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A Professor of Medical Ethics and a theatre director, also mother and daughter, talk about health, illness, suffering, performance and practice. Using the lenses of ethical and performance theory, they explore what it means to be a patient, a spectator and a practitioner and cover many plays, texts and productions: Samuel Beckett's "Not I" and "All That Fall," Sarah Kane's Crave, Tim Crouch's "An Oak Tree," Enda Walsh's "Ballyturk," Annie Ryan's adaptation of Eimear McBride's novel "A Girl Is a Half-Formed Thing," Duncan MacMillan's "People, Place and Things" and Henrik Ibsen's "Hedda Gabler." These were selected because first we have seen, studied or worked with each and they have continued to inspire us. Second, they offer rich and revealing insights into the ways in which meaning(s) is/are both negotiated and contested in relation to health and illness. It is the iterative negotiation of meaning(s) that, it is argued, is the essence of narrative practice, be it in medicine or in the theatre. The difference and divergence of perception, response and interpretation to dramatic performance can test relationships, be they professional, creative or familial. Yet, the capacity to understand, and embrace, disagreement and uncertainty is vital; fundamental to a flourishing life. For it is by recognising our part in creating narratives, broken and otherwise, that we can begin to recognise the necessary interactionism and humanity of both medicine and theatre.

Abstract Freezing of gait (FOG) in Parkinson’s disease (PD) can be triggered by sensomotor, cognitive or limbic factors. The limbic system’s impact on FOG is attributed to elevated limbic load, characterized by aversive stimuli, potentially depleting cognitive resources for movement control, resulting in FOG episodes. However, to date, PD patients with and without FOG have not shown alterations of anticipatory postural adjustments during gait initiation after exposure to emotional images, possibly because visual stimuli are less immediately disruptive than auditory stimuli, which can more directly affect attention and the limbic system. This study aims to determine if step initiation is influenced by ecological auditory stimuli with emotional content in patients with FOG compared to those without. Forty-five participants, divided into 3 groups (15 PD with FOG, 15 PD without FOG in ON state, and 15 healthy subjects), stood on a force platform and were asked to step forward in response to neutral, pleasant, or unpleasant ecological auditory stimuli. Anticipatory postural adjustments were investigated in imbalance and unloading phases, while spatio-temporal parameters, including center of pressure (CoP) displacements, were computed for step initiation. PD with FOG showed a reduction of CoP displacements after listening to unpleasant stimuli. Conversely, pleasant stimuli facilitated CoP displacements in these subjects. No influence of affective stimuli on CoP displacements was found in the other two groups. Multiple regression analysis revealed that the behavioral pattern in PD with FOG, modulated by stimuli with affective valence, was mainly associated with the limbic area (i.e., depression). The findings showed that the emotional network plays a crucial role in the pathophysiology of freezing, generating probably interference with attentional reserves that trigger FOG.

Abstract Rice (Oryza sativa L.) producers in the Mid-south are experiencing difficulties with herbicide-resistant weeds such as barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.]. As a result, methods that can alter E. crus-galli ecology are needed. This research evaluated the ecological implications of rice cultivar and row widths on crop and E. crus-galli growth. Overall, for E. crus-galli, as the row width increased, greater density, panicle counts, and seed production occurred. Echinochloa crus-galli density was 120% greater in a 38-cm row width than the 13-cm row width at the preflood rice stage. Reduced early-season rice canopy coverage in the wider row widths allowed for increased E. crus-galli densities. At the preharvest stage, E. crus-galli panicle counts were similar for the 13- and 19-cm rows. Row width did not affect rice yield, indicating wider row widths could be feasible agronomically, but additional weed management efforts would be needed because greater ecological advantages were obtained in narrower rows. Less E. crus-galli seed production occurred in competition with hybrid cultivars compared to inbred cultivars. Overall, the standard row width (19-cm) and hybrid cultivars would provide the greatest ecological advantage over E. crus-galli.

Abstract Oral fluids provide ready detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to evaluate relationships between oral virus, oral and systemic anti-SARS-CoV-2-specific antibodies, and symptoms. Oral fluids (saliva/throat wash (saliva/TW)) and serum were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+ human participants (n = 45). SARS-CoV-2 RT-qPCR and N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR for subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA and ELISA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. At time of enrollment (baseline, BL), LFA-detected N-antigen in 86% of TW and was immunoblot-confirmed. However, only 3/17 were saliva/TW qPCR+ . Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three anti-spike sero-negative participants suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19–29aa, RMSD 1–1.5 Angstroms). At enrollment, symptomatic participants demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (63%/54%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral and serum IgG correlated 100% with NP+ PCR status. Cough and fatigue severity (p = 0.010 and 0.018 respectively), and presence of weakness, nausea, and composite upper respiratory symptoms (p = 0.037, 0.005, and 0.017, respectively) were negatively associated with saliva IgM but not TW or serum IgM. Throat wash IgM levels were higher in women compared to men, although the association did not reach statistical significance (median: 290 (female) versus 0.697, p = 0.056). Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms and early oral IgM responses during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.

Background Entity resolution (ER) is the process of identifying and linking records that refer to the same real-world entity. ER is a fundamental challenge in data science, and a common barrier to ER research and development is that the data fields used for this fuzzy matching are personally identifiable information, such as name, address, and date of birth. The necessary restrictions on accessing and sharing these authentic data have slowed the work in developing, testing, and adopting new methods and software for ER. We recently released pseudopeople, a Python package that allows users to generate simulated datasets with configurable noise approaching the scale and complexity of the data on which large organizations and federal agencies, like the US Census Bureau regularly perform ER. With pseudopeople, researchers can develop new algorithms and software for ER of US population data without needing access to personal and confidential information. Methods We created the simulated population data available for noising with pseudopeople using our Vivarium simulation platform. Our model simulates individuals and their families, households, and employment dynamics over time, which we observe through simulated censuses, surveys, and administrative data collection systems. Results Our simulation process produced over 900 gigabytes of simulated censuses, surveys, and administrative data for pseudopeople, representing hundreds of millions of simulants. A sample simulated population of thousands of simulants is now openly available to all users of the pseudopeople package, and large-scale simulated populations of millions and hundreds of millions of simulants are also available by online request through GitHub. These simulated population data are structured for use by the pseudopeople package, which includes additional affordances to add various kinds of noise to the data to provide realistic, sharable challenges for ER researchers.

Abstract Lymphedema is a progressive lymphatic disease that potentiates physical and psychosocial distress. Despite its impact, patients reportedly encounter lymphatic ignorance throughout the healthcare system. This cross-sectional study aims to summarize clinical characteristics and interactions of lymphedema patients within the healthcare system. Two lymphedema patient cohorts were included: The Global Registry Analysis Cohort included lymphedema patients who contributed to an international digital lymphatic registry and the Interactions Cohort included patients who initiated a questionnaire about interactions with the medical system. The global registry was used to obtain demographic and clinical characteristics from affiliated lymphedema patients. A 23-item online questionnaire on healthcare experiences and satisfaction with lymphatic healthcare was then distributed to the Interactions Cohort. Complete responses were obtained from 2474 participants. Participants were a mean age of 57.5 ± 16.1 years and 51.4% had a cancer history. Participants reported substantial delays in diagnosis and treatment. Cancer-related and non-cancer-related lymphedema patients reported similar levels of perceived physician disinterest in their lymphedema; however, non-cancer-related lymphedema patients reported more care dissatisfaction. Ultimately, patients continue to face delays in lymphedema diagnosis and treatment. We developed an evidence-based model highlighting areas of reform needed to improve lymphatic education and healthcare.

Background: The United States is increasingly recognizing period product insecurity, insufficient access to menstrual products and limited private spaces for managing menstruation due to financial constraints, as an issue impacting the well-being and dignity of Americans. One strategy to address period product insecurity has been distributing free period products via period supply banks. The outcomes of period product distribution outside the school setting are absent from the literature. Objectives: This study, a formative evaluation of the free period product distribution efforts of the Alliance for Period Supplies, aims to identify (1) characteristics of individuals receiving products from period supply banks and their experiences of period product insecurity and (2) health and social outcomes experienced by recipients of free period products. Design: Survey data collection occurred at two points: baseline and one-year follow-up. All study participants provided verbal consent. Methods: Between Fall 2018 and Spring 2020, 1863 baseline and 80 follow-up surveys were administered. Participants received free period products for themselves and/or a household member from one of 20 participating Alliance for Period Supplies period supply banks directly or from one of their 64 partner agencies. Results: At baseline, 72.4% of participants had to choose between buying period products and another basic need. One year after accessing a period supply bank, 36.3% of participants reported this experience ( p = 0.018). Participants reported at baseline, on average, 7.8 days in the past year of avoiding seeing others, canceling appointments, or skipping work or school because they did not have access to period products. At follow-up, this was reduced to 1.2 days, on average, t (68) = 2.214, p

Abstract This study evaluated the tolerability and efficacy of the topical rho-kinase inhibitor netarsudil for canine primary corneal endothelial degeneration (PCED). Twenty-six eyes of 21 client-owned dogs with PCED were enrolled in a prospective, randomized, vehicle control clinical trial and received topical netarsudil 0.02% (Rhopressa®) or vehicle control twice daily (BID) for the first 4 months. Then, all patients received netarsudil for the next 4 or 8 months. Complete ophthalmic examination, ultrasonic pachymetry, Fourier-domain optical coherence tomography, and in vivo confocal microscopy were performed at baseline and 1, 2, 4, 6, 8 and 12 months. Effect of netarsudil on central corneal thickness (CCT), percentage of cornea with edema, and endothelial cell density (ECD) were evaluated by repeated measures ANOVA. Kaplan–Meier curves and log-rank test were used to compare corneal edema and clinical progression of eyes in netarsudil versus vehicle control groups. All dogs developed conjunctival hyperemia in at least one eye while receiving netarsudil. Unilateral transient reticulated intraepithelial bullae and stromal hemorrhage were observed respectively in 2 dogs in the netarsudil group. Two dogs showed persistently decreased tear production while receiving netarsudil, requiring topical immunomodulatory treatment. No significant differences in CCT, ECD, corneal edema or clinical progression were observed between netarsudil or vehicle treated eyes. When comparing efficacy of topical netarsudil BID and topical ripasudil 0.4% administered four times daily from our previous study, dogs receiving ripasudil had significantly less progression than those receiving netarsudil.

Background: Period products like pads, tampons, and a variety of disposable and reusable hygiene supplies constitute just one facet essential for effectively managing menstruation in a way that allows an individual to feel comfortable carrying out their daily routine. Given the absence of comprehensive public policies ensuring access to period products for economically disadvantaged menstruating individuals, community-based basic needs banks, particularly period supply banks, were established to alleviate the challenges faced by those experiencing period product insecurity. These initiatives specifically aim to furnish essential menstrual products to individuals in need. Objectives: This study aimed to assess any organizational benefits experienced by community-based organizations distributing period products on behalf of period supply banks. Design: This cross-sectional study is a formative evaluation of the distribution of free period products by period supply banks, which provide access to essential menstrual hygiene products for individuals in need through partnership with community-based partner agencies. Methods: Staff members at agencies distributing period products were invited to complete an anonymous web-based survey at two different time points about their agency’s experiences distributing free period products. All participants provided informed consent via the web-based survey. Results: Agency staff reported that since they started providing period products, clients were more likely to: start a conversation about other needs (66.7%), extend the length of their relationship with the agency (60.0%), keep scheduled appointments (62.1%), ask for assistance with another need (75.0%), communicate between visits (42.4%), participate in other agency programming (55.9%), and seek other agency services (73.5%). Due to a shortage of period products, 41% of agencies have had to turn away clients needing period supplies. Conclusion: Period supply banks, in collaboration with community-based partner agencies, enhance access to both menstrual products and the associated support, fostering engagement with diverse resources and opportunities among recipients. However, addressing unmet needs necessitates policies and additional funding to ensure universal access to essential hygiene supplies for everyone to thrive and actively participate in society.

Abstract Objective Clinician-scientists are critical to medical innovation and research. However, the number of clinician scientists in the UK has been declining steadily over the last decade. One of the cited reasons is poor student recruitment to academic training pathways. The SMART study aims to assess current student perceptions on research and identify key factors influencing whether a student is interested in research. Design We conducted a cross-sectional survey study between January and May 2022. Setting This was a multi-centre national study with data collected across 40 universities offering medical courses in the UK. Participants Participants were UK medical students enrolled in medicine for 21/22 academic year. Main outcome and measure The main outcomes were related to participant perceptions on research and whether they were interested in engaging with research in their future career. These measures were correlated with demographic and non-demographic details using regression analyses. Results One thousand seven hundred seventy-four individuals participated in the SMART survey from 40 medical schools. Nearly half the participants felt there were barriers preventing them from doing research (46.67%) and almost three-quarters felt it was at least somewhat difficult to combine research with medical school (73.49%). Of the options available, most commonly students did not want to pursue an academic career (43.11%) or training pathway (42.49%). However, most participants felt it was useful to do research at medical school (59.54%) and were also interested in doing more research in the future (69.16%). Regression analysis identified many factors influencing student’s perceptions of research including year of study, gender, socioeconomic status, family background, research exposure at medical school, ethnicity, and country of pre-university education. Conclusions The SMART study is the first of its kind in the UK, shedding light on medical student perceptions. While some express strong interest in academic careers, a larger proportion show a broader interest in research. Demographic factors like gender, parental occupation, and socioeconomic status play a role. Further exploration is needed for specific groups to address barriers, promote research, and boost academic pathway recruitment.

Abstract Commercial citrus trees are composed of a scion grafted onto a rootstock. Because grafting is one of the most expensive methods of plant propagation, grafting efficiency is of large practical importance. The purpose of this study was to improve citrus bud-grafting efficiency. The effects of six factors that included BA, Tween-20, DMSO, type of solvent (water or EtOH), cardinal orientation of grafted bud, and type of supplemental light (LED, metal halide, none) on forty-four bud-grafting measures were determined using a multifactor design of experiment approach. Four measures useful for identifying treatments of practical value included the number of rootstock axial buds that formed shoots, the percentage of grafted buds that formed shoots, the length of the longest shoot formed from the grafted buds, and the total leaf area of the grafted bud shoots. The factors that most affected these responses were no supplemental light to minimize the number of shoots from rootstock axial buds, a south orientation and 5 mM BA to maximize the percentage of grafted buds that formed shoots, a north orientation and 5 mM BA to maximize the length of the longest grafted bud shoot, and 5 mM BA to maximize the leaf area of the grafted bud shoots.

The rise of mRNA as a novel vaccination strategy presents new opportunities to confront global disease. Double-stranded RNA (dsRNA) is an impurity byproduct of the in vitro transcription reaction used to manufacture mRNA that may affect the potency and safety of the mRNA vaccine in patients. Careful quantitation of dsRNA during manufacturing is critical to ensure that residual dsRNA is minimized in purified mRNA drug substances. In this work, we describe the development and implementation of a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) to quantitate nanogram quantities of residual dsRNA contaminants in mRNA process intermediates using readily available commercial reagents. This sandwich ELISA developed in this study follows a standard protocol and can be easily adapted to most research laboratory environments. Additionally, a liquid handler coupled with an automated robotics system was utilized to increase assay throughput, improve precision, and reduce the analyst time requirement. The final automated sandwich ELISA was able to measure

Abstract The compound 2,4-diacetylphloroglucinol (DAPG) is a broad-spectrum antibiotic that is primarily produced by Pseudomonas spp. DAPG plays an important role in the biocontrol disease suppressing activity of Pseudomonas spp. In the current study, we report the discovery of the DAPG biosynthetic cluster in strains of Chromobacterium vaccinii isolated from Brazilian aquatic environments and the distribution of the biosynthetic cluster in the Chromobacterium genus. Phylogenetic analysis of the phlD protein suggests the biosynthetic cluster probably entered the genus of Chromobacterium after a horizontal gene transfer event with a member of the Pseudomonas fluorescens group. We were able to detect trace amounts of DAPG in wild type cultures and confirm the function of the cluster with heterologous expression in Escherichia coli. In addition, we identified and verified the presence of other secondary metabolites in these strains. We also confirmed the ability of C. vaccinii strains to produce bioactive pigment violacein and bioactive cyclic depsipeptide FR900359. Both compounds have been reported to have antimicrobial and insecticidal activities. These compounds suggest strains of C. vaccinii should be further explored for their potential as biocontrol agents.

Gliding is only present in six extant groups of mammals—interestingly, despite divergent evolutionary histories, all mammalian gliders are strictly nocturnal. Gliding mammals also seem to have relatively high rates of ultrasound use and ultraviolet-induced photoluminescence (UVP) in contrast with their close relatives. Therefore, we hypothesized that, despite diverging lineages, gliding mammals use similar modes of cryptic communication compared to their non-gliding counterparts. We developed two datasets containing the vocal range (minimum-maximum of the dominant harmonic; kHz) and UVP of 73 and 82 species, respectively; we report four novel vocal repertoires and 57 novel observations of the presence or absence of UVP. We complemented these datasets with information about body size, diel activity patterns, habitat openness, and sociality to explore possible covariates related to vocal production and UVP. We found that the maximum of the dominant harmonic was significant higher in gliding mammals when vocalizing than their non-gliding relatives. Additionally, we found that nocturnality was the only significant predictor of UVP, consistent with the previous hypothesis that luminophores primarily drive UVP in mammal fur. In contrast, however, we did not find UVP ubiquitous in nocturnal mammals, suggesting that some unknown process may contribute to variation in this trait.

Abstract Background and Aims Patients with end stage kidney disease on hemodialysis are vulnerable to SARS‐CoV‐2 infection. Current guidelines recommend boosters of SARS‐CoV‐2 mRNA‐based vaccines. The long‐term humoral response of hemodialysis patients infected with SARS‐CoV‐2 after receiving a booster of SARS‐CoV‐2 mRNA‐based vaccines has been incompletely characterized. Here, we determined the long‐term humoral response of hemodialysis patients to two and three doses of the Pfizer BioNTech (BNT162b2) mRNA SARS‐CoV‐2 vaccine and investigated the effect of postbooster SARS‐CoV‐2 infection on antibody levels over time. Methods Samples were collected on a monthly basis and tested for anti‐SARS‐CoV‐2 antibodies against anti‐spike S1 domain. Thirty‐five hemodialysis patients were enrolled in the original study and 27 of these received a booster. Patients were followed up to 6 months after the first two doses and an additional 7 months after the third BNT162b2 dose. Results are presented as the internationally harmonized binding antibody units (BAU/mL). Results Antibody level significantly increased from prebooster to 2 weeks postbooster, with a median [25th, 75th percentile] rise from 52.72 [28.55, 184.7] to 6216 [3806, 11,730] BAU/mL in the total population. Of patients with a negative or borderline detectable antibody level 6 months after vaccination who received a third dose, 89% developed positive antibody levels 2 weeks postbooster. Postbooster antibody levels declined an average rate of 29% per month in infection‐naïve patients. Antibody levels spiked in patients infected with SARS‐CoV‐2 after receiving a booster but declined rapidly. No patients infected postbooster required hospitalization. Conclusions A third dose of BNT162b2 restores antibody levels to high levels in dialysis patients but levels decline over time. A third dose did not necessarily prevent infection, but no patients suffered severe infection or required hospitalization. SARS‐CoV‐2 recovered patients appear to have a blunted rise in antibody levels after a third dose. Although patients infected with SARS‐CoV‐2 postbooster had an immediate spike in antibody levels, these declined over time.

BackgroundTo date, there has been no medication that has prevented the progression of Parkinson's disease (PD). Many benefits of intensive and multidisciplinary rehabilitation program for PD are supported by clinical, epidemiological, and experimental data. The main question is whether high-intensity motor and cognitive exercises have an effect on the disease's biological mechanisms.ObjectiveThis study protocol is a Randomized Controlled Trial (RCT) designed to determine the efficacy of an experimental, intensive, and multidisciplinary treatment in comparison to a home-based self-treatment in improving biomolecular and functional parameters in PD.MethodsA total of 72 participants will be randomly allocated to two different groups, experimental (n = 36) and control group (n = 36). The rehabilitation program will last 6 consecutive weeks and will involve the execution of a total of 30 sessions, one for each day of the week from Monday to Friday. Participants allocated to the control group will carry out a home-based self-treatment program that includes muscle-stretching and active mobilization exercises for 40'/day for 6 consecutive weeks. The primary outcome measure is the effects of both treatments on a new set of molecular biomarkers such as oligomeric alpha-synuclein and neurotrophic factors measured in peripheral neural derived extracellular vesicles (NDEVs). Secondary outcomes will include changes of motor and non-motor symptoms, balance and gait performance and cognitive functioning. This RCT has been registered as "Intensive Multidisciplinary Rehabilitation and Biomarkers in Parkinson's Disease" on 30 May, 2022 to ClinicalTrials.gov with the Study ID number: NCT05452655.DiscussionThis rehabilitation program is believed to be crucial in modifying biomolecular and functional parameters in people with PD. We expect that this study will provide additional evidence to understand the impact of an aerobic and intensive rehabilitation program on brain plasticity in patients with PD.

Environmental DNA (eDNA) is an increasingly useful method for detecting pelagic animals in the ocean but typically requires large water volumes to sample diverse assemblages. Ship-based pelagic sampling programs that could implement eDNA methods generally have restrictive water budgets. Studies that quantify how eDNA methods perform on low water volumes in the ocean are limited, especially in deep-sea habitats with low animal biomass and poorly described species assemblages. Using 12S rRNA and COI gene primers, we quantified assemblages comprised of micronekton, coastal forage fishes, and zooplankton from low volume eDNA seawater samples (n = 436, 380-1800 mL) collected at depths of 0-2200 m in the southern California Current. We compared diversity in eDNA samples to concurrently collected pelagic trawl samples (n = 27), detecting a higher diversity of vertebrate and invertebrate groups in the eDNA samples. Differences in assemblage composition could be explained by variability in size-selectivity among methods and DNA primer suitability across taxonomic groups. The number of reads and amplicon sequences variants (ASVs) did not vary substantially among shallow (600 m), but the proportion of invertebrate ASVs that could be assigned a species-level identification decreased with sampling depth. Using hierarchical clustering, we resolved horizontal and vertical variability in marine animal assemblages from samples characterized by a relatively low diversity of ecologically important species. Low volume eDNA samples will quantify greater taxonomic diversity as reference libraries, especially for deep-dwelling invertebrate species, continue to expand.

Sustainable crop production requires adequate and efficient management practices to reduce the negative environmental impacts of excessive nitrogen (N) fertilization. Remote sensing has gained traction as a low-cost and time-efficient tool for monitoring and managing cropping systems. In this study, vegetation indices (VIs) obtained from an unmanned aerial vehicle (UAV) were used to detect corn (Zea mays L.) response to varying N rates (ranging from 0 to 208 kg N ha-1) and fertilizer application methods (liquid urea ammonium nitrate (UAN), urea side-dressing and slow-release fertilizer). Four VIs were evaluated at three different growth stages of corn (V6, R3, and physiological maturity) along with morphological traits including plant height and leaf chlorophyll content (SPAD) to determine their predictive capability for corn yield. Our results show no differences in grain yield (average 13.2 Mg ha-1) between furrow-applied slow-release fertilizer at ≥156 kg N ha-1 and 208 kg N ha-1 side-dressed urea. Early season remote-sensed VIs and morphological data collected at V6 were least effective for grain yield prediction. Moreover, multivariate grain yield prediction was more accurate than univariate. Late-season measurements at the R3 and mature growth stages using a combination of normalized difference vegetation index (NDVI) and green normalized difference vegetation index (GNDVI) in a multilinear regression model showed effective prediction for corn yield. Additionally, a combination of NDVI and normalized difference red edge index (NDRE) in a multi-exponential regression model also demonstrated good prediction capabilities.

People with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grown Pseudomonas aeruginosa biofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models.

Thioredoxin-interacting protein (TXNIP) has emerged as a key player in cancer and diabetes since it targets thioredoxin (TRX)-mediated redox regulation and glucose transporter (GLUT)-mediated metabolism. TXNIP consists of two arrestin (ARR, N-ARR and C-ARR) domains at its amino-terminus and two PPxY (PY) motifs and a di-leucine (LL) motif for endocytosis at its carboxyl-terminus. Here, we report that TXNIP shuffles between TRX and GLUTs to regulate homeostasis of intracellular oxidative stress and glucose metabolism. While TXNIP functions as a gatekeeper of TRX by default, it robustly interacted with class I GLUTs through its C-ARR domain upon increase of intracellular reactive oxygen species. This interaction prompted the surface expression downregulation and lysosomal degradation of GLUTs by its carboxyl-terminal LL endocytic signaling motif to attenuate glucose uptake. Consequently, TXNIP expression significantly limited glucose uptake, leading to the suppression of glycolysis, hexosamine biosynthesis, and the pentose phosphate pathway. Our findings establish a fundamental link between ROS and glucose metabolism through TXNIP and provide a promising target for the drug development against GLUT-related metabolic disorders.

Abstract Background MLH1 epimutation is characterised by constitutional monoallelic MLH1 promoter hypermethylation, which can cause colorectal cancer (CRC). Tumour molecular profiles of MLH1 epimutation CRCs were used to classify germline MLH1 promoter variants of uncertain significance and MLH1 methylated early-onset CRCs (EOCRCs). Genome-wide DNA methylation and somatic mutational profiles of tumours from two germline MLH1: c.-11C > T and one MLH1: c.-[28A > G; 7C > T] carriers and three MLH1 methylated EOCRCs ( T carriers and MLH1 methylated EOCRCs clustered with the constitutional MLH1 epimutation CRCs but not with the sporadic MLH1 methylated CRCs. Furthermore, monoallelic MLH1 methylation and APC promoter hypermethylation in tumour were observed in both MLH1 epimutation and germline MLH1: c.-11C > T carriers and MLH1 methylated EOCRCs. Mosaic constitutional MLH1 methylation in MLH1: c.-11C > T carriers and 1 of 3 MLH1 methylated EOCRCs was identified by methylation-sensitive ddPCR. Conclusions Mosaic MLH1 epimutation underlies the CRC aetiology in MLH1: c.-11C > T germline carriers and a subset of MLH1 methylated EOCRCs. Tumour profiling and ultra-sensitive ddPCR methylation testing can be used to identify mosaic MLH1 epimutation carriers.

Abstract Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

Summary: Background: Autochthonous transmission of Zika virus (ZIKV) has been reported in 87 countries since 2015. Although most infections are mild, there is risk of Guillain-Barré syndrome and adverse pregnancy outcomes. Vaccines are urgently needed to prevent Zika, but sufficient understanding of humoral responses and tools to assess ZIKV-specific immunity are lacking. Methods: We developed a blockade-of-binding (BOB) ELISA using A9E and G9E, two strongly neutralising ZIKV-specific monoclonal antibodies, which do not react with dengue virus. Receiver operating characteristic curve analysis assessed A9E and G9E BOB serodiagnostic performance. BOB was then applied to samples from a surveillance cohort in Risaralda, Colombia, and phase 1 ZIKV vaccine trial samples, comparing results against traditional serologic tests. Findings: In the validation sample set (n = 120), A9E BOB has a sensitivity of 93.5% (95% CI: 79.3, 98.9) and specificity 97.8 (95% CI: 92.2, 99.6). G9E BOB had a sensitivity of 100% (95% CI: 89.0, 100.0) and specificity 100% (95% CI: 95.9, 100). Serum from natural infections consistently tested positive in these assays for up to one year, and reactivity tracks well with ZIKV infection status among sera from endemic areas with complicated flavivirus exposures. Interestingly, a leading ZIKV vaccine candidate elicited minimal BOB reactivity despite generating neutralising antibody responses. Interpretation: In conclusion, A9E and G9E BOB assays are sensitive and specific assays for detecting antibodies elicited by recent or remote ZIKV infections. Given the additional ability of these BOB assays to detect immune responses that target different epitopes, further development of these assays is well justified for applications including flavivirus surveillance, translational vaccinology research and as potential serologic correlates of protective immunity against Zika. Funding: R21 AI129532 (PI: S. Becker-Dreps), CDC BAA 2017-N-18041 (PI: A. M. de Silva), Thrasher Fund (PI: M. H. Collins), K22 AI137306 (PI: M. H. Collins).

It has been recognized that well-connected networks of protected areas are needed to halt the continued loss of global biodiversity. The recently signed Kunming-Montreal biodiversity agreement commits countries to protecting 30% of terrestrial lands in well-connected networks of protected areas by 2030. To meet these ambitious targets, land-use planners and conservation practitioners will require tools to identify areas important for connectivity and track future changes. In this study we present methods using circuit theoretic models with a subset of sentinel park nodes to evaluate connectivity for a protected areas network. We assigned a lower cost to natural areas within protected areas, under the assumption that animal movement within parks should be less costly given the regulation of activities. We found that by using mean pairwise effective resistance (MPER) as an indicator of overall network connectivity, we were able to detect changes in a parks network in response to simulated land-use changes. As expected, MPER increased with the addition of high-cost developments and decreased with the addition of new, low-cost protected areas. We tested our sentinel node method by evaluating connectivity for the protected area network in the province of Ontario, Canada. We also calculated a node isolation index, which highlighted differences in protected area connectivity between the north and the south of the province. Our method can help provide protected areas ecologists and planners with baseline estimates of connectivity for a given protected area network and an indicator that can be used to track changes in connectivity in the future.

Abstract Colonialism has disrupted Indigenous socioecological systems around the globe, including those supported by intentional landscape burning. Because most disruptions happened centuries ago, our understanding of Indigenous fire management is largely inferential and open to debate. Here, we investigate the ecological consequences of the loss of traditional Aboriginal fire management on fire-exposed savannas on the Arnhem Plateau, northern Australia, using the fire-sensitive conifer Callitris intratropica as a bio-indicator. We contrast Kakadu National Park, where traditional Aboriginal fire management was severely disrupted during the early twentieth century following Aboriginal relocation to surrounding settlements, and an adjacent Aboriginal estate where traditional Aboriginal fire management endures. Since 2006, traditional Aboriginal fire management at this site has been overlaid by a program of broad-scale institutionalized burning in the early dry season, designed to reduce greenhouse emissions. Using remote sensing, field survey, and dendrochronology, we show that on the Aboriginal estate, C. intratropica populations depend on the creation of a shifting patch mosaic of long unburned areas necessary for the recruitment of C. intratropica. However, the imposition of broad-scale fire management is disrupting this population patch dynamic. In Kakadu, there have been extreme declines of C. intratropica associated with widespread fires since the mid twentieth century and consequent proliferation of grass fuels. Fire management in Kakadu since 2007, designed to increase the size and abundance of patches of unburned vegetation, has not been able to reverse the population collapse of C. intratropica. Our study demonstrates that colonial processes including relocation of Indigenous people and institutional fire management can have deleterious consequences that are nearly irreversible because of hysteresis in C. intratropica population dynamics.

Abstract The regulated translocation of the glucose transporter, GLUT4, to the surface of adipocytes and muscle is a key action of insulin. This is underpinned by the delivery and fusion of GLUT4-containing vesicles with the plasma membrane. Recent studies have revealed that a further action of insulin is to mediate the dispersal of GLUT4 molecules away from the site of GLUT4 vesicle fusion with the plasma membrane. Although shown in adipocytes, whether insulin-stimulated dispersal occurs in other cells and/or is exhibited by other proteins remains a matter of debate. Here we show that insulin stimulates GLUT4 dispersal in the plasma membrane of adipocytes, induced pluripotent stem cell-derived cardiomyocytes and HeLa cells, suggesting that this phenomenon is specific to GLUT4 expressed in all cell types. By contrast, insulin-stimulated dispersal of TfR was not observed in HeLa cells, suggesting that the mechanism may be unique to GLUT4. Consistent with dispersal being an important physiological mechanism, we observed that insulin-stimulated GLUT4 dispersal is reduced under conditions of insulin resistance. Adipocytes of different sizes have been shown to exhibit distinct metabolic properties: larger adipocytes exhibit reduced insulin-stimulated glucose transport compared to smaller cells. Here we show that both GLUT4 delivery to the plasma membrane and GLUT4 dispersal are reduced in larger adipocytes, supporting the hypothesis that larger adipocytes are refractory to insulin challenge compared to their smaller counterparts, even within a supposedly homogeneous population of cells.

Abstract Background Squamous cell carcinoma (SqCC) is a subtype of non-small cell lung cancer for which patient prognosis remains poor. The extracellular matrix (ECM) is critical in regulating cell behavior; however, its importance in tumor aggressiveness remains to be comprehensively characterized. Methods Multi-omics data of SqCC human tumor specimens was combined to characterize ECM features associated with initiation and recurrence. Penalized logistic regression was used to define a matrix risk signature for SqCC tumors and its performance across a panel of tumor types and in SqCC premalignant lesions was evaluated. Consensus clustering was used to define prognostic matreotypes for SqCC tumors. Matreotype-specific tumor biology was defined by integration of bulk RNAseq with scRNAseq data, cell type deconvolution, analysis of ligand-receptor interactions and enriched biological pathways, and through cross comparison of matreotype expression profiles with aging and idiopathic pulmonary fibrosis lung profiles. Results This analysis revealed subtype-specific ECM signatures associated with tumor initiation that were predictive of premalignant progression. We identified an ECM-enriched tumor subtype associated with the poorest prognosis. In silico analysis indicates that matrix remodeling programs differentially activate intracellular signaling in tumor and stromal cells to reinforce matrix remodeling associated with resistance and progression. The matrix subtype with the poorest prognosis resembles ECM remodeling in idiopathic pulmonary fibrosis and may represent a field of cancerization associated with elevated cancer risk. Conclusions Collectively, this analysis defines matrix-driven features of poor prognosis to inform precision medicine prevention and treatment strategies towards improving SqCC patient outcome.

Abstract The United States’ swine industry is under constant threat of foreign animal diseases, which may emerge without warning due to the globalized transportation networks moving people, animals, and products. Therefore, having disease control and elimination protocols in place prior to pathogen introduction is paramount for business continuity and economic recovery. During extraordinary circumstances, it may become necessary to depopulate large populations of animals, including swine, as a disease containment measure. Currently approved depopulation methods for swine present significant logistical challenges when scaled to large populations or performed in field conditions. In the United States, water-based foam is currently approved for poultry depopulation, and recent field studies demonstrate water-based foam is an effective depopulation alternative for swine. While effective, the speed at which water-based foam induces loss of consciousness prior to death, a major welfare consideration, has not been adequately investigated. In this study, 12 nursery pigs were terminated using water-based medium-expansion foam to quantify the time to induce loss of consciousness and ultimately brain death. Each pig was implanted with subdermal electrodes to capture electroencephalographic data, placed in a body sling, and suspended in a plastic bulk container that was subsequently filled with water-based foam. Electroencephalographic data was recorded for 15 min, during which the pigs remained immersed in the water-based foam. Conservatively, average (± SD) time to unconsciousness and brain death was 1 min, 53 s ± 36 s and 3 min, 3 s ± 56 s, respectively. The relatively rapid loss of consciousness compared to other methods limits the amount of distress and is overall a positive finding for the welfare of the pigs that might be depopulated with water-based foam. The findings of this study add additional evidence supporting the use of water-based medium-expansion foam for an emergency depopulation of swine.

Reducing zoonotic influenza A virus (IAV) risk in the United States necessitates mitigation of IAV in exhibition swine. We evaluated the effectiveness of shortening swine exhibitions to

RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematologic malignancies. We previously reported that coexpression of Sf3b1 mutation and Atm deletion in B cells, but not either lesion alone, leads to the onset of chronic lymphocytic leukemia (CLL) with CLL cells harboring chromosome amplification. However, the exact role of Sf3b1 mutation and Atm deletion in chromosomal instability (CIN) remains unclear. Here, we demonstrated that SF3B1 mutation promotes centromeric R-loop (cen-R-loop) accumulation, leading to increased chromosome oscillation, impaired chromosome segregation, altered spindle architecture, and aneuploidy, which could be alleviated by removal of cen-R-loop and exaggerated by deletion of ATM. Aberrant splicing of key genes involved in R-loop processing underlay augmentation of cen-R-loop, as overexpression of the normal isoform, but not the altered form, mitigated mitotic stress in SF3B1-mutant cells. Our study identifies a critical role of splice variants in linking RNA splicing dysregulation and CIN and highlights cen-R-loop augmentation as a key mechanism for leukemogenesis.

Abstract Background People with a mental health condition experience an elevated risk of chronic disease and greater prevalence of health and behaviours. Lifestyle interventions aim to reduce this risk by modifying health behaviours such as physical activity and diet. Previous reviews exploring the efficacy of such interventions for this group have typically limited inclusion to individuals with severe mental illness (SMI), with a focus of impact on weight. This review assessed the efficacy of lifestyle interventions delivered in community or outpatient settings to people with any mental health condition, on weight, physical activity and diet. Methods Eligible studies were randomised or cluster-randomised controlled trials published between January 1999 and February 2019 aiming to improve weight, physical activity or diet, for people with any mental health condition. Two reviewers independently completed study screening, data extraction and assessment of methodological quality. Primary outcome measures were weight, physical activity and diet. Secondary outcome measures were body mass index (BMI), waist circumference, sedentary behaviour and mental health. Where possible, meta-analyses were conducted. Narrative synthesis using vote counting based on direction of effect was used where studies were not amenable to meta-analysis. Results Fifty-seven studies were included (49 SMI only), with 46 contributing to meta-analyses. Meta-analyses revealed significant (< 0.05) effect of interventions on mean weight loss (−1.42 kg), achieving 5% weight loss (OR 2.48), weight maintenance (−2.05 kg), physical activity (IPAQ MET minutes: 226.82) and daily vegetable serves (0.51), but not on fruit serves (0.01). Significant effects were also seen for secondary outcomes of BMI (−0.48 units) and waist circumference (−0.87cm), but not mental health (depression: SMD −0.03; anxiety: SMD −0.49; severity of psychological symptoms: SMD 0.72). Studies reporting sedentary behaviour were not able to be meta-analysed. Most trials had high risk of bias, quality of evidence for weight and physical activity were moderate, while quality of evidence for diet was low. Conclusion Lifestyle interventions delivered to people with a mental health condition made statistically significant improvements to weight, BMI, waist circumference, vegetable serves and physical activity. Further high-quality trials with greater consistency in measurement and reporting of outcomes are needed to better understand the impact of lifestyle interventions on physical activity, diet, sedentary behaviour and mental health and to understand impact on subgroups. Systematic review registration PROSPERO CRD42019137197

Objectives. To describe the presence and persistence of neurological and neuropsychological sequelae among children with acquired Zika virus infection and assess whether those sequelae were more common in children infected with Zika virus compared to uninfected children. Methods. We conducted a prospective cohort study of children with and without Zika virus infection in León, Nicaragua, using a standard clinical assessment tool and questionnaire to collect data on symptoms at three visits, about 6 months apart, and a battery of standardized instruments to evaluate neurocognitive function, behavior, depression, and anxiety at the last two visits. Results. Sixty-two children were enrolled, with no significant differences in demographics by infection group. Children infected with Zika virus had a range of neurological symptoms, some of which persisted for 6 to 12 months; however, no consistent pattern of symptoms was observed. At baseline a small percentage of children infected with Zika virus had an abnormal finger-to-nose test (13%), cold touch response (13%), and vibration response (15%) versus 0% in the uninfected group. Neurocognitive deficits and behavioral problems were common in both groups, with no significant differences between the groups. Children infected with Zika virus had lower cognitive efficiency scores at the 6-month visit. Anxiety and depression were infrequent in both groups. Conclusions. Larger studies are needed to definitively investigate the relationship between Zika virus infection and neurological symptoms and neurocognitive problems, with adjustment for factors affecting cognition and behavior, including mood and sleep disorders, home learning environment, history of neuroinvasive infections, and detailed family history of neuropsychological problems.

Abstract Avian influenza viruses can pose serious risks to agricultural production, human health, and wildlife. An understanding of viruses in wild reservoir species across time and space is important to informing surveillance programs, risk models, and potential population impacts for vulnerable species. Although it is recognized that influenza A virus prevalence peaks in reservoir waterfowl in late summer through autumn, temporal and spatial variation across species has not been fully characterized. We combined two large influenza databases for North America and applied spatiotemporal models to explore patterns in prevalence throughout the annual cycle and across the continental United States for 30 waterfowl species. Peaks in prevalence in late summer through autumn were pronounced for dabbling ducks in the genera Anas and Spatula, but not Mareca. Spatially, areas of high prevalence appeared to be related to regional duck density, with highest predicted prevalence found across the upper Midwest during early fall, though further study is needed. We documented elevated prevalence in late winter and early spring, particularly in the Mississippi Alluvial Valley. Our results suggest that spatiotemporal variation in prevalence outside autumn staging areas may also represent a dynamic parameter to be considered in IAV ecology and associated risks.

Thalattosuchian crocodylomorphs were a diverse clade that lived from the Early Jurassic to the Early Cretaceous. The subclade Metriorhynchoidea underwent a remarkable transition, evolving from semi-aquatic ambush predators into fully aquatic forms living in the open oceans. Thalattosuchians share a peculiar palatal morphology with semi-aquatic and aquatic fossil cetaceans: paired anteroposteriorly aligned grooves along the palatal surface of the bony secondary palate. In extant cetaceans, these grooves are continuous with the greater palatine artery foramina, arteries that supply their oral thermoregulatory structures. Herein, we investigate the origins of thalattosuchian palatal grooves by examining CT scans of six thalattosuchian species (one teleosauroid, two early-diverging metriorhynchoids and three metriorhynchids), and CT scans of eleven extant crocodylian species. All thalattosuchians had paired osseous canals, enclosed by the palatines, that connect the nasal cavity to the oral cavity. These osseous canals open into the oral cavity via foramina at the posterior terminus of the palatal grooves. Extant crocodylians lack both the external grooves and the internal canals. We posit that in thalattosuchians these novel palatal canals transmitted hypertrophied medial nasal vessels (artery and vein), creating a novel heat exchange pathway connecting the palatal vascular plexus to the endocranial region. Given the general hypertrophy of thalattosuchian cephalic vasculature, and their increased blood flow and volume, thalattosuchians would have required a more extensive suite of thermoregulatory pathways to maintain stable temperatures for their neurosensory tissues.

Summary: Background: Tuberculosis (TB) is a leading infectious cause of death worldwide and treating latent TB infection (LTBI) with TB preventative therapy is a global priority. This study aimed to measure interferon gamma (IFN-γ) release assay (IGRA) positivity (the current reference standard for LTBI diagnosis) and Mtb-specific IgG antibodies in otherwise healthy adults without HIV and those living with HIV (PLWH). Methods: One-hundred and eighteen adults (65 without HIV and 53 antiretroviral-naïve PLWH), from a peri-urban setting in KwaZulu-Natal, South Africa were enrolled. IFN-γ released following stimulation with ESAT-6/CFP-10 peptides and plasma IgG antibodies specific for multiple Mtb antigens were measured using the QuantiFERON-TB Gold Plus (QFT) and customized Luminex assays, respectively. The relationships between QFT status, relative concentrations of anti-Mtb IgG, HIV-status, sex, age and CD4 count were analysed. Findings: Older age, male sex and higher CD4 count were independently associated with QFT positivity (p = 0.045, 0.05 and 0.002 respectively). There was no difference in QFT status between people with and without HIV infection (58% and 65% respectively, p = 0.06), but within CD4 count quartiles, people with HIV had higher QFT positivity than people without HIV (p = 0.008 (2nd quartile),

Abstract The importance of metal biology in neurodegenerative diseases such as Huntingtin Disease is well documented with evidence of direct interactions between metals such as copper, zinc, iron and manganese and mutant Huntingtin pathobiology. To date, it is unclear whether these interactions are observed in humans, how this impacts other metals, and how mutant Huntington alters homeostatic mechanisms governing levels of copper, zinc, iron and manganese in cerebrospinal fluid and blood in HD patients. Plasma and cerebrospinal fluid from control, pre-manifest, manifest and late manifest HD participants were collected as part of HD-Clarity. Levels of cerebrospinal fluid and plasma copper, zinc, iron and manganese were measured as well as levels of mutant Huntingtin and neurofilament in a sub-set of cerebrospinal fluid samples. We find that elevations in cerebrospinal fluid copper, manganese and zinc levels are altered early in disease prior to alterations in canonical biomarkers of HD although these changes are not present in plasma. We also evidence that CSF iron is elevated in manifest patients. The relationships between plasma and cerebrospinal fluid metal are altered based on disease stage. These findings demonstrate that there are alterations in metal biology selectively in the CSF which occur prior to changes in known canonical biomarkers of disease. Our work indicates that there are pathological changes related to alterations in metal biology in individuals without elevations in neurofilament and mutant Huntingtin.

Abstract Zika virus (ZIKV) infection at the maternal–placental interface is associated with adverse pregnancy outcomes including fetal demise and pregnancy loss. To determine how infection impacts placental trophoblasts, we utilized rhesus macaque trophoblast stem cells (TSC) that can be differentiated into early gestation syncytiotrophoblasts (ST) and extravillous trophoblasts (EVT). TSCs and STs, but not EVTs, were highly permissive to productive infection with ZIKV strain DAK AR 41524. The impact of ZIKV on the cellular transcriptome showed that infection of TSCs and STs increased expression of immune related genes, including those involved in type I and type III interferon responses. ZIKV exposure altered extracellular vesicle (EV) mRNA, miRNA and protein cargo, including ZIKV proteins, regardless of productive infection. These findings suggest that early gestation macaque TSCs and STs are permissive to ZIKV infection, and that EV analysis may provide a foundation for identifying non-invasive biomarkers of placental infection in a highly translational model.

Abstract Background Recent investigations of the meninges have highlighted the importance of the dura layer in central nervous system immune surveillance beyond a purely structural role. However, our understanding of the meninges largely stems from the use of pre-clinical models rather than human samples. Methods Single-cell RNA sequencing of seven non-tumor-associated human dura samples and six primary meningioma tumor samples (4 matched and 2 non-matched) was performed. Cell type identities, gene expression profiles, and T cell receptor expression were analyzed. Copy number variant (CNV) analysis was performed to identify putative tumor cells and analyze intratumoral CNV heterogeneity. Immunohistochemistry and imaging mass cytometry was performed on selected samples to validate protein expression and reveal spatial localization of select protein markers. Results In this study, we use single-cell RNA sequencing to perform the first characterization of both non-tumor-associated human dura and primary meningioma samples. First, we reveal a complex immune microenvironment in human dura that is transcriptionally distinct from that of meningioma. In addition, we characterize a functionally diverse and heterogenous landscape of non-immune cells including endothelial cells and fibroblasts. Through imaging mass cytometry, we highlight the spatial relationship among immune cell types and vasculature in non-tumor-associated dura. Utilizing T cell receptor sequencing, we show significant TCR overlap between matched dura and meningioma samples. Finally, we report copy number variant heterogeneity within our meningioma samples. Conclusions Our comprehensive investigation of both the immune and non-immune cellular landscapes of human dura and meningioma at single-cell resolution builds upon previously published data in murine models and provides new insight into previously uncharacterized roles of human dura.

The American Association of Equine Practitioners strongly advocates evidence-based intestinal strongyle control in horses. It recommends targeted treatment of all heavy egg shedders (>500 eggs per gram (EPG) of feces), while the low shedders (0–200 EPG) are left untreated. As 50–75% of adult horses in a herd are low shedders, preventing them from unnecessary anthelmintic exposure is critical for tackling resistance. There are various fecal egg count (FEC) techniques with many modifications and variations in use, but none is identified as a gold standard. The hypothesis of the study was that the diagnostic performance of 12 commonly used quantitation methodologies (three techniques with four variants) differs. In this regard, method comparison studies were performed using polystyrene beads as proxy for intestinal strongyle eggs. Mini-FLOTAC-based variants had the lowest coefficient of variation (CV%) in bead recovery, whereas McMaster variants had the highest. All four variants of Mini-FLOTAC and the NaNO3 1.33 specific gravity variant of modified Wisconsin followed a linear fit with R2 > 0.95. In contrast, the bead standard replicates for modified McMaster variants dispersed from the regression curve, causing a lower R2. The Mini-FLOTAC method seems less influenced by the choice of floatation solution and has better repeatability parameters and linearity for bead standard recovery. For FEC tests with high R2 (>0.95) but that underestimated the true bead count, a correction factor (CF) was determined to estimate the true count. Finally, the validity of CF was analyzed for 5 tests with R2 > 0.95 to accurately quantify intestinal strongyle eggs from 40 different horses. Overall, this study identified FEC methodologies with the highest diagnostic performance. The limitations in standardizing routine FEC tests are highlighted, and the importance of equalization of FEC results is emphasized for promoting uniformity in the implementation of parasite control guidelines.

Abstract The nutritional integrity of wheat is jeopardized by rapidly rising atmospheric carbon dioxide (CO2) and the associated emergence and enhanced virulence of plant pathogens. To evaluate how disease resistance traits may impact wheat climate resilience, 15 wheat cultivars with varying levels of resistance to Fusarium Head Blight (FHB) were grown at ambient and elevated CO2. Although all wheat cultivars had increased yield when grown at elevated CO2, the nutritional contents of FHB moderately resistant (MR) cultivars were impacted more than susceptible cultivars. At elevated CO2, the MR cultivars had more significant differences in plant growth, grain protein, starch, fructan, and macro and micro-nutrient content compared with susceptible wheat. Furthermore, changes in protein, starch, phosphorus, and magnesium content were correlated with the cultivar FHB resistance rating, with more FHB resistant cultivars having greater changes in nutrient content. This is the first report of a correlation between the degree of plant pathogen resistance and grain nutritional content loss in response to elevated CO2. Our results demonstrate the importance of identifying wheat cultivars that can maintain nutritional integrity and FHB resistance in future atmospheric CO2 conditions.

Objectives Dryness, fatigue and joint/muscle pain are typically assessed in Sjögren’s trials using European Alliance of Associations for Rheumatology Sjögren’s Syndrome Patient Reported Index (ESSPRI). A Patient Acceptable Symptom State of 90%), white (90%), with mean age of 50 years. Receiver operating characteristic estimates supported an MCII threshold of 1.5–1.6 in the phase II data, whereas correlation-weighted mean change estimates supported a low/minimal symptom severity threshold of ≥2. A low/minimal symptom severity of ≤3 showed the greatest sensitivity/specificity balance. Analyses in the pooled data supported these thresholds (MCII: 1.5–2.1; low/minimal symptom severity: 2.7–3.7). Unblinded analyses confirmed the revised RD reduced placebo rates.Conclusions Completing a trial with an improvement of ≥1.5 points compared with baseline and an ESSPRI score of ≤3 points is a relevant RD for moderate-to-severe systemic Sjögren’s and reduces placebo rates.

Tuberculosis remains one of the leading causes of death worldwide, especially in low- and middle-income countries. Tuberculosis treatment and control efforts are hindered by the difficulty in making the diagnosis, as currently available diagnostic tests are too slow, too expensive, or not sufficiently sensitive. Recombinase polymerase amplification (RPA) is a novel technique that allows for the amplification of DNA rapidly, at constant temperature, and with minimal expense. We calculated and compared the limit of detection, sensitivity, and specificity of two RPA-based assays for the diagnosis of pulmonary tuberculosis, using two sets of published primers. We also calculated and compared the assays' limits of detection and compared their performance using two different DNA extraction methods prior to amplification (a commercially available DNA extraction kit vs. the chelex method). The RPA-lateral flow assay had a limit of detection of 5 fg/μL of DNA, a sensitivity of 53.2%, and a specificity of 93.3%, while the real time-RPA assay had a limit of detection of 25 fg/μL of DNA, a sensitivity of 85.1%, and a specificity of 93.3%. There was no difference in assay performance when DNA extraction was carried out using the commercial kit vs. the chelex method. The real-time RPA assay has adequate sensitivity and specificity for the diagnosis of pulmonary tuberculosis and could be a viable diagnostic tool in resource-limited settings, but the lateral flow assay did not perform as well, perhaps due to the fact we used stored sputum specimens from a biorepository. More work is needed to optimize the RPA-lateral flow assay, to get a more accurate estimate of its specificity and sensitivity using prospectively collected specimens, and to develop both assays into point-of-care tests that can be easily deployed in the field.

The U.S. pork supply chain is vulnerable to various internal and external threats and in need of prompt, comprehensive response plans. Under urgent circumstances, for example in the case of foreign disease incursions, swine farms will have to perform on-farm animal depopulation to prevent disease spread. Several animal depopulation methods including water-based foam (WBF) have been proposed and are under evaluation for feasibility in the field. However, the psychological/emotional impacts of applying depopulation methods for personnel managing and carrying on the tasks are not currently well understood. Thus, this study aimed to investigate WBF as an alternative for depopulation compared to existing methods approved by the American Veterinary Medical Association. Swine industry stakeholders were invited to voluntarily observe a WBF depopulation trial and to provide their self-reported perspectives before and after the observation. A survey was designed to explore key areas on expected and perceived method effectiveness, efficiency, and animal welfare considerations, as well as to evaluate short-term post-observation psychological impacts. Among 42 recruited stakeholders, 31.0% (13/42) were educators/researchers followed by animal health officials (26.2%, 11/42) and veterinarians (23.8%, 10/42), with an average of 11.7 ± 12.6 (n = 39) years of work experience. After the trial, respondents' positive perception of WBF depopulation increased specifically regarding the animal loading process being less stressful than restrained in-barn depopulation options (P = 0.003) and by the observation of fewer swine escape attempts and vocalizations than expected (P < 0.001). Respondents' positive perception of WBF also increased regarding to the time required to fill the trailer with foam, to stop hearing animal vocalization, and stop hearing animal movement, as the observed trial times were faster than their pre-observation estimates (P < 0.001). Additionally, 79.5% (31/39) of respondents agreed that the rapid destruction of animal populations had priority over animal welfare under urgent scenarios. Minor post-traumatic stress disorder-like (PTSD-like) symptoms from the observed trials were reported (26.7%, 4/15 respondents) one month after the observation. This study showed that the WBF depopulation process was perceived positively by swine stakeholders and may have limited short-term psychological impacts on personnel involved in animal depopulation.

The hyphal tips of Neurospora crassa have prevacuolar compartments (PVCs) of unusual size and shape. They appear to function as late endosomes/multivesicular bodies. PVCs are highly variable in size (1-3 microns) and exhibit rapid changes in structure. When visualized with tagged integral membrane proteins of the vacuole the PVCs appear as ring or horseshoe-shaped structures. Some soluble molecules that fill the lumen of mature spherical vacuoles do not appear in the lumen of the PVC but are seen in the ring or horseshoe-shaped structures. By using super-resolution microscopy I have achieved a better understanding of the structure of the PVCs. The PVC appears to form a pouch with an open end. The walls of the pouch are composed of small vesicles or tubules, approximately 250 nm in diameter. The shape of the PVC can change in a few seconds, caused by the apparent movement of the vesicles/tubules. In approximately 85% of the PVCs dynein and dynactin were observed as poorly defined lumps inside the pouch-shaped PVCs. Within the PVCs they were not attached to microtubules nor did they appear to be in direct contact with the vesicles and tubules that formed the PVCs. In the future, the structure and relatively large size of the Neurospora PVC may allow us to visualize protein-sorting events that occur in the formation of vacuoles.

BackgroundWeight loss through lifestyle modification can produce health benefits and may reduce cancer risk. The goal of this study was to examine the feasibility of and adherence to a 15-week telephone-based weight loss intervention in rural Ohio, an area with high rates of obesity.MethodsThis pilot 2-arm randomized controlled study was designed for rural Ohio residents who were overweight or obese. Eligible participants were 2:1 randomly assigned to either a 15-week weight loss intervention group or active control group. The weight loss intervention group received weekly telephone sessions to improve healthy diet and increase physical activity. The active control group received education brochures with information on physical activity and dietary guidelines. Feasibility was defined as at least 80% of participants completing the follow-up surveys, and acceptable adherence was defined as the percentage of participants in the weight loss group who attend ≥75% of weekly telephone sessions.ResultsA total of 423 individuals entered the online screening survey, 215 (50.8%) completed the survey, and 98 (45.6%) of those were eligible. Forty eligible individuals were enrolled and randomly assigned to the weight loss group (n = 27) or active control group (n = 13). The average age of the weight loss group was 49 (SD = 10) years, and 89% were female. The average age of the active control group was 51 (SD = 9) years, and 92% were female. Feasibility was demonstrated: 90% of participants completed the online follow-up surveys at 15-weeks. Among participants in the weight loss group, 22 out of 27 (81.5%) completed the 15-week intervention, the average number of sessions attended was 9.7 (64.9%). Adherence to the intervention was rated as acceptable among almost half of the group (48.1%).ConclusionsFeasibility of a 15-week telephone-based weight loss study among rural residents with overweight/obesity were determined. A future study will test this intervention for weight loss efficacy.

The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least six of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 ms of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatin’s binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R2=0.82). In a blind prediction for an isoform (Myh7b) whose blebbistatin sensitivity was unknown, we find good agreement between predicted and measured IC50s (0.67 μM vs. 0.36 μM). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets.

Governments around the world have acknowledged that urgent action is needed to conserve and restore ecological connectivity to help reverse the decline of biodiversity. In this study we tested the hypothesis that functional connectivity for multiple species can be estimated across Canada using a single, upstream connectivity model. We developed a movement cost layer with cost values assigned using expert opinion to anthropogenic land cover features and natural features based on their known and assumed effects on the movement of terrestrial, non-volant fauna. We used Circuitscape to conduct an omnidirectional connectivity analysis for terrestrial landscapes, in which the potential contribution of all landscape elements to connectivity were considered and where source and destination nodes were independent of land tenure. Our resulting map of mean current density provided a seamless estimate of movement probability at a 300 m resolution across Canada. We tested predictions in our map using a variety of independently collected wildlife data. We found that GPS data for individual caribou, wolves, moose, and elk that traveled longer distances in western Canada were all significantly correlated with areas of high current densities. The frequency of moose roadkill in New Brunswick was also positively associated with current density, but our map was not able to predict areas of high road mortality for herpetofauna in southern Ontario. The results demonstrate that an upstream modelling approach can be used to characterize functional connectivity for multiple species across a large study area. Our national connectivity map can help governments in Canada prioritize land management decisions to conserve and restore connectivity at both national and regional scales.