Lentiviral transduction facilitates RNA interference in the nematode parasite Nippostrongylus brasiliensis.

Animal-parasitic nematodes have thus far been largely refractory to genetic manipulation, and methods employed to effect RNA interference (RNAi) have been ineffective or inconsistent in most cases. We describe here a new approach for genetic manipulation of Nippostrongylus brasiliensis, a widely used laboratory model of gastrointestinal nematode infection. N. brasiliensis was successfully transduced with Vesicular Stomatitis Virus glycoprotein G (VSV-G)-pseudotyped lentivirus. The virus was taken up via the nematode intestine, RNA reverse transcribed into proviral DNA, and transgene transcripts produced stably in infective larvae, which resulted in expression of the reporter protein mCherry. Improved transgene expression was achieved by incorporating the C. elegans hlh11 promoter and the tbb2 3´-UTR into viral constructs. MicroRNA-adapted short hairpin RNAs delivered in this manner were processed correctly and resulted in partial knockdown of ß-tubulin isotype-1 (tbb-iso-1) and secreted acetylcholinesterase B (ache-B). The system was further refined by lentiviral delivery of double stranded RNAs, which acted as a trigger for RNAi following processing and generation of 22G-RNAs. Virus-encoded sequences were detectable in F1 eggs and third stage larvae, demonstrating that proviral DNA entered the germline and was heritable. Lentiviral transduction thus provides a new means for genetic manipulation of parasitic nematodes, including gene silencing and expression of exogenous genes.

MicroRNA-142 Critically Regulates Group 2 Innate Lymphoid Cell Homeostasis and Function.

Innate lymphoid cells are central to the regulation of immunity at mucosal barrier sites, with group 2 innate lymphoid cells (ILC2s) being particularly important in type 2 immunity. In this study, we demonstrate that microRNA(miR)-142 plays a critical, cell-intrinsic role in the homeostasis and function of ILC2s. Mice deficient for miR-142 expression demonstrate an ILC2 progenitor-biased development in the bone marrow, and along with peripheral ILC2s at mucosal sites, these cells display a greatly altered phenotype based on surface marker expression. ILC2 proliferative and effector functions are severely dysfunctional following Nippostrongylus brasiliensis infection, revealing a critical role for miR-142 isoforms in ILC2-mediated immune responses. Mechanistically, Socs1 and Gfi1 expression are regulated by miR-142 isoforms in ILC2s, impacting ILC2 phenotypes as well as the proliferative and effector capacity of these cells. The identification of these novel pathways opens potential new avenues to modulate ILC2-dependent immune functions.

Articular Surface Involvement With Retrograde Headless Screw Fixation of the Proximal Phalanx.

PURPOSE: Intramedullary (IM) screw fixation of proximal phalanx (P1) fractures is a treatment option increasing in popularity. This study aimed to quantify the articular surface loss after retrograde screw insertion and to determine the range of motion (ROM) of the proximal interphalangeal (PIP) joint while the defect in the P1 head is engaged with the base of the middle phalanx (P2). METHODS: Twelve fresh frozen cadaver hand specimens were analyzed for prefixation ROM of the PIP joint. A retrograde screw was placed using a percutaneous technique under fluoroscopic guidance. Following screw insertion, specimens were dissected to determine size of the extensor mechanism defect, evaluate the lateral bands with passive ROM of the PIP joint, and determine the angle at which the dorsal aspect of P2 ceases to engage with the defect and the amount of articular surface loss. The percentage of articular surface loss was calculated using a digital image software program. RESULTS: The angle at which P2 ceased to engage with the articular surface defect was an average of 36.8° of PIP joint flexion. In full PIP joint flexion, the average extensor mechanism defect was 8.8%. The average total articular surface loss was 4.4% across all digits. CONCLUSION: Percutaneous retrograde P1 intramedullary screw fixation results in minimal damage to the extensor mechanism and articular surface. The arc during which the defect in the head of P1 engages the base of the P2 is almost entirely outside the functional ROM of the PIP joint. CLINICAL RELEVANCE: Quantifying the amount of articular surface loss through the P1 head and extensor apparatus damage in IM screw fixation can inform surgeons of the consequences of this technique. This study supports the use of a retrograde intramedullary screw as a safe option for fixation of P1 fractures.

The presence and potential impact of psychological safety in the healthcare setting: an evidence synthesis.

INTRODUCTION: Psychological safety is the shared belief that the team is safe for interpersonal risk taking. Its presence improves innovation and error prevention. This evidence synthesis had 3 objectives: explore the current literature regarding psychological safety, identify methods used in its assessment and investigate for evidence of consequences of a psychologically safe environment. METHODS: We searched multiple trial registries through December 2018. All studies addressing psychological safety within healthcare workers were included and reviewed for methodological limitations. A thematic analysis approach explored the presence of psychological safety. Content analysis was utilised to evaluate potential consequences. RESULTS: We included 62 papers from 19 countries. The thematic analysis demonstrated high and low levels of psychological safety both at the individual level in study participants and across the studies themselves. There was heterogeneity in responses across all studies, limiting generalisable conclusions about the overall presence of psychological safety. A wide range of methods were used. Twenty-five used qualitative methodology, predominantly semi-structured interviews. Thirty quantitative or mixed method studies used surveys. Ten studies inferred that low psychological safety negatively impacted patient safety. Nine demonstrated a significant relationship between psychological safety and team outcomes. The thematic analysis allowed the development of concepts beyond the content of the original studies. This analytical process provided a wealth of information regarding facilitators and barriers to psychological safety and the development of a model demonstrating the influence of situational context. DISCUSSION: This evidence synthesis highlights that whilst there is a positive and demonstrable presence of psychological safety within healthcare workers worldwide, there is room for improvement. The variability in methods used demonstrates scope to harmonise this. We draw attention to potential consequences of both high and low psychological safety. We provide novel information about the influence of situational context on an individual's psychological safety and offer more detail about the facilitators and barriers to psychological safety than seen in previous reviews. There is a risk of participation bias - centres involved in safety research may be more aligned to these ideals. The data in this synthesis are useful for institutions looking to improve psychological safety by providing a framework from which modifiable factors can be identified.

A Comparison of Three Airborne Laser Scanner Types for Species Identification of Individual Trees.

Species identification is a critical factor for obtaining accurate forest inventories. This paper compares the same method of tree species identification (at the individual crown level) across three different types of airborne laser scanning systems (ALS): two linear lidar systems (monospectral and multispectral) and one single-photon lidar (SPL) system to ascertain whether current individual tree crown (ITC) species classification methods are applicable across all sensors. SPL is a new type of sensor that promises comparable point densities from higher flight altitudes, thereby increasing lidar coverage. Initial results indicate that the methods are indeed applicable across all of the three sensor types with broadly similar overall accuracies (Hardwood/Softwood, 83-90%; 12 species, 46-54%; 4 species, 68-79%), with SPL being slightly lower in all cases. The additional intensity features that are provided by multispectral ALS appear to be more beneficial to overall accuracy than the higher point density of SPL. We also demonstrate the potential contribution of lidar time-series data in improving classification accuracy (Hardwood/Softwood, 91%; 12 species, 58%; 4 species, 84%). Possible causes for lower SPL accuracy are (a) differences in the nature of the intensity features and (b) differences in first and second return distributions between the two linear systems and SPL. We also show that segmentation (and field-identified training crowns deriving from segmentation) that is performed on an initial dataset can be used on subsequent datasets with similar overall accuracy. To our knowledge, this is the first study to compare these three types of ALS systems for species identification at the individual tree level.

Prior muscle activation affects the compound muscle action potential.

INTRODUCTION: This study was performed to evaluate the effect of prior voluntary activation of a muscle on the subsequently-recorded compound muscle action potential (CMAP). METHODS: The CMAPs from the hypothenar, thenar, and extensor digitorum brevis muscles were recorded in 6 healthy volunteers at rest and for up to 30 min following 5 separate epochs of up to 20 s of voluntary muscle activation. RESULTS: There was consistent, significant (P < 0.02) enhancement of the negative area, amplitude, and duration of the CMAP after activation. The enhancement was maximal, up to 144% of baseline, within about 1 min post-activation; thereafter, the CMAP gradually returned to baseline over about 15 min. DISCUSSION: Activation of a muscle within several minutes prior to testing enhances the subsequently-recorded CMAP. This observation highlights prior muscle activation as a physiological variable that influences the size of the CMAP during motor nerve conduction studies. Muscle Nerve, 2019.

'I'm not a real boozer': a qualitative study of primary care patients' views on drinking and its consequences.

BACKGROUND: The public health message around alcohol is complex, with benefits versus harms, the confusing concept of risk and drinking guidance changing over time. This provides a difficult context for alcohol screening in primary care, with established barriers from the practitioner perspective, but less is known about the patients' perspective. This study explores patients' views on drinking. METHODS: Eligible participants were recorded as drinking above low risk levels in primary care. Six practices in North London participated. Interviews were in-depth, semi-structured, transcribed verbatim and underwent detailed thematic analysis. FINDINGS: Interviews were conducted with 8 women and 12 men, aged 26-83 years, mostly educated to undergraduate level and of 'White' ethnicity. UK drinking guidance was viewed as irrelevant for reasons related to life stage, lifestyle and absence of harm. Dependence, loss of functionality and control were perceived as key features of problematic drinking. Healthy lifestyles, in terms of diet, exercise and not smoking, were thought to mitigate potential problems associated with alcohol intake. CONCLUSION: The findings suggest that public health messages and brief advice should focus on harm experienced at different life stages, among people with different lifestyles, to challenge the ubiquitous view that 'I'm not a real boozer'.

The effects of denitrification parameterization and potential benefits of spatially targeted regulation for the reduction of N-discharges from agriculture.

Diffuse nitrate leaching from agricultural areas is a major environmental problem in many parts of the world. Understanding where in a catchment nitrate is removed is key for designing effective land use management strategies that protect water quality, while minimizing the impact on economic development. In this study we assess the effects of spatially targeted nitrate leaching regulation in a basin with limited knowledge of the complexity of chemical heterogeneity. Three alternative nitrate reactivity spatial parameterizations were incorporated in a catchment-scale flow and transport model and used to evaluate the effectiveness of four possible spatially targeted regulation options. Our findings confirm that denitrification parameterization cannot be numerically determined based on model inversion alone. Detailed field based characterization using physical and geochemical methods should be considered and incorporated in the numerical inversion scheme. We also demonstrate that there are potential benefits of implementing spatially targeted regulation compared to spatially uniform regulation. Focusing regulation in areas where nitrate residence time is short, such as riparian zones or areas with low natural N-reduction, results in greater reduction of N-discharges through groundwater. Significantly improved efficiencies can be expected when delineation of management zones considers the chemical heterogeneity and groundwater flow paths. These improved efficiencies are achieved by adopting management rules that regulate land use in discharge sensitive areas, where leaching changes contribute the most to the catchment nitrate discharges. In our case study, regulation in discharge sensitive zones was twice as efficient compared to other management options.

Bacterial community structure and response to nitrogen amendments in Lake Shenandoah (VA, USA).

Microbial processes are critical to the function of freshwater ecosystems, yet we still do not fully understand the factors that shape freshwater microbial communities. Furthermore, freshwater ecosystems are particularly susceptible to effects of environmental change, including influx of exogenous nutrients such as nitrogen and phosphorus. To evaluate the impact of nitrogen loading on the microbial community structure of shallow freshwater lakes, water samples collected from Lake Shenandoah (Virginia, USA) were incubated with two concentrations of either ammonium, nitrate, or urea as a nitrogen source. The potential impact of these nitrogen compounds on the bacterial community structure was assessed via 16S rRNA amplicon sequencing. At the phylum level, the dominant taxa in Lake Shenandoah were comprised of Actinobacteria and Proteobacteria, which were not affected by exposure to the various nitrogen treatments. Overall, there was not a significant shift in the diversity of the bacterial community of Lake Shenandoah with the addition of nitrogen sources, indicating this shallow system may be constrained by other environmental factors.

Phase I study of the checkpoint kinase 1 inhibitor GDC-0575 in combination with gemcitabine in patients with refractory solid tumors.

BACKGROUND: Checkpoint kinase 1 (Chk1) inhibition following chemotherapy-elicited DNA damage overrides cell cycle arrest and induces mitotic catastrophe and cell death. GDC-0575 is a highly-selective oral small-molecule Chk1 inhibitor that results in tumor shrinkage and growth delay in xenograft models. We evaluated the safety, tolerability, and pharmacokinetic properties of GDC-0575 alone and in combination with gemcitabine. Antitumor activity and Chk1 pathway modulation were assessed. PATIENTS AND METHODS: In this phase I open-label study, in the dose escalation stage, patients were enrolled in a GDC-0575 monotherapy Arm (1) or GDC-0575 combination with gemcitabine Arm (2) to determine the maximum tolerated dose. Patients in arm 2 received either i.v. gemcitabine 1000 mg/m2 (arm 2a) or 500 mg/m2 (arm 2b), followed by GDC-0575 (45 or 80 mg, respectively, as RP2D). Stage II enrolled disease-specific cohorts. RESULTS: Of 102 patients treated, 70% were female, the median age was 59 years (range 27-85), and 47% were Eastern Cooperative Oncology Group PS 0. The most common tumor type was breast (37%). The most frequent adverse events (all grades) related to GDC-0575 and/or gemcitabine were neutropenia (68%), anemia (48%), nausea (43%), fatigue (42%), and thrombocytopenia (35%). Maximum concentrations of GDC-0575 were achieved within 2 hours of dosing, and half-life was ∼23 hours. No pharmacokinetic drug-drug interaction was observed between GDC-0575 and gemcitabine. Among patients treated with GDC-0575 and gemcitabine, there were four confirmed partial responses, three occurring in patients with tumors harboring TP53 mutation. Pharmacodynamic data were consistent with GDC-0575 inhibition of gemcitabine-induced expression of pCDK1/2. CONCLUSION: GDC-0575 can be safely administered as a monotherapy and in combination with gemcitabine; however, overall tolerability with gemcitabine was modest. Hematological toxicities were frequent but manageable. Preliminary antitumor activity was observed but limited to a small number of patients with a variety of refractory solid tumors treated with GDC-0575 and gemcitabine. CLINICAL TRIAL NUMBER: NCT01564251.

Modulation of the Immune Response by Nematode Secreted Acetylcholinesterase Revealed by Heterologous Expression in Trypanosoma musculi.

Nematode parasites secrete molecules which regulate the mammalian immune system, but their genetic intractability is a major impediment to identifying and characterising the biological effects of these molecules. We describe here a novel system for heterologous expression of helminth secreted proteins in the natural parasite of mice, Trypanosoma musculi, which can be used to analyse putative immunomodulatory functions. Trypanosomes were engineered to express a secreted acetylcholinesterase from Nippostrongylus brasiliensis. Infection of mice with transgenic parasites expressing acetylcholinesterase resulted in truncated infection, with trypanosomes cleared early from the circulation. Analysis of cellular phenotypes indicated that exposure to acetylcholinesterase in vivo promoted classical activation of macrophages (M1), with elevated production of nitric oxide and lowered arginase activity. This most likely occurred due to the altered cytokine environment, as splenocytes from mice infected with T. musculi expressing acetylcholinesterase showed enhanced production of IFNγ and TNFα, with diminished IL-4, IL-13 and IL-5. These results suggest that one of the functions of nematode secreted acetylcholinesterase may be to alter the cytokine environment in order to inhibit development of M2 macrophages which are deleterious to parasite survival. Transgenic T. musculi represents a valuable new vehicle to screen for novel immunoregulatory proteins by extracellular delivery in vivo to the murine host.

Ubiquitin-Dependent Modification of Skeletal Muscle by the Parasitic Nematode, Trichinella spiralis.

Trichinella spiralis is a muscle-specific parasitic worm that is uniquely intracellular. T. spiralis reprograms terminally differentiated skeletal muscle cells causing them to de-differentiate and re-enter the cell cycle, a process that cannot occur naturally in mammalian skeletal muscle cells, but one that holds great therapeutic potential. Although the host ubiquitin pathway is a common target for viruses and bacteria during infection, its role in parasite pathogenesis has been largely overlooked. Here we demonstrate that the secreted proteins of T. spiralis contain E2 Ub-conjugating and E3 Ub-ligase activity. The E2 activity is attributed to TsUBE2L3, a novel and conserved T. spiralis enzyme located in the secretory organ of the parasite during the muscle stages of infection. TsUBE2L3 cannot function with any T.spiralis secreted E3, but specifically binds to a panel of human RING E3 ligases, including the RBR E3 ARIH2 with which it interacts with a higher affinity than the mammalian ortholog UbcH7/UBE2L3. Expression of TsUBE2L3 in skeletal muscle cells causes a global downregulation in protein ubiquitination, most predominantly affecting motor, sarcomeric and extracellular matrix proteins, thus mediating their stabilization with regards to proteasomal degradation. This effect is not observed in the presence of the mammalian ortholog, suggesting functional divergence in the evolution of the parasite protein. These findings demonstrate the first example of host-parasite interactions via a parasite-derived Ub conjugating enzyme; an E2 that demonstrates a novel muscle protein stabilization function.

Surfactant Protein-D Is Essential for Immunity to Helminth Infection.

Pulmonary epithelial cell responses can enhance type 2 immunity and contribute to control of nematode infections. An important epithelial product is the collectin Surfactant Protein D (SP-D). We found that SP-D concentrations increased in the lung following Nippostrongylus brasiliensis infection; this increase was dependent on key components of the type 2 immune response. We carried out loss and gain of function studies of SP-D to establish if SP-D was required for optimal immunity to the parasite. N. brasiliensis infection of SP-D-/- mice resulted in profound impairment of host innate immunity and ability to resolve infection. Raising pulmonary SP-D levels prior to infection enhanced parasite expulsion and type 2 immune responses, including increased numbers of IL-13 producing type 2 innate lymphoid cells (ILC2), elevated expression of markers of alternative activation by alveolar macrophages (alvM) and increased production of the type 2 cytokines IL-4 and IL-13. Adoptive transfer of alvM from SP-D-treated parasite infected mice into naïve recipients enhanced immunity to N. brasiliensis. Protection was associated with selective binding by the SP-D carbohydrate recognition domain (CRD) to L4 parasites to enhance their killing by alvM. These findings are the first demonstration that the collectin SP-D is an essential component of host innate immunity to helminths.

Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages.

Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs) are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs). Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements.

Trauma to the Superior Mesenteric Artery and Superior Mesenteric Vein: A Narrative Review of Rare but Lethal Injuries.

Mesenteric vessels, including the superior mesenteric artery (SMA) and vein (SMV), provide and drain the rich blood supply of the midgut and hindgut. SMA and SMV injuries are rare and often lethal. Clinical management of these injuries is not well established, but treatment options include operative, non-operative, and endovascular strategies. A narrative review of the literature was conducted using MEDLINE Complete-EBSCO. Relevant studies, specifically those focusing on diagnosis and management of SMA and SMV injuries, were selected. Only original reports and collected series were selected to prevent duplication of cases. A search of the literature for mesenteric arterial injuries yielded 87 studies. Vessel-specific breakdown of the studies yielded 40 with SMA injuries and 41 with SMV injuries. These searches were winnowed to 26 individual studies, which were included in this collective review. Limitations of this study are similar to all narrative literature reviews: the dependence on previously published research and availability of references as outlined in our methodology. Although historically rare, mesenteric vessel injuries are seen with increasing incidence and continue to present a challenge to trauma surgeons due to their daunting mortality rates. Currently, universal treatment guidelines do not exist, but the various options for their management have been extensively reviewed in the literature.

Natural genomic amplification of cholinesterase genes in animals.

Tight control of the concentration of acetylcholine at cholinergic synapses requires precise regulation of the number and state of the acetylcholine receptors, and of the synthesis and degradation of the neurotransmitter. In particular, the cholinesterase activity has to be controlled exquisitely. In the genome of the first experimental models used (man, mouse, zebrafish and drosophila), there are only one or two genes coding for cholinesterases, whereas there are more genes for their closest relatives the carboxylesterases. Natural amplification of cholinesterase genes was first found to occur in some cancer cells and in insect species subjected to evolutionary pressure by insecticides. Analysis of the complete genome sequences of numerous representatives of the various metazoan phyla show that moderate amplification of cholinesterase genes is not uncommon in molluscs, echinoderms, hemichordates, prochordates or lepidosauria. Amplification of acetylcholinesterase genes is also a feature of parasitic nematodes or ticks. In these parasites, over-production of cholinesterase-like proteins in secreted products and the saliva are presumed to have effector roles related to host infection. These amplification events raise questions about the role of the amplified gene products, and the adaptation processes necessary to preserve efficient cholinergic transmission. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

The M3 muscarinic receptor is required for optimal adaptive immunity to helminth and bacterial infection.

Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R) plays an important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-γ respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection.

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.