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DNA metabarcoding of benthic diatoms has been successfully applied for biomonitoring at the national scale and can now be considered technically ready for routine application. However, protocols and methods still vary between and within countries, limiting their transferability and the comparability of results. In order to overcome this, routine use of DNA metabarcoding for diatom biomonitoring requires knowledge of the sources of variability introduced by the different steps of the procedure. Here, we examine how elements of routine procedures contribute to variability between European laboratories. A set of four experiments were performed focusing on DNA extraction and PCR amplification steps to evaluate their reproducibility between different laboratories and the variability introduced by different protocols currently applied by the scientific community. Under the guidance of a reference laboratory, 17 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using the same fixed protocol and their own choice of protocol. Experiments were performed by each participant on a set of standardised DNA and biofilm samples (river, lake and mock community) to investigate potential systematic and random errors. Our results revealed the successful transferability of a protocol amongst labs and a highly similar and consistent ecological assessment outcome obtained regardless of the protocols used by each participant. We propose an “all for one but prove them all” strategy, suggesting that distinct protocols can be used within the scientific community, as long as their consistency is be proven by following minimum standard requirements.

Abstract Background While high-grade serous ovarian cancer (HGSC) has proven largely resistant to immunotherapy, sporadic incidents of partial and complete response have been observed in clinical trials and case reports. These observations suggest that a molecular basis for effective immunity may exist within a subpopulation of HGSC. Herein, we developed an algorithm, CONSTRU (Computing Prognostic Marker Dependencies by Successive Testing of Gene-Stratified Subgroups), to facilitate the discovery and characterization of molecular backgrounds of HGSC that confer resistance or susceptibility to protective anti-tumor immunity. Methods We used CONSTRU to identify genes from tumor expression profiles that influence the prognostic power of an established immune cytolytic activity signature (CYTscore). From the identified genes, we developed a stratification signature (STRATsig) that partitioned patient populations into tertiles that varied markedly by CYTscore prognostic power. The tertile groups were then analyzed for distinguishing biological, clinical and immunological properties using integrative bioinformatics approaches. Results Patient survival and molecular measures of immune suppression, evasion and dysfunction varied significantly across STRATsig tertiles in validation cohorts. Tumors comprising STRATsig tertile 1 (S-T1) showed no immune-survival benefit and displayed a hyper-immune suppressed state marked by activation of TGF-β, Wnt/β-catenin and adenosine-mediated immunosuppressive pathways, with concurrent T cell dysfunction, reduced potential for antigen presentation, and enrichment of cancer-associated fibroblasts. By contrast, S-T3 tumors exhibited diminished immunosuppressive signaling, heightened antigen presentation machinery, lowered T cell dysfunction, and a significant CYTscore-survival benefit that correlated with mutational burden in a manner consistent with anti-tumor immunoediting. These tumors also showed elevated activity of DNA damage/repair, cell cycle/proliferation and oxidative phosphorylation, and displayed greater proportions of Th1 CD4 + T cells. In these patients, but not those of S-T1 or S-T2, validated predictors of immunotherapy response were prognostic of longer patient survival. Further analyses showed that STRATsig tertile properties were not explained by known HGSC molecular or clinical subtypes or singular immune mechanisms. Conclusions STRATsig is a composite of parallel immunoregulatory pathways that mirrors tumor immunogenic potential. Approximately one-third of HGSC cases classify as S-T3 and display a hypo-immunosuppressed and antigenic molecular composition that favors immunologic tumor control. These patients may show heightened responsiveness to current immunotherapies.

Abstract The vertebrate heart tube extends by progressive addition of epithelial second heart field (SHF) progenitor cells from the dorsal pericardial wall. The interplay between epithelial mechanics and genetic mechanisms during SHF deployment is unknown. Here, we present a quantitative single-cell morphometric analysis of SHF cells during heart tube extension, including force inference analysis of epithelial stress. Joint spatial Principal Component Analysis reveals that cell orientation and stress direction are the main parameters defining apical cell morphology and distinguishes cells adjacent to the arterial and venous poles. Cell shape and mechanical forces display a dynamic relationship during heart tube formation. Moreover, while the T-box transcription factor Tbx1 is necessary for cell orientation towards the arterial pole, activation of Tbx5 in the posterior SHF correlates with the establishment of epithelial stress and SHF deletion of Tbx5 relaxes the progenitor epithelium. Integrating findings from cell-scale feature patterning and mechanical stress provides new insights into cardiac morphogenesis.

Abstract Micro-segregation and resulting nanoscale microstructural heterogeneities are unavoidable in wrought 7xxx aluminum alloys produced using current casting and thermomechanical processes. This study demonstrates that these nanoscale heterogeneities significantly impact pit growth in an extruded, age-hardened aluminum alloy 7075-T651. The alloy exhibits a complex microstructure with heterogeneously distributed E-Al18Mg3Cr2 dispersoids, which promote the precipitation of coarse ƞ-Mg(ZnAlCu)2 nanoparticles resulting in the formation of solute-depleted regions both within the grain interior and along the grain boundaries in dispersoid-rich areas. These alterations affect resistance to pit growth at the nanoscale, governing the transition of micro-galvanic sites into large pits with potential for crack initiation. This work underscores the necessity for modifying alloy composition and casting processes to develop superior aluminum alloys for critical applications.

This research investigates the relationship between neighbourhood parks and crime, exploring park conditions, activities, landscaping, and socioeconomic factors. Evaluating 351 parks across 45 states using the BRAT-direct observation instrument and national crime data, the study aims to discern links between parks and crime, emphasizing the impact of park attributes and surrounding income levels. Contrary to some prevailing theories, no significant correlation was found between park conditions or activities and crime rates. However, a moderate correlation emerges between burglary rates and the poverty levels of the surrounding area, and a negative correlation between burglary and median income of the area, suggesting that while parks themselves may not be crime generators, they may facilitate burglaries of opportunity due to increased foot traffic. The findings prompt a re-evaluation of existing theories, emphasizing the need for tailored safety measures during peak park usage times.

Abstract Networks of nanowires, nanotubes, and nanosheets are important for many applications in printed electronics. However, the network conductivity and mobility are usually limited by the resistance between the particles, often referred to as the junction resistance. Minimising the junction resistance has proven to be challenging, partly because it is difficult to measure. Here, we develop a simple model for electrical conduction in networks of 1D or 2D nanomaterials that allows us to extract junction and nanoparticle resistances from particle-size-dependent DC network resistivity data. We find junction resistances in porous networks to scale with nanoparticle resistivity and vary from 5 Ω for silver nanosheets to 24 GΩ for WS2 nanosheets. Moreover, our model allows junction and nanoparticle resistances to be obtained simultaneously from AC impedance spectra of semiconducting nanosheet networks. Through our model, we use the impedance data to directly link the high mobility of aligned networks of electrochemically exfoliated MoS2 nanosheets (≈ 7 cm2 V−1 s−1) to low junction resistances of ∼2.3 MΩ. Temperature-dependent impedance measurements also allow us to comprehensively investigate transport mechanisms within the network and quantitatively differentiate intra-nanosheet phonon-limited bandlike transport from inter-nanosheet hopping.

Objective: Minimally invasive surgery (MIS) is the standard approach for the staging and treatment of early-stage endometrial cancer (EC) and often includes use of a uterine manipulator. Uterine perforation is a known risk in this setting, and the impact of perforation and tumor spillage on cancer recurrence is largely unknown. The aim of this study was to assess the association between uterine perforation and/or tumor spillage at the time of MIS for low-grade, early-stage EC on disease recurrence. Methods: A retrospective single-center cohort study was conducted including patients who underwent MIS for management of low-grade and early-stage EC with use of a uterine manipulator. Rates of disease recurrence were compared between patients with and without documented uterine perforation and/or tumor spillage at the time of surgery. Statistical significance was defined as p

Inflammasome activation is a robust innate immune mechanism that promotes inflammatory responses through the release of alarmins and leaderless cytokines, including IL-1α, IL-1β, and IL-18. Various stimuli, including infectious agents and cellular stress, cause inflammasomes to assemble and activate caspase-1. Then, caspase-1 cleaves targets that lead to pore formation and leaderless cytokine activation and release. Toxoplasma gondii has been shown to promote inflammasome formation, but the cell types utilizing caspase-1 and the downstream effects on immunological outcomes during acute in vivo infection have not been explored. Here, using knockout mice, we examine the role of caspase-1 responses during acute T. gondii infection globally and in Cx3cr1-positive populations. We provide in vivo evidence that caspase-1 expression is critical for, IL-18 release, optimal interferon-γ (IFN-γ) production, monocyte and neutrophil recruitment to the site of infection, and parasite control. Specifically, we find that caspase-1 expression in Cx3cr1-positive cells drives IL-18 release, which potentiates CD4+ T cell IFN-γ production and parasite control. Notably, our Cx3cr1-Casp1 knockouts exhibited a selective T cell defect, mirroring the phenotype observed in Il18 knockouts. In further support of this finding, treatment of Cx3cr1-Casp1 knockout mice with recombinant IL-18 restored CD4+ T cell IFN-γ responses and parasite control. Additionally, we show that neutrophil recruitment is dependent on IL-1 receptor accessory protein (IL-1RAP) signaling but is dispensable for parasite control. Overall, these experiments highlight the multifaceted role of caspase-1 in multiple cell populations contributing to specific pathways that collectively contribute to caspase-1 dependent immunity to T. gondii.

Honey bee (Apis mellifera) population declines have been associated with the parasitic mite, Varroa destructor, which is currently primarily controlled by the use of acaricides. An alternative is to breed for resistance to Varroa, which was conducted in this study by bidirectional selection for mite fall to obtain colonies with low (resistant) or high (susceptible) Varroa population growth (LVG and HVG, respectively). Selection for three generations resulted in approx. 90% lower Varroa population growth in LVG than in HVG colonies. In addition, late summer Varroa infestation rates of brood and adults were both significantly lower in LVG colonies (p < 0.01), which was also significantly associated with lower Deformed Wing Virus (DWV) infection levels (p < 0.01). Survival of Varroa-parasitized bees was almost 50% higher for LVG bees compared to HVG bees (p < 0.01). Also, colony winter survivorship was significantly higher for LVG colonies than for HVG colonies (p < 0.05). However, the higher colony populations observed for LVG colonies were not significantly different from those of HVG colonies. Overall, individual and colony health was improved by selecting colonies for LVG, demonstrating its effectiveness as a means of breeding for controlling Varroa populations in honey bee colonies.

Background Adherence to pharmacotherapy and use of the correct inhaler technique are important basic principles of asthma management. Video- or remote-direct observation of therapy (v-DOT) could be a feasible approach to facilitate monitoring and supervising therapy, supporting the delivery of standard care.Objective To explore the utility and the feasibility of v-DOT to monitor inhaler technique and adherence to treatment in adults attending the asthma outpatient service in a tertiary hospital in Northern Ireland.Method The project evaluated use of the technology with 10 asthma patients. Patient and clinician feedback was obtained, in addition to measures of patient engagement and disease-specific clinical markers to assess the feasibility and utility of v-DOT technology in this group of patients.Results The engagement rate with v-DOT for participating patients averaged 78% (actual video uploads vs expected video uploads) over a median 7 week usage period. Although 50% of patients reported a technical issue at some stage during the usage period, all patients and clinicians reported that the technology was easy to use and that they were satisfied with the outcomes. A range of positive impacts were observed, including optimised inhaler technique and an observed improvement in lung function. An increase in asthma control test scores aligned with clinical aims to promote adherence and alleviate symptoms.Conclusion The v-DOT technology was shown to be a feasible method of assessing inhaler technique and monitoring adherence in this small group of adult asthma patients. A range of positive impacts for participating patients and clinicians were observed. Not all patients invited to join the project agreed to participate or engage with using the technology, highlighting that in this setting, digital modes of delivering care provide only one of the approaches in the necessary “tool kit” for clinicians and patients.

Abstract Networks of solution-processed nanomaterials are becoming increasingly important across applications in electronics, sensing and energy storage/generation. Although the physical properties of these devices are often completely dominated by network morphology, the network structure itself remains difficult to interrogate. Here, we utilise focused ion beam – scanning electron microscopy nanotomography (FIB-SEM-NT) to quantitatively characterise the morphology of printed nanostructured networks and their devices using nanometre-resolution 3D images. The influence of nanosheet/nanowire size on network structure in printed films of graphene, WS2 and silver nanosheets (AgNSs), as well as networks of silver nanowires (AgNWs), is investigated. We present a comprehensive toolkit to extract morphological characteristics including network porosity, tortuosity, specific surface area, pore dimensions and nanosheet orientation, which we link to network resistivity. By extending this technique to interrogate the structure and interfaces within printed vertical heterostacks, we demonstrate the potential of this technique for device characterisation and optimisation.

JC polyomavirus (JCPyV) infects the majority of the population and initially establishes a persistent but asymptomatic infection of the kidneys. In healthy individuals, the infection remains controlled by the host immune system, but for individuals experiencing prolonged immunosuppression, the infection can reactivate and spread to the brain, where it causes progressive multifocal leukoencephalopathy (PML), which is a fatal neurodegenerative disease. Currently, there are no approved therapies to treat PML, and affected individuals suffer rapid motor weakness and cognitive deterioration. To identify novel therapeutic treatments for JCPyV infection, receptor agonists/antagonists identified in a previously published drug screen were evaluated for their antiviral properties. Seven drugs were selected and validated using infectivity assays, and the mechanism of inhibition was further explored for G protein coupled receptor (GPCR)-associated inhibitors due to the role of the GPCR 5-hydroxytryptamine 2 receptors (5-HT2Rs) in JCPyV entry. The inhibitors cetirizine and paroxetine both reduced infection early in the JCPyV infectious cycle. Paroxetine specifically reduced viral internalization through altering the receptor density of 5-HT2CR, inhibiting β-arrestin recruitment to the receptor, and reducing MAPK signaling through ERK. These findings highlight the potential of receptor signaling and viral entry mechanisms as possible targets for antiviral drug development. Further, this research suggests that FDA-approved receptor agonists/antagonists currently used to treat other medical conditions could be repurposed into antivirals for the possible treatment of JCPyV infection and the fatal disease PML.

Abstract Background In recent years, the length of elite sporting competitions has raised concerns regarding player well-being, highlighting a need to review current match calendars. Therefore, this study aimed to explore the perceptions of elite National Rugby League (NRL) players and staff on the annual training and competition calendar from a player workload and well-being perspective. Methods This study adopted a mixed-methods approach, using a sequential explanatory design. Phase one implemented a cross-sectional survey, and phase two utilised semi-structured interviews. Four hundred and thirty-nine elite rugby league players and 46 staff completed the survey. Eighteen elite professional NRL players and six football staff were interviewed, and verbal data were analysed into pre-defined topic summaries using qualitative coding reliability methods. Topics included in-season, off-season, pre-season and well-being. Results Data analysis suggests that elite NRL players and staff believe players appear particularly comfortable with the current number of games; however, they are at their maximum capacity. Importantly, this study identified several minority groups that may require support to enhance player well-being. Players believe reducing the pre-season would negate fatigue experienced later in the subsequent season. Players and staff believe this timeframe still provides sufficient time to prepare for the upcoming season. Further, players were open to extending the off-season to 8–10 weeks and believed that extra time would allow for greater recovery from the previous season. Mid-season congested scheduling affects players following the intensified period and requires attention to alleviate fatigue. Conclusion The results of this study convey important implications for the NRL, emphasising a need to review their annual training and competitive calendar, or to implement specific strategies to enhance the well-being of minority groups. The findings from this study should be considered when discussing the ideal length and structure of the match calendar to support players’ physical and mental welfare.

A global survey of 179 restoration practitioners spanning 65 countries identified the extent of stakeholder engagement as a key factor determining the success or failure of restoration projects. Lack of support across sectors and for funding, policy, monitoring, governance and knowledge assessment of pressures and their effects were most frequently cited as factors contributing to restoration failure.The responses indicate that, although nutrient enrichment is perceived to be the primary issue for lakes globally, the impacts of climate change, hydrological modifications and invasive species are widely recognized as pervasive anthropogenic pressures of global importance. Practitioners recognized that the ecosystem services most impacted by these pressures were recreation and tourism, although in low income countries the provisioning service, aquaculture, was considered most impacted. Ecology-based and/or pressure-related restoration targets had been set for most restoration programs in our survey. However, the strength of the evidence underpinning these targets was often weak and the effects of climate change were rarely considered when setting targets.The most effective and widely used restoration measures target nutrient loading (both catchment and in-lake) while hydrological modifications and the implementation of nature-based solutions are used to a lesser extent. Measures for the control of non-native invasive species are rarely applied and are viewed as being largely ineffective.The results of the survey provide direction for future work. New and emerging pressures, singly and in combination, may require new approaches to lake restoration: for both setting restoration targets and devising restoration strategies. The future of lake restoration depends on joined-up thinking that better integrates science into policy and practice and, most importantly, ensures strong and inclusive stakeholder engagement and collaboration across multiple sectors.

In this article, I consider the interoperation of twin contemporary governmental imperatives, fostering economic growth and ensuring biopolitical security, in the face of the COVID-19 pandemic. At a theoretical level, I thereby consider the question of the applicability of a Marxist analysis vis-à-vis a Foucauldian one in understanding state responses to the pandemic. Despite the apparent prioritization of preserving life over economic activity by governments around the world in this context, I will argue that the basic problem that COVID-19 posed for the state was one of sheer unknowability and that the fundamental motive for the governmental response was a concern for security in Foucault’s sense, that is, ensuring a baseline predictability in the social field, upon which economic activity, like myriad other social activity, is premised. I argue that this drive for security motivated states to appeal to medical experts to determine the direction of their response, who in turn applied a default model of quarantine. While we cannot be certain that the medically-guided response was optimal in terms either of health outcomes or economically, I argue it served its essential purpose by providing a structured framework for social action in the face of the unknown. While this is vital for the maintenance of the basic coordinates of capitalist society, I argue it nonetheless cannot entirely be explained simply by an appeal to Marxist categories and instead requires Foucault’s insights into the medicalization of society.

Involving patients as co-leaders and co-creators in research is key to reflecting the patient’s voice in decision-making. However, co-creation of patient-centered data to inform decisions is rare, especially in early drug development where patient input is critical to prioritizing patient-relevant outcomes and endpoints for use in clinical trials. Despite the industry's growing commitment to patient centricity, most patients are excluded from sharing their expertise in research; more inclusive methods of engaging patients as research partners are needed. We describe a collaboration between a pharmaceutical company and a patient organization in co-leading and co-creating a program to understand priorities of patients and caregivers for treatment features and outcomes in sickle cell disease to inform endpoint selection in clinical development. The results of this program will be used as a basis for continued interaction between patients and the sponsor and to inform ongoing clinical development and evidence-generation activities. This case study demonstrates an approach to meaningful collaborations between patient organizations and pharmaceutical companies aimed at including the patient's voice early in the medical product lifecycle.

Phytobenthos, major primary producers in freshwater, may be periodically exposed to herbicides through runoff, spray drift, and leaching, but experimental work on their response to herbicides is limited. Outdoor flumes were used to measure the recovery of intact phytobenthic biofilms, following a short-term pulse to a glyphosate-based herbicide (GBH) and chlorotoluron (CLT), singly or as a mixture (GBHC). Two days following the removal of the herbicides, significant declines in biomass and rates of areal gross maximum photosynthesis were recorded in GBH and GBHC-treated flumes. Herbicide pulses negatively impacted the biomass of phytobenthos, measured as chlorophyll a, which in turn may have alleviated resource limitation, evidenced by significant increases in biomass-specific rates of gross maximum photosynthesis. After 4.5 days, biomass concentrations were similar in controls and treatments, indicating biofilm recovery in the GBH and GBHC biofilms, though the proportion of green algae relative to diatoms was significantly higher in GBH and GBHC-impacted biofilms. Significant increases were recorded in the Trophic Diatom Index (TDI), indicating benthic diatom development along different trajectories, following the herbicide pulse. Collectively, these results indicate greater insights into herbicide impacts on phytobenthos may be gained by working with the whole phytobenthic community.

Abstract Background There is evidence of sex differences in the physiology of endurance exercise, yet most of the advice and guidelines on training, racing, nutrition, and recovery for ultramarathons are based on research that has largely excluded female athletes. The objective was therefore to review the current knowledge of sex differences in ultramarathon runners and determine if sufficient evidence exists for providing separate guidelines for males and females. Methods This systematic review was carried out in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Three databases were searched for studies investigating differences in elite and recreational male and female ultramarathon runners. Studies were included if they compared males and females and looked at outcomes relating to the performance or health of ultramarathon runners. The quality of the included studies was determined using the Grading of Recommendations Assessment Development and Evaluation (GRADE) approach. Results The search strategy identified 45 studies that met the inclusion criteria. Most studies were observational in design, with only three papers based on randomised controlled trials. The overall quality of the evidence was low. Sex differences in the predictors of ultramarathon performance; physiological responses to training, racing, and recovery; chronic and acute health issues; and pacing strategies were found. There were areas with contradictory findings, and very few studies examined specific interventions. Conclusion The results from this review suggest that the development of sex-specific guidelines for ultramarathon coaches and athletes could have a significant effect on the performance and health of female runners. At present, there is insufficient high-quality evidence on which to formulate these guidelines, and further research is required.

Monitoring population size at ecosystem scales is difficult for most species of conservation concern. While assessing site occupancy at broad scales has proven feasible, rigorous tracking of changes in population size over time has not – even though it can provide a stronger basis for assessing population status and conservation-decision making. Therefore, we demonstrate how relatively low-intensity, ecosystem-scale passive acoustic monitoring (PAM) can be linked to local-density monitoring to estimate the population size of native California spotted owls (Strix occidentalis occidentalis) and invasive barred owls (S. varia) across the western Sierra Nevada, California. Based on a PAM sampling grid with 400 ha cells (the approximate home range size of these species), we estimated site occupancy to be between 0.42 (SE = 0.02) and 0.30 (SE = 0.02) for California spotted owls using relatively liberal and strict criteria, respectively, for considering a cell occupied. PAM-based site occupancy estimates within local-scale density monitoring study areas (range = 0.41–0.78 and 0.28–0.76 for liberal and strict criteria, respectively) were strongly and positively correlated with local density (range = 0.08–0.31 owl/km2) for this species. In contrast, ecosystem-wide site occupancy of barred owls was very low based on PAM (0.034, SE

Background: IgA vasculitis in adults has not been thoroughly studied. This has left a practice gap related to the management and follow-up of a population that is at an increased risk of comorbidities and potentially poor outcomes. For this reason, it is important to synthesize evidence from the current literature because this can help direct the movement for more robust studies to clarify best practice recommendations. Objective: We sought to create a narrative review for the practicing dermatologist when diagnosing and leading the care of IgA vasculitis in adult patients. Methods: A broad literature search was performed with a focus on articles that were published after the introduction of the most updated European Alliance of Associations for Rheumatology/Pediatric Rheumatology International Trials Organization/Pediatric Rheumatology European Society criteria. Results: The characteristics and management guidelines for IgA vasculitis in adults have been refined, although more rigorous studies are needed to develop best practice recommendations. Limitations: Because of the lack of sufficient randomized controlled trials on IgA vasculitis in adults, this narrative review is composed of mostly observational, descriptive studies. Conclusion: Adults with IgA vasculitis are at an increased risk of complicated disease course, necessitating formal diagnostic assessment and clear-cut follow-up recommendations to manage and prevent poor health outcomes related to various comorbidities.

Background Hospitals have improved stroke thrombolysis times through rigorous assessment of care delays. However, this same rigor has not yet been applied to the novel setting of mobile stroke units (MSUs). Methods We reviewed all cases of intravenous tPA (tissue‐type plasminogen activator) administration onboard our MSU in Rochester, NY, since its first complete year of operation in 2019. The dispatch timeline was divided into 6 intervals: (1) response time, (2) onboarding time, (3) head computed tomography collection, (4) head computed tomography reading, (5) decision time, and (6) intravenous tPA administration. Results The mean±SD total time between MSU dispatch and tPA was 42±8.4 minutes (range, 26–60 minutes; N=53). The largest mean duration was in (1), between dispatch and arrival (12.0±4.7 minutes). However, the intercase variation was greatest in (5), between head computed tomography reading and treatment decision (6.3±6.2 minutes). After ranking cases by total time, the mean decision times of first and fourth quartile cases varied the most, by a factor of 2.45, whereas the mean times for the other intervals varied by

Perturbations of the cardiopharyngeal mesoderm can lead to congenital defects in individuals with 22q11.2 deletion syndrome. Here the authors use single cell RNA-sequencing to identify a multilineage primed population within the mesoderm, marked by Tbx1, which has bipotent properties to form cardiac and branchiomeric muscle cells.

Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, β-glucan–binding receptor, Dectin-1. The patient’s PBMCs failed to produce TNF-α and IL-1β in response to β-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1β and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1–dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α– and IL-1β–mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi.

The use of toothbrushes and similar devices for sampling diatoms from hard surfaces is a well-established approach. Toothbrushes are routinely cleaned and reused when sampling for analysis by light microscopy. This paper looks at the scale of contamination encountered when this technique is used to sample diatoms for metabarcoding analyses, as well as at the scale of contamination to be expected if stream, rather than distilled water, is used to wash diatoms from stones. Although some contamination attributable to toothbrushes was detected, read numbers were low and had no effect on index calculation or ecological status estimates. However, if the primary focus of a study is to thoroughly document diversity in a sample, then even this small level of contamination may be unacceptable and more stringent measures may be required.

Antibody responses to SARS-CoV-2 are critical in the immune response to infection, but the potential cross-reactivity to other human corona viruses is poorly appreciated. Here the authors apply a systems based approach to characterise the antibody response in pre-pandemic cohorts and assess heterotypic reactivity to SARS-CoV-2.

Current vaccine strategies for SARS-CoV-2 focus on eliciting neutralising antibodies to the spike protein (S), but differences in immunogenicity of full-length S versus receptor binding domain (RBD) only aren’t fully understood. Here, the authors show immunogenicity of different prime-boost strategies with S and/or RBD in mice and macaques.

Longitudinal analyses are needed to show how the immune response to Sars-Cov-2 infection changes over time. Here, the authors use multiple strategies to profile the change in immune cell responses from patients with convalescent COVID-19 over the course of ~5 months, showing that although neutralizing antibody responses drop off after ~4 months, B cell immune responses strengthen.

BackgroundThe available literature on mobile stroke units (MSU) has focused on clinical outcomes, rather than operational performance. Our objective was to establish normalized metrics and to conduct a meta-analysis of the current literature on MSU performance.MethodsOur MSU in upstate New York serves 741,000 people. We present prospectively collected, retrospectively analyzed data from the inception of our MSU in October of 2018, through March of 2021. Rates of transportation/dispatch and MSU utilization were reported. We also performed a meta-analysis using MEDLINE, SCOPUS, and Cochrane Library databases, calculating rates of tPA/dispatch, tPA-per-24-operational-hours (“per day”), mechanical thrombectomy (MT)/dispatch and MT/day.ResultsOur MSU was dispatched 1,719 times in 606 days (8.5 dispatches/24-operational-hours) and transported 324 patients (18.8%) to the hospital. Intravenous tPA was administered in 64 patients (3.7% of dispatches) and the rate of tPA/day was 0.317 (95% CI 0.150–0.567). MT was performed in 24 patients (1.4% of dispatches) for a MT/day rate of 0.119 (95% CI 0.074–0.163). The MSU was in use for 38,742 minutes out of 290,760 total available minutes (13.3% utilization rate). Our meta-analysis included 14 articles. Eight studies were included in the analysis of tPA/dispatch (342/5,862) for a rate of 7.2% (95% CI 4.8–9.5%, I2 = 92%) and 11 were included in the analysis of tPA/day (1,858/4,961) for a rate of 0.358 (95% CI 0.215–0.502, I2 = 99%). Seven studies were included for MT/dispatch (102/5,335) for a rate of 2.0% (95% CI 1.2–2.8%, I2 = 67%) and MT/day (103/1,249) for a rate of 0.092 (95% CI 0.046–0.138, I2 = 91%).ConclusionsIn this single institution retrospective study and meta-analysis, we outline the following operational metrics: tPA/dispatch, tPA/day, MT/dispatch, MT/day, and utilization rate. These metrics are useful for internal and external comparison for institutions with or considering developing mobile stroke programs.

Abstract The ferret is a key animal model for investigating the pathogenicity and transmissibility of important human viruses, and for the pre‐clinical assessment of vaccines. However, relatively little is known about the ferret immune system, due in part to a paucity of ferret‐reactive reagents. In particular, T follicular helper (Tfh) cells are critical in the generation of effective humoral responses in humans, mice and other animal models but to date it has not been possible to identify Tfh in ferrets. Here, we describe the screening and development of ferret-reactive BCL6, CXCR5 and PD-1 monoclonal antibodies. We found two commercial anti-BCL6 antibodies (clone K112-91 and clone IG191E/A8) had cross-reactivity with lymph node cells from influenza-infected ferrets. We next developed two murine monoclonal antibodies against ferret CXCR5 (clone feX5-C05) and PD-1 (clone fePD-CL1) using a single B cell PCR-based method. We were able to clearly identify Tfh cells in lymph nodes from influenza infected ferrets using these antibodies. The development of ferret Tfh marker antibodies and the identification of ferret Tfh cells will assist the evaluation of vaccine-induced Tfh responses in the ferret model and the design of novel vaccines against the infection of influenza and other viruses, including SARS-CoV2.

Here, the authors apply genetic fate mapping and temporal clonal analysis to study progenitor recruitment and network morphogenesis of murine cardiac Purkinje fibers. Additionally, they characterize how transcription factor dosage regulates cell fate divergence during distinct phases of this process.

The ventricular conduction or His-Purkinje system (VCS) mediates the rapid propagation and precise delivery of electrical activity essential for the synchronization of heartbeats. Mutations in the transcription factor Nkx2-5 have been implicated in a high prevalence of developing ventricular conduction defects or arrhythmias with age. Nkx2-5 heterozygous mutant mice reproduce human phenotypes associated with a hypoplastic His-Purkinje system resulting from defective patterning of the Purkinje fiber network during development. Here, we investigated the role of Nkx2-5 in the mature VCS and the consequences of its loss on cardiac function. Neonatal deletion of Nkx2-5 in the VCS using a Cx40-CreERT2 mouse line provoked apical hypoplasia and maturation defects of the Purkinje fiber network. Genetic tracing analysis demonstrated that neonatal Cx40-positive cells fail to maintain a conductive phenotype after Nkx2-5 deletion. Moreover, we observed a progressive loss of expression of fast-conduction markers in persistent Purkinje fibers. Consequently, Nkx2-5-deleted mice developed conduction defects with progressively reduced QRS amplitude and RSR’ complex associated with higher duration. Cardiac function recorded by MRI revealed a reduction in the ejection fraction in the absence of morphological changes. With age, these mice develop a ventricular diastolic dysfunction associated with dyssynchrony and wall-motion abnormalities without indication of fibrosis. These results highlight the requirement of postnatal expression of Nkx2-5 in the maturation and maintenance of a functional Purkinje fiber network to preserve contraction synchrony and cardiac function.

COVID-19 has become a major cause of global mortality and driven massive health and economic disruptions. Mass global vaccination offers the most efficient pathway towards ending the pandemic. The development and deployment of first-generation COVID-19 vaccines, encompassing mRNA or viral vectors, has proceeded at a phenomenal pace. Going forward, nanoparticle-based vaccines which deliver SARS-CoV-2 antigens will play an increasing role in extending or improving vaccination outcomes against COVID-19. At present, over 26 nanoparticle vaccine candidates have advanced into clinical testing, with ∼60 more in pre-clinical development. Here, we discuss the emerging promise of nanotechnology in vaccine design and manufacturing to combat SARS-CoV-2, and highlight opportunities and challenges presented by these novel vaccine platforms.

Introduction and objectives: The success of direct-acting antivirals (DAA) has transformed the management of hepatitis C virus (HCV) infection and has led to the expansion of the deceased donor organ pool for liver transplantation. Material and methods: We present a single center retrospective review of liver transplantations performed on HCV-seronegative recipients from HCV-seropositive organs from 11/2017 to 05/2020. HCV nucleic acid testing (NAT) was performed on HCV-seropositive donors to assess active HCV infection. Results: 42 HCV-seronegative recipients underwent a liver transplant from a HCV-seropositive donor, including 21 NAT negative (20 liver, 1 simultaneous liver kidney transplant) and 21 NAT positive liver transplants. Two (9.5%) HCV antibody positive/NAT negative recipients developed HCV viremia and achieved sustained virologic response with DAA therapy. The remaining patients with available data (19 patients) remained polymerase chain reaction (PCR) negative at 6 months. 20 (95%) of HCV antibody positive/NAT positive recipients had a confirmed HCV viremia. 100% of patients with available data (15 patients) achieved SVR. Observed events include 1 mortality and graft loss and equivalent rates of post-transplant complications between NAT positive and NAT negative recipients. Conclusions: HCV-seropositive organs can be safely transplanted into HCV-seronegative patients with minimal complications post-transplant.

Passive acoustic monitoring (PAM) is an increasingly common method for studying populations of vocally active species. However, the detection of individuals not resident to a site may obfuscate inferences about occurrence and population change. Here, we provide a framework for distinguishing resident from non-resident individuals to estimate territory—rather than site—occupancy in PAM programs by leveraging datasets on vocal behavior and acoustic detections for spotted owls (Strix occidentalis) in the Sierra Nevada, California, USA. Based on acoustic/GPS tags, the extent over which individuals typically vocalized (the “vocal home range”) was small relative to space use, such that the likelihood of double counting resident territory-holders across multiple survey sites was low. However, comparing passive acoustic detections to known resident owl locations revealed that detections occurred at PAM survey sites known to be unoccupied, possibly because of the presence of non-territorial individuals. Strict thresholds that required acoustic detections over multiple survey periods successfully removed all detections of non-resident individuals but increased the probability of not detecting resident individuals. Conversely, relatively liberal thresholds that minimized the probability of missing resident individuals increased the probability of detecting non-residents. Thus, a tradeoff exists between error types, with optimal threshold criteria dependent on conservation objectives. Our study highlights the importance of examining patterns in individual vocal behavior and acoustic detections to minimize inferential errors in PAM. It also provides a generalizable framework that can be tailored according to specific conservation objectives to strengthen inferences from PAM as it becomes standard practice in conservation science.

Summary: In type 1 diabetes, a renewable source of human pancreatic β cells, in particular from human induced pluripotent stem cell (hiPSC) origin, would greatly benefit cell therapy. Earlier work showed that pancreatic progenitors differentiated from human embryonic stem cells in vitro can further mature to become glucose responsive following macroencapsulation and transplantation in mice. Here we took a similar approach optimizing the generation of pancreatic progenitors from hiPSCs. This work demonstrates that hiPSCs differentiated to pancreatic endoderm in vitro can be efficiently and robustly generated under large-scale conditions. The hiPSC-derived pancreatic endoderm cells (HiPECs) can further differentiate into glucose-responsive islet-like cells following macroencapsulation and in vivo implantation. The HiPECs can protect mice from streptozotocin-induced hyperglycemia and maintain normal glucose homeostasis and equilibrated plasma glucose concentrations at levels similar to the human set point. These results further validate the potential use of hiPSC-derived islet cells for application in clinical settings. : In this article, Kraus and colleagues show that human induced pluripotent stem cells can robustly and efficiently be differentiated to pancreatic endoderm in a large-scale setup. Moreover, after macroencapsulation and further in vivo maturation, hiPSC-derived pancreatic endoderm cells can give rise to glucose-responsive β cells, which protect mice from STZ-induced hyperglycemia. Keywords: β cell, diabetes mellitus, differentiation, human, stem cell, iPSC, encapsulation, therapy

Introduction: Immunization has been a remarkably successful public health intervention; however, new approaches to vaccine design are essential to counter existing and emerging infectious diseases which have defied traditional vaccination efforts to date. Nanoparticles (ordered structures with dimensions in the range of 1–1000 nm) have great potential to supplement traditional vaccines based upon pathogen subunits, or killed or attenuated microorganisms, as exemplified by the successful licensure of virus-like particle vaccines for human papillomavirus and hepatitis B. However, the immunological mechanisms that underpin the potent immunity of nanoparticle vaccines are poorly defined. Areas covered: Here, we review the immunity of nanoparticle immunization. The display of antigen in a repetitive, ordered array mimics the surface of a pathogen, as does their nanoscale size. These properties facilitate enhanced innate immune activation, improved drainage and retention in lymph nodes, stronger engagement with B cell receptors, and augmented T cell help in driving B cell activation. Expert opinion: In the near future, increasingly complex nanoparticle vaccines displaying multiple antigens and/or co-delivered adjuvants will reach clinical trials. An improved mechanistic understanding of nanoparticle vaccination will ultimately facilitate the rational design of improved vaccines for human health.