic and free-living adult life cycle stages. This is an almost unique feature amongst nematodes and comparison of these two stages can provide insights into the genetic basis and evolution of Strongyloides nematode parasitism. Here, we present RNAseq data for S. venezuelensis, a parasite of rodents, and identify genes that are differentially expressed in parasitic and free-living life cycle stages. Comparison of these data with analogous RNAseq data for three other Strongyloides spp., has identified key protein-coding gene families with a putative role in parasitism including WAGO-like Argonautes (at the genus level) and speckle-type POZ-like coding genes (S. venezuelensis-S. papillosus phylogenetic subclade level). Diverse gene families are uniquely upregulated in the parasitic stage of all four Strongyloides species, including a distinct upregulation of genes encoding cytochrome P450 in S. venezuelensis, suggesting some diversification of the molecular tools used in the parasitic life cycle stage among individual species. Together, our results identify key gene families with a putative role in Strongyloides parasitism or features of the parasitic life cycle stage, and deepen our understanding of parasitism evolution among Strongyloides species.

Infection does not increase long-term mortality in patients with acute severe alcoholic hepatitis treated with corticosteroids.

AIM: To determine whether infection in patients with acute severe alcoholic hepatitis (AAH) treated with corticosteroids is associated with increased mortality. METHODS: Consecutive patients with AAH were treated with steroids and recruited to the study. Clinically relevant infections (body temperature > 38 °C or < 36 °C for more than 4 h, ascitic neutrophil count > 0.25 ×109/L, consolidation on chest radiograph or clinically relevant positive microbiological culture of bodily fluid) were recorded prospectively. Clinical and laboratory parameters were recorded and survival at 90 d and 6 mo was determined. Univariate analysis of factors associated with 90-d mortality was performed and significant variables included in a multivariate analysis. RESULTS: Seventy-two patients were included in the final analysis (mean age 47.9 years, 26% female, mean discriminant function 53.0). Overall mortality in the group occurred in 15 (21%), 23 (32%) and 31 (43%) at day 28, day 90 and 1 year respectively. 36 (50%) had a clinically relevant infection during their hospitalisation (23 after initiation of steroids). The median time to development of incident infection after commencement of steroids was 10 d. The commonest site of infection was ascites (31%) and bacteraemia (31%) followed by urinary tract (19%) and respiratory tract (8%). Forty-one separate organisms were isolated in 33 patients; the most frequent genus was Escherichia (22%) and Enterococcus (20%). Infection was not associated with 90-d or 1 year mortality but was associated with higher creatinine, model for end-stage liver disease and Lille score. Baseline urea was the only independent predictor of 90-d mortality. CONCLUSION: Clinically relevant infections are common in patients with AAH but are not associated with increased 90-d or 1 year mortality.

The use of Oxford Nanopore native barcoding for complete genome assembly.

Background: The Oxford Nanopore Technologies MinION(TM) is a mobile DNA sequencer that can produce long read sequences with a short turn-around time. Here we report the first demonstration of single contig genome assembly using Oxford Nanopore native barcoding when applied to a multiplexed library of 12 samples and combined with existing Illumina short read data. This paves the way for the closure of multiple bacterial genomes from a single MinION(TM) sequencing run, given the availability of existing short read data. The strain we used, MHO_001, represents the important community-acquired methicillin-resistant Staphylococcus aureus lineage USA300. Findings: Using a hybrid assembly of existing short read and barcoded long read sequences from multiplexed data, we completed a genome of the S. aureus USA300 strain MHO_001. The long read data represented only ∼5% to 10% of an average MinION(TM) run (∼7x genomic coverage), but, using standard tools, this was sufficient to complete the circular chromosome of S. aureus strain MHO_001 (2.86 Mb) and two complete plasmids (27 Kb and 3 Kb). Minor differences were noted when compared to USA300 reference genome, USA300_FPR3757, including the translocation, loss, and gain of mobile genetic elements. Conclusion: Here we demonstrate that MinION(TM) reads, multiplexed using native barcoding, can be used in combination with short read data to fully complete a bacterial genome. The ability to complete multiple genomes, for which short read data is already available, from a single MinION(TM) run is set to impact our understanding of accessory genome content, plasmid diversity, and genome rearrangements.

The genome of Strongyloides spp. gives insights into protein families with a putative role in nematode parasitism.

Parasitic nematodes are important and abundant parasites adapted to live a parasitic lifestyle, with these adaptations all aimed at facilitating their survival and reproduction in their hosts. The recently sequenced genomes of four Strongyloides species, gastrointestinal parasites of humans and other animals, alongside transcriptomic and proteomic analysis of free-living and parasitic stages of their life cycles have revealed a number of protein families with a putative role in their parasitism. Many of these protein families have also been associated with parasitism in other parasitic nematode species, suggesting that these proteins may play a fundamental role in nematode parasitism more generally. Here, we review key protein families that have a putative role in Strongyloides' parasitism - acetylcholinesterases, astacins, aspartic proteases, prolyl oligopeptidases, proteinase inhibitors (trypsin inhibitors and cystatins), SCP/TAPS and transthyretin-like proteins - and the evidence for their key, yet diverse, roles in the parasitic lifestyle.

The genomic basis of parasitism in the Strongyloides clade of nematodes.

Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families--families encoding astacin-like and SCP/TAPS proteins--is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.

Cold-seeking behaviour mitigates reproductive losses from fungal infection in Drosophila.

Animals must tailor their life-history strategies to suit the prevailing conditions and respond to hazards in the environment. Animals with lethal infections are faced with a difficult choice: to allocate more resources to reproduction and suffer higher mortality or to reduce reproduction with the expectation of enhanced immunity and late-age reproduction. However, the strategies employed to mediate shifts in life-history traits are largely unknown. Here, we investigate the temperature preference of the fruit fly, Drosophila melanogaster, during infection with the fungal pathogen, Metarhizium robertsii, and the consequence of temperature preference on life-history traits. We have measured the temperature preference of fruit flies under different pathogen conditions. We conducted multiple fitness assays of the host and the pathogen under different thermal conditions. From these data, we estimated standard measures of fitness and used age-specific methodologies to test for the fitness trade-offs that are thought to underlie differences in life-history strategy. We found that fungus-infected fruit flies seek out cooler temperatures, which facilitates an adaptive shift in their life-history strategy. The colder temperatures preferred by infected animals were detrimental to the pathogen because it increased resistance to infection. But, it did not provide net benefits that were specific to infected animals, as cooler temperatures increased lifetime reproductive success and survival whether or not the animals were infected. Instead, we find that cold-seeking benefits infected animals by increasing their late-age reproductive output, at a cost to their early-age reproductive output. In contrast, naive control flies prefer warmer temperatures that optimize early-age reproductive, at a cost to reproductive output at late ages. These findings show that infected animals exhibit fundamentally different reproductive strategies than their healthy counterparts. Temperature preference can facilitate shifts in strategy, but not without inevitable trade-offs.

Hormesis results in trade-offs with immunity.

Many have argued that we may be able to extend life and improve human health through hormesis, the beneficial effects of low-level toxins and other stressors. But, studies of hormesis in model systems have not yet established whether stress-induced benefits are cost free, artifacts of inbreeding, or come with deleterious side effects. Here, we provide evidence that hormesis results in trade-offs with immunity. We find that a single topical dose of dead spores of the entomopathogenic fungus, Metarhizium robertsii, increases the longevity of the fruit fly, Drosophila melanogaster, without significant decreases in fecundity. We find that hormetic benefits of pathogen challenge are greater in lines that lack key components of antifungal immunity (Dif and Turandot M). And, in outbred fly lines, we find that topical pathogen challenge enhances both survival and fecundity, but reduces ability to fight off live infections. The results provide evidence that hormesis is manifested by stress-induced trade-offs with immunity, not cost-free benefits or artifacts of inbreeding. Our findings illuminate mechanisms underlying pathogen-induced life-history trade-offs, and indicate that reduced immune function may be an ironic side effect of the "elixirs of life."

Randomized study comparing a Basal-bolus with a basal plus correction insulin regimen for the hospital management of medical and surgical patients with type 2 diabetes: basal plus trial.

OBJECTIVE: Effective and easily implemented insulin regimens are needed to facilitate hospital glycemic control in general medical and surgical patients with type 2 diabetes (T2D). RESEARCH DESIGN AND METHODS: This multicenter trial randomized 375 patients with T2D treated with diet, oral antidiabetic agents, or low-dose insulin (≤ 0.4 units/kg/day) to receive a basal-bolus regimen with glargine once daily and glulisine before meals, a basal plus regimen with glargine once daily and supplemental doses of glulisine, and sliding scale regular insulin (SSI). RESULTS: Improvement in mean daily blood glucose (BG) after the first day of therapy was similar between basal-bolus and basal plus groups (P = 0.16), and both regimens resulted in a lower mean daily BG than did SSI (P = 0.04). In addition, treatment with basal-bolus and basal plus regimens resulted in less treatment failure (defined as >2 consecutive BG >240 mg/dL or a mean daily BG >240 mg/dL) than did treatment with SSI (0 vs. 2 vs. 19%, respectively; P < 0.001). A BG <70 mg/dL occurred in 16% of patients in the basal-bolus group, 13% in the basal plus group, and 3% in the SSI group (P = 0.02). There was no difference among the groups in the frequency of severe hypoglycemia (<40 mg/dL; P = 0.76). CONCLUSIONS: The use of a basal plus regimen with glargine once daily plus corrective doses with glulisine insulin before meals resulted in glycemic control similar to a standard basal-bolus regimen. The basal plus approach is an effective alternative to the use of a basal-bolus regimen in general medical and surgical patients with T2D.