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Long-billed Curlews ( Numenius americanus ) are declining throughout North America, and the loss of grassland breeding habitat is one of the primary threats to the species. Intermountain West, in particular, has been identified as the most important region in North America for breeding curlews. Nevertheless, the density and abundance of Long-billed Curlews in this region is not well understood. Lands managed for military training can provide habitat for wildlife species of conservation concern, and increasingly these lands are becoming relevant to sustaining biodiversity. We conducted point count surveys of Long-billed Curlews on Department of Defense lands in the Columbia Basin near Boardman, Oregon, USA during two consecutive breeding seasons. We used multinomial-Poisson mixture models to estimate detection probability and density of curlews and to investigate environmental correlates of those metrics. Mean detection probability at a distance of 400 m was 0.45 and 0.61 in 2015 and 2016, respectively. In 2015, the clarity of skies increased detection probability, but in 2016, none of the variables we measured influenced detection probability. Mean predicted density was 3.3 (95% confidence interval: 2.4–4.7) and 1.8 (1.2–2.7) curlews/km² in 2015 and 2016, respectively. In both years, curlew density was higher in lower-elevation or topographically smoother areas. Estimated abundance of curlews in the study area was 639 (456–912) and 350 (237–520) birds in 2015 and 2016, respectively. The number of curlews appeared to fluctuate across the two years of our study, a demographic trend that may have been influenced by a wildfire in our study area in June 2015. The results of our study indicate that federal grasslands, including areas where military operations are conducted, can provide conservation benefit to breeding Long-billed Curlews.

New York, USA, is a regional hub for processing carrot (Daucus carota ssp. sativus) production and Nantes-type cultivars are preferred for slicing. Diameter is critical in carrots for slicing, with roots larger than 1 5/8 inch being rejected. The potential to manipulate carrot root diameter and hence suitability for slicing by foliar-applied gibberellic acid (GA3) was tested in four small plot replicated field trials over 3 years (2020, 2021, and 2022). In the most efficacious treatments, GA3 resulted in a 23.1% to 135.4% increase in foliar biomass at the expense of root weight and diameter. Increases in foliar biomass are beneficial to facilitate top-pulling harvest. Reductions in root diameter from GA3 ranged from 9.5% to 19.6%, and in 2020 and 2022, increased the proportion of roots suitable for slicing. GA3 did not significantly affect root length and number. In two 2021 trials, GA3 increased color intensity quantified by a colorimeter, but this change was not noticeable to the naked eye. The optimal number of GA3 applications was seasonally dependent, ranging from a single application at 107 to 108 days after planting (DAP) in 2021, to two applications at 74 + 92 DAP in 2022. Three GA3 applications per season or late applications (up to 14 days prior to harvest) were not beneficial.

Abstract Background Cardiac magnetic resonance (CMR) imaging is important for diagnosis and risk stratification of hypertrophic cardiomyopathy (HCM) patients. However, collection of information from large numbers of CMR reports by manual review is time-consuming, error-prone and costly. Natural language processing (NLP) is an artificial intelligence method for automated extraction of information from narrative text including text in CMR reports in electronic health records (EHR). Our objective was to assess whether NLP can accurately extract diagnosis of HCM from CMR reports. Methods An NLP system with two tiers was developed for information extraction from narrative text in CMR reports; the first tier extracted information regarding HCM diagnosis while the second extracted categorical and numeric concepts for HCM classification. We randomly allocated 200 HCM patients with CMR reports from 2004 to 2018 into training (100 patients with 185 CMR reports) and testing sets (100 patients with 206 reports). Results NLP algorithms demonstrated very high performance compared to manual annotation. The algorithm to extract HCM diagnosis had accuracy of 0.99. The accuracy for categorical concepts included HCM morphologic subtype 0.99, systolic anterior motion of the mitral valve 0.96, mitral regurgitation 0.93, left ventricular (LV) obstruction 0.94, location of obstruction 0.92, apical pouch 0.98, LV delayed enhancement 0.93, left atrial enlargement 0.99 and right atrial enlargement 0.98. Accuracy for numeric concepts included maximal LV wall thickness 0.96, LV mass 0.99, LV mass index 0.98, LV ejection fraction 0.98 and right ventricular ejection fraction 0.99. Conclusions NLP identified and classified HCM from CMR narrative text reports with very high performance.

New York state is among the largest producers of table beets in the United States, which, by extension, has placed a new focus on precision crop management. For example, an operational unmanned aerial system (UAS)-based yield forecasting tool could prove helpful for the efficient management and harvest scheduling of crops for factory feedstock. The objective of this study was to evaluate the feasibility of predicting the weight of table beet roots from spectral and textural features, obtained from hyperspectral images collected via UAS. We identified specific wavelengths with significant predictive ability, e.g., we down-select >200 wavelengths to those spectral indices sensitive to root yield (weight per unit length). Multivariate linear regression was used, and the accuracy and precision were evaluated at different growth stages throughout the season to evaluate temporal plasticity. Models at each growth stage exhibited similar results (albeit with different wavelength indices), with the LOOCV (leave-one-out cross-validation) R2 ranging from 0.85 to 0.90 and RMSE of 10.81–12.93% for the best-performing models in each growth stage. Among visible and NIR spectral regions, the 760–920 nm-wavelength region contained the most wavelength indices highly correlated with table beet root yield. We recommend future studies to further test our proposed wavelength indices on data collected from different geographic locations and seasons to validate our results.

Abstract Background Many Plasmodium infections in endemic regions exist at densities below the limit of detection of standard diagnostic tools. These infections threaten control efforts and may impact vaccine and therapeutic drug studies. Simple, cost-effective methods are needed to study the natural history of asymptomatic submicroscopic parasitaemia. Self-collected dried blood spots (DBS) analysed using pooled and individual quantitative reverse transcription polymerase chain reaction (qRT-PCR) provide such a solution. Here, the feasibility and acceptability of daily at-home DBS collections for qRT-PCR was studied to better understand low-density infections. Methods Rapid diagnostic test (RDT)-negative individuals in Katakwi District, northeastern Uganda, were recruited between April and May 2021. Venous blood samples and clinic-collected DBS were taken at enrollment and at four weekly clinic visits. Participants were trained in DBS collection and asked to collect six DBS weekly between clinic visits. Opinions about the collection process were solicited using daily Diary Cards and a Likert scale survey at the final study visit. Venous blood and DBS were analysed by Plasmodium 18S rRNA qRT-PCR. The number of participants completing the study, total DBS collected, and opinions of the process were analysed to determine compliance and acceptability. The human internal control mRNA and Plasmodium 18S rRNA were evaluated for at-home vs. clinic-collected DBS and venous blood to assess quality and accuracy of at-home collected samples. Results One-hundred two adults and 29 children were enrolled, and 95 and 26 completed the study, respectively. Three individuals withdrew due to pain or inconvenience of procedures. Overall, 96% of participants collected ≥ 16 of 24 at-home DBS, and 87% of DBS contained ≥ 40 µL of blood. The procedure was well tolerated and viewed favourably by participants. At-home collected DBS were acceptable for qRT-PCR and showed less than a one qRT-PCR cycle threshold shift in the human control mRNA compared to clinic-collected DBS. Correlation between Plasmodium falciparum 18S rRNA from paired whole blood and DBS was high (R = 0.93). Conclusions At-home DBS collection is a feasible, acceptable, and robust method to obtain blood to evaluate the natural history of low-density Plasmodium infections by qRT-PCR.

Background: Naloxone distributed to people at risk for opioid overdose has been associated with reduced overdose death rates; however, associations of retail pharmacy-distributed naloxone with overdose mortality have not been evaluated. Methods: Our analytic cohort uses retail pharmacy claims data; three health departments’ community distribution data; federal opioid overdose data; and American Community Survey data. Data were analyzed by 3-digit ZIP Code and calendar quarter-year (2016Q1–2018Q4), and weighted by population. We regressed opioid-related overdose mortality on retail-pharmacy and community naloxone distribution, and community-level demographics using a linear model, hypothesizing that areas with high overdose rates would have higher current levels of naloxone distribution but that increasing naloxone distribution from one quarter to the next would be associated with lower overdose. Results: From Q1–2016 to Q4–2018, the unadjusted naloxone distribution rate increased from 97 to 257 kits per 100,000 persons, while the unadjusted opioid overdose mortality rate fell from 8.1 to 7.2 per 100,000 persons. The concurrent level of naloxone distribution (both pharmacy and community) was positively and significantly associated with fatal opioid overdose rates. We did not detect associations between change in naloxone distribution rates and overdose mortality. Conclusion: Naloxone distribution volumes were correlated with fatal opioid overdose, suggesting medication was getting to communities where it was needed most. Amid high rates of overdose driven by fentanyl in the drug supply, our findings suggest additional prevention, treatment, and harm reduction interventions are required—and dramatically higher naloxone volumes needed—to reverse the opioid overdose crisis in the US.

Summary: Plasmodium sporozoites invade hepatocytes and transform into liver stages within a parasitophorous vacuole (PV). The parasites then grow and replicate their genome to form exoerythrocytic merozoites that infect red blood cells. We report that the human malaria parasite Plasmodium falciparum (Pf) expresses a C-type ATP-binding cassette transporter, Pf ABCC2, which marks the transition from invasive sporozoite to intrahepatocytic early liver stage. Using a humanized mouse infection model, we show that Pf ABCC2 localizes to the parasite plasma membrane in early and mid-liver stage parasites but is not detectable in late liver stages. Pf abcc2— sporozoites invade hepatocytes, form a PV, and transform into liver stage trophozoites but cannot transition to exoerythrocytic schizogony and fail to transition to blood stage infection. Thus, Pf ABCC2 is an expression marker for early phases of parasite liver infection and plays an essential role in the successful initiation of liver stage replication.

Abstract Wildlife managers require defensible and transparent population estimates to justify species management. Statistical population reconstruction (SPR) is being widely adopted to estimate wildlife population sizes from hunter harvest data. We assessed factors influencing variation in SPR population estimates produced for bobcats in Wyoming, USA. Specifically, we tested whether prey availability, hunter/trapper effort, the number of bobcats killed or the methods used to classify the age and sex of bobcats (‘classification protocol’) best explained changing SPR abundance estimates. We then quantified the relative magnitude of these effects on SPR model outputs. Classification protocol had the strongest impact on SPR abundance estimates, such that a shift to visual age and sex classifications by trappers/hunters resulted in overestimates of bobcat abundance. The Wyoming bobcat SPR population estimates were likely unreliable and we suggest that spatially explicit integrated population models may be a better approach to obtaining defensible estimates upon which to establish scientific management of this charismatic carnivore.

Abstract Background Sport-related concussion (SRC) is a heterogenous injury that often presents with varied symptoms and impairment. Recently, research has focused on identifying subtypes, or clinical profiles of concussion to be used in assessing and treating athletes with SRC. The purpose of this study was to investigate sex differences in clinical profiles, recovery duration, and initial symptom severity after SRC in a cohort of collegiate athletes in the Pacific-12 Conference (Pac-12). Methods This prospective cohort study examined post-SRC symptoms, recovery, and return-to-play times using data from the Pac-12 CARE Affiliated Program and Pac-12 Health Analytics Program. Clinical profiles reported by student-athletes were defined by the number (> 50%) of specific symptoms frequently reported for each profile. Generalized linear mixed models were used to examine associations among sex, clinical profiles, time-to-recovery, and return-to-play times. Results 479 concussion incidents met inclusion criteria. The probabilities of initial presentation of each clinical profile, initial injury severity scores, and recovery times within a profile did not differ between sexes (p = 0.33–0.98). However, both males and females had > 0.75 probabilities of exhibiting cognitive and ocular profiles. Initial injury severity score was a strong nonlinear predictor of initial number of clinical profiles (p

Abstract Background Communities That HEAL (CTH) is a novel, data-driven community-engaged intervention designed to reduce opioid overdose deaths by increasing community engagement, adoption of an integrated set of evidence-based practices, and delivering a communications campaign across healthcare, behavioral-health, criminal-legal, and other community-based settings. The implementation of such a complex initiative requires up-front investments of time and other expenditures (i.e., start-up costs). Despite the importance of these start-up costs in investment decisions to stakeholders, they are typically excluded from cost-effectiveness analyses. The objective of this study is to report a detailed analysis of CTH start-up costs pre-intervention implementation and to describe the relevance of these data for stakeholders to determine implementation feasibility. Methods This study is guided by the community perspective, reflecting the investments that a real-world community would need to incur to implement the CTH intervention. We adopted an activity-based costing approach, in which resources related to hiring, training, purchasing, and community dashboard creation were identified through macro- and micro-costing techniques from 34 communities with high rates of fatal opioid overdoses, across four states—Kentucky, Massachusetts, New York, and Ohio. Resources were identified and assigned a unit cost using administrative and semi-structured-interview data. All cost estimates were reported in 2019 dollars. Results State-level average and median start-up cost (representing 8–10 communities per state) were $268,657 and $175,683, respectively. Hiring and training represented 40%, equipment and infrastructure costs represented 24%, and dashboard creation represented 36% of the total average start-up cost. Comparatively, hiring and training represented 49%, purchasing costs represented 18%, and dashboard creation represented 34% of the total median start-up cost. Conclusion We identified three distinct CTH hiring models that affected start-up costs: hospital-academic (Massachusetts), university-academic (Kentucky and Ohio), and community-leveraged (New York). Hiring, training, and purchasing start-up costs were lowest in New York due to existing local infrastructure. Community-based implementation similar to the New York model may have lower start-up costs due to leveraging of existing infrastructure, relationships, and support from local health departments.

Abstract Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2 – /linup – or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.

Abstract Radiation-attenuated sporozoite (RAS) vaccines can completely prevent blood stage Plasmodium infection by inducing liver-resident memory CD8+ T cells to target parasites in the liver. Such T cells can be induced by ‘Prime-and-trap’ vaccination, which here combines DNA priming against the P. yoelii circumsporozoite protein (CSP) with a subsequent intravenous (IV) dose of liver-homing RAS to “trap” the activated and expanding T cells in the liver. Prime-and-trap confers durable protection in mice, and efforts are underway to translate this vaccine strategy to the clinic. However, it is unclear whether the RAS trapping dose must be strictly administered by the IV route. Here we show that intradermal (ID) RAS administration can be as effective as IV administration if RAS are co-administrated with the glycolipid adjuvant 7DW8-5 in an ultra-low inoculation volume. In mice, the co-administration of RAS and 7DW8-5 in ultra-low ID volumes (2.5 µL) was completely protective and dose sparing compared to standard volumes (10–50 µL) and induced protective levels of CSP-specific CD8+ T cells in the liver. Our finding that adjuvants and ultra-low volumes are required for ID RAS efficacy may explain why prior reports about higher volumes of unadjuvanted ID RAS proved less effective than IV RAS. The ID route may offer significant translational advantages over the IV route and could improve sporozoite vaccine development.

Traumatic brain injury (TBI) disrupts the blood?brain barrier (BBB), which may exacerbate neuroinflammation post-injury. Few translational studies have examined BBB dysfunction and subsequent neuroinflammation post-TBI in juveniles. We hypothesized that BBB dysfunction positively predicts microglial activation and that vulnerability to BBB dysfunction and associated neuroinflammation are dependent on age at injury. Post-natal day (PND)17 and PND35 rats (n?=?56) received midline fluid percussion injury or sham surgery, and immunoglobulin-G (IgG) stain was quantified as a marker of extravasated blood in the brain and BBB dysfunction. We investigated BBB dysfunction and the microglial response in the hippocampus, hypothalamus, and motor cortex relative to age at injury and days post-injury (DPI; 1, 7, and 25). We measured the morphologies of ionized calcium-binding adaptor molecule 1?labeled microglia using cell body area and perimeter, microglial branch number and length, end-points/microglial cell, and number of microglia. Data were analyzed using generalized hierarchical models. In PND17 rats, TBI increased levels of IgG compared to shams. Independent of age at injury, IgG in TBI rats was higher at 1 and 7 DPI, but resolved by 25 DPI. TBI activated microglia (more cells and fewer end-points) in PND35 rats compared to respective shams. Independent of age at injury, TBI induced morphological changes indicative of microglial activation, which resolved by 25 DPI. TBI rats had fewer cells and end-points per cell at 1 and 7 DPI than 25 DPI. Independent of TBI, PND17 rats had larger, more activated microglia than PND35 rats; PND17 TBI rats had larger cell body areas and perimeters than PND35 TBI rats. Importantly, we found support in both ages that IgG quantification predicted microglial activation after TBI. The number of microglia increased with increasing IgG, whereas branch length decreased with increasing IgG, which together indicate microglial activation. Our results suggest that stabilization of the BBB after pediatric TBI may be an important therapeutic strategy to limit neuroinflammation and promote recovery.

Abstract Malaria, caused by Plasmodium parasites, remains one of the most devastating infectious diseases worldwide, despite control efforts to lower morbidity and mortality. Both advanced candidate vaccines, RTS,S and R21, are subunit (SU) vaccines that target a single Plasmodium falciparum (Pf) pre-erythrocytic (PE) sporozoite (spz) surface protein known as circumsporozoite (CS). These vaccines induce humoral immunity but fail to elicit CD8 + T-cell responses sufficient for long-term protection. In contrast, whole-organism (WO) vaccines, such as Radiation Attenuated Sporozoites (RAS), achieved sterile protection but require a series of intravenous doses administered in multiple clinic visits. Moreover, these WO vaccines must be produced in mosquitos, a burdensome process that severely limits their availability. To reduce reliance on WO while maintaining protection via both antibodies and Trm responses, we have developed an accelerated vaccination regimen that combines two distinct agents in a prime-and-trap strategy. The priming dose is a single dose of self-replicating RNA encoding the full-length P. yoelii CS protein, delivered via an advanced cationic nanocarrier (LIONTM). The trapping dose consists of one dose of WO RAS. Our vaccine induces a strong immune response when administered in an accelerated regimen, i.e., either 5-day or same-day immunization. Additionally, mice after same-day immunization showed a 2-day delay of blood patency with 90% sterile protection against a 3-week spz challenge. The same-day regimen also induced durable 70% sterile protection against a 2-month spz challenge. Our approach presents a clear path to late-stage preclinical and clinical testing of dose-sparing, same-day regimens that can confer sterilizing protection against malaria.

Abstract Background Plasmodium knowlesi is an established experimental model for basic and pre-clinical malaria vaccine research. Historically, rhesus macaques have been the most common host for malaria vaccine studies with P. knowlesi parasites. However, rhesus are not natural hosts for P. knowlesi, and there is interest in identifying alternative hosts for vaccine research. The study team previously reported that pig-tailed macaques (PTM), a natural host for P. knowlesi, could be challenged with cryopreserved P. knowlesi sporozoites (PkSPZ), with time to blood stage infection equivalent to in rhesus. Here, additional exploratory studies were performed to evaluate PTM as potential hosts for malaria vaccine studies. The aim was to further characterize the parasitological and veterinary health outcomes after PkSPZ challenge in this macaque species. Methods Malaria-naïve PTM were intravenously challenged with 2.5 × 103 PkSPZ and monitored for blood stage infection by Plasmodium 18S rRNA RT-PCR and thin blood smears. Disease signs were evaluated by daily observations, complete blood counts, serum chemistry tests, and veterinary examinations. After anti-malarial drug treatment, a subset of animals was re-challenged and monitored as above. Whole blood gene expression analysis was performed on selected animals to assess host response to infection. Results In naïve animals, the kinetics of P. knowlesi blood stage replication was reproducible, with parasite burden rising linearly during an initial acute phase of infection from 6 to 11 days post-challenge, before plateauing and transitioning into a chronic low-grade infection. After re-challenge, infections were again reproducible, but with lower blood stage parasite densities. Clinical signs of disease were absent or mild and anti-malarial treatment was not needed until the pre-defined study day. Whole blood gene expression analysis identified immunological changes associated with acute and chronic phases of infection, and further differences between initial challenge versus re-challenge. Conclusions The ability to challenge PTM with PkSPZ and achieve reliable blood stage infections indicate this model has significant potential for malaria vaccine studies. Blood stage P. knowlesi infection in PTM is characterized by low parasite burdens and a benign disease course, in contrast with the virulent P. knowlesi disease course commonly reported in rhesus macaques. These findings identify new opportunities for malaria vaccine research using this natural host-parasite combination. Graphical Abstract

Abstract Background Perinatal depression affects an estimated 1 in 5 women in North America during the perinatal period, with annualized lifetime costs estimated at $20.6 billion CAD in Canada and over $45.9 billion USD in the US. Access to psychological treatments remains limited for most perinatal women suffering from depression and anxiety. Some barriers to effective care can be addressed through task-sharing to non-specialist providers and through telemedicine platforms. The cost-effectiveness of these strategies compared to traditional specialist and in-person models remains unknown. This protocol describes an economic evaluation of non-specialist providers and telemedicine, in comparison to specialist providers and in-person sessions within the ongoing Scaling Up Maternal Mental healthcare by Increasing access to Treatment (SUMMIT) trial. Methods The economic evaluation will be undertaken alongside the SUMMIT trial. SUMMIT is a pragmatic, randomized, non-inferiority trial across five North American study sites (N = 1,226) of the comparable effectiveness of two types of providers (specialist vs. non-specialist) and delivery modes (telemedicine vs. in-person) of a behavioural activation treatment for perinatal depressive and anxiety symptoms. The primary economic evaluation will be a cost-utility analysis. The outcome will be the incremental cost-effectiveness ratio, which will be expressed as the additional cost required to achieve an additional quality-adjusted life-year, as assessed by the EuroQol 5-Dimension 5-Level instrument. A secondary cost-effectiveness analysis will use participants’ depressive symptom scores. A micro-costing analysis will be conducted to estimate the resources/costs required to implement and sustain the interventions; healthcare resource utilization will be captured via self-report. Data will be pooled and analysed using uniform price and utility weights to determine cost-utility across all trial sites. Secondary country-specific cost-utility and cost-effectiveness analyses will also be completed. Sensitivity analyses will be conducted, and cost-effectiveness acceptability-curves will be generated, in all instances. Discussion Results of this study are expected to inform key decisions related to dissemination and scale up of evidence-based psychological interventions in Canada, the US, and possibly worldwide. There is potential impact on real-world practice by informing decision makers of the long-term savings to the larger healthcare setting in services to support perinatal women with common mental health conditions.

Abstract Introduction We evaluated racial/ethnic differences in the receipt of naloxone distributed by opioid overdose prevention programs (OOPPs) in New York City (NYC). Methods We used naloxone recipient racial/ethnic data collected by OOPPs from April 2018 to March 2019. We aggregated quarterly neighborhood-specific rates of naloxone receipt and other covariates to 42 NYC neighborhoods. We used a multilevel negative binomial regression model to assess the relationship between neighborhood-specific naloxone receipt rates and race/ethnicity. Race/ethnicity was stratified into four mutually exclusive groups: Latino, non-Latino Black, non-Latino White, and non-Latino Other. We also conducted racial/ethnic-specific geospatial analyses to assess whether there was within-group geographic variation in naloxone receipt rates for each racial/ethnic group. Results Non-Latino Black residents had the highest median quarterly naloxone receipt rate of 41.8 per 100,000 residents, followed by Latino residents (22.0 per 100,000), non-Latino White (13.6 per 100,000) and non-Latino Other residents (13.3 per 100,000). In our multivariable analysis, compared with non-Latino White residents, non-Latino Black residents had a significantly higher receipt rate, and non-Latino Other residents had a significantly lower receipt rate. In the geospatial analyses, both Latino and non-Latino Black residents had the most within-group geographic variation in naloxone receipt rates compared to non-Latino White and Other residents. Conclusions This study found significant racial/ethnic differences in naloxone receipt from NYC OOPPs. We observed substantial variation in naloxone receipt for non-Latino Black and Latino residents across neighborhoods, indicating relatively poorer access in some neighborhoods and opportunities for new approaches to address geographic and structural barriers in these locations.

Abstract The human airways are complex structures with important interactions between cells, extracellular matrix (ECM) proteins and the biomechanical microenvironment. A robust, well-differentiated in vitro culture system that accurately models these interactions would provide a useful tool for studying normal and pathological airway biology. Here, we report the development and characterization of a physiologically relevant air–liquid interface (ALI) 3D airway ‘organ tissue equivalent’ (OTE) model with three novel features: native pulmonary fibroblasts, solubilized lung ECM, and hydrogel substrate with tunable stiffness and porosity. We demonstrate the versatility of the OTE model by evaluating the impact of these features on human bronchial epithelial (HBE) cell phenotype. Variations of this model were analyzed during 28 days of ALI culture by evaluating epithelial confluence, trans-epithelial electrical resistance, and epithelial phenotype via multispectral immuno-histochemistry and next-generation sequencing. Cultures that included both solubilized lung ECM and native pulmonary fibroblasts within the hydrogel substrate formed well-differentiated ALI cultures that maintained a barrier function and expressed mature epithelial markers relating to goblet, club, and ciliated cells. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. This study highlights the feasibility and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment.

Abstract Background\Objectives Concomitant with low rates of pharmacotherapy for incarcerated individuals with OUD, there is a high rate of opioid overdose following re-entry into the community. Our research objective was to develop a better understanding of the factors that influence health-related quality-of-life (HRQoL) among this population during the high-risk transition period from incarceration to community. Few studies have assessed health-related quality-of-life (HRQoL) among individuals with OUD who are involved with the criminal-legal system, let alone over the period directly surrounding release from incarceration. Methods Secondary longitudinal analysis of data from a clinical trial where participants were randomized 1:1 to pre-release extended-release naltrexone (XR-NTX) + referral to community XR-NTX, vs. referral only. We conducted individual, multivariable regressions of EQ-5D domains (mobility, pain/discomfort, anxiety/depression; usual activities and self-care were excluded due to insufficient variation in scores), and the overall preference/utility score. HRQoL data were subset to timepoints immediately before release (baseline) and 12 weeks post-release; treatment groups were collapsed across condition. Multiple imputation by chained equations was conducted to handle missing 3-month data in the dependent variables and covariates, ad hoc. Results Greater severity in the psychiatric composite score was associated with substantially lower HRQoL, across all measures, following release from incarceration. Greater severity in the medical composite score was associated with lower pain/discomfort-related HRQoL. Conclusions Our findings highlight the importance of ensuring individuals with OUD are linked not only to MOUD, but also treatment for their comorbid conditions upon release from incarceration.

To investigate microglial mechanisms in central and peripheral inflammation after experimental traumatic brain injury (TBI), we inhibited the colony-stimulating factor-1 receptor (CSF-1R) with PLX5622 (PLX). We hypothesized that microglia depletion would attenuate central inflammation acutely with no effect on peripheral inflammation. After randomization, male mice (n?=?105) were fed PLX or control diets (21 days) and then received midline fluid percussion injury or sham injury. Brain and blood were collected at 1, 3, or 7 days post-injury (DPI). Immune cell populations were quantified in the brain and blood by flow cytometry. Cytokines (interleukin [IL]-6, IL-1?, tumor necrosis factor-?, interferon-?, IL-17A, and IL-10) were quantified in the blood using a multi-plex enzyme-linked immunosorbent assay. Data were analyzed using Bayesian multi-variate, multi-level models. PLX depleted microglia at all time points and reduced neutrophils in the brain at 7 DPI. PLX also depleted CD115+ monocytes, reduced myeloid cells, neutrophils, and Ly6Clow monocytes in blood, and elevated IL-6. TBI induced a central and peripheral immune response. TBI elevated leukocytes, microglia, and macrophages in the brain and elevated peripheral myeloid cells, neutrophils, Ly6Cint monocytes, and IL-1? in the blood. TBI lowered peripheral CD115+ and Ly6Clow monocytes in the blood. TBI PLX mice had fewer leukocytes and microglia in the brain at 1 DPI, with elevated neutrophils at 7 DPI compared to TBI mice on a control diet. TBI PLX mice also had fewer peripheral myeloid cells, CD115+, and Ly6Clow monocytes in the blood at 3 DPI, but elevated Ly6Chigh, Ly6Cint, and CD115+ monocyte populations at 7 DPI, compared to TBI mice on a control diet. TBI PLX mice had elevated proinflammatory cytokines and lower anti-inflammatory cytokines in the blood at 7 DPI compared to TBI mice on a control diet. CSF-1R inhibition reduced the immune response to TBI at 1 and 3 DPI, but elevated peripheral inflammation at 7 DPI.

Summary: Liver stage (LS) Plasmodia mature in 2–2.5 days in rodents compared to 5–6 days in humans. Plasmodium-specific CD8+ T cell expansion differs across these varied timespans. To mimic the kinetics of CD8+ T cells of human Plasmodium infection, a two-dose challenge mouse model that achieved 4–5 days of LS antigen exposure was developed. In this model, mice were inoculated with a non-protective, low dose of late-arresting, genetically attenuated sporozoites to initiate T cell activation and then re-inoculated 2–3 days later with wild-type sporozoites. Vaccines that partially protected against traditional challenge completely protected against two-dose challenge. During the challenge period, CD8+ T cell frequencies increased in the livers of two-dose challenged mice but not in traditionally challenged mice, further suggesting that this model better recapitulates kinetics of CD8+ T cell expansion in humans during the P. falciparum LS. Vaccine development and antigen discovery efforts may be aided by using the two-dose challenge strategy.

Introduction Malaria, a devastating febrile illness caused by protozoan parasites, sickened 247,000,000 people in 2021 and killed 619,000, mostly children and pregnant women in sub-Saharan Africa. A highly effective vaccine is urgently needed, especially for Plasmodium falciparum (Pf), the deadliest human malaria parasite. Areas covered Sporozoites (SPZ), the parasite stage transmitted by Anopheles mosquitoes to humans, are the only vaccine immunogen achieving > 90% efficacy against Pf infection. This review describes > 30 clinical trials of PfSPZ vaccines in the U.S.A., Europe, Africa, and Asia, based on first-hand knowledge of the trials and PubMed searches of ‘sporozoites,’ ‘malaria,’ and ‘vaccines.’ Expert opinion First generation (radiation-attenuated) PfSPZ vaccines are safe, well tolerated, 80-100% efficacious against homologous controlled human malaria infection (CHMI) and provide 18-19 months protection without boosting in Africa. Second generation chemo-attenuated PfSPZ are more potent, 100% efficacious against stringent heterologous (variant strain) CHMI, but require a co-administered drug, raising safety concerns. Third generation, late liver stage-arresting, replication competent (LARC), genetically-attenuated PfSPZ are expected to be both safe and highly efficacious. Overall, PfSPZ vaccines meet safety, tolerability, and efficacy requirements for protecting pregnant women and travelers, with licensure for these populations possible within five years. Protecting children and mass vaccination programs to block transmission and eliminate malaria are long-term objectives.

IntroductionScreening for G6PD deficiency can inform disease management including malaria. Treatment with the antimalarial drugs primaquine and tafenoquine can be guided by point-of-care testing for G6PD deficiency.Methods and findingsData from similar clinical studies evaluating the performance of the STANDARD G6PD Test (SD Biosensor, South Korea) conducted in Bangladesh, Brazil, Ethiopia, India, Thailand, the United Kingdom, and the United States were pooled. Test performance was assessed in a retrospective analysis on capillary and venous specimens. All study sites used spectrophotometry for reference G6PD testing, and either the HemoCue or complete blood count for reference hemoglobin measurement. The sensitivity of the STANDARD G6PD Test using the manufacturer thresholds for G6PD deficient and intermediate cases in capillary specimens from 4212 study participants was 100% (95% Confidence Interval (CI): 97.5%-100%) for G6PD deficient cases with 60% on the reference assay. The negative predictive value for females with G6PD activity >60% was 99.6% (95% CI 99.1%-99.8%) on capillary specimens. Sensitivity among 396 P. vivax malaria cases was 100% (69.2%-100.0%) for both deficient and intermediate cases. Across the full dataset, 37% of those classified as G6PD deficient or intermediate resulted from true normal cases. Despite this, over 95% of cases would receive correct treatment with primaquine, over 87% of cases would receive correct treatment with tafenoquine, and no true G6PD deficient cases would be treated inappropriately based on the result of the STANDARD G6PD Test.ConclusionsThe STANDARD G6PD Test enables safe access to drugs which are contraindicated for individuals with G6PD deficiency. Operational considerations will inform test uptake in specific settings.

Abstract Background Pragmatic primary care trials aim to test interventions in “real world” health care settings, but clinics willing and able to participate in trials may not be representative of typical clinics. This analysis compared patients in participating and non-participating clinics from the same health systems at baseline in the PRimary care Opioid Use Disorders treatment (PROUD) trial. Methods This observational analysis relied on secondary electronic health record and administrative claims data in 5 of 6 health systems in the PROUD trial. The sample included patients 16–90 years at an eligible primary care visit in the 3 years before randomization. Each system contributed 2 randomized PROUD trial clinics and 4 similarly sized non-trial clinics. We summarized patient characteristics in trial and non-trial clinics in the 2 years before randomization (“baseline”). Using mixed-effect regression models, we compared trial and non-trial clinics on a baseline measure of the primary trial outcome (clinic-level patient-years of opioid use disorder (OUD) treatment, scaled per 10,000 primary care patients seen) and a baseline measure of the secondary trial outcome (patient-level days of acute care utilization among patients with OUD). Results Patients were generally similar between the 10 trial clinics (n = 248,436) and 20 non-trial clinics (n = 341,130), although trial clinics’ patients were slightly younger, more likely to be Hispanic/Latinx, less likely to be white, more likely to have Medicaid/subsidized insurance, and lived in less wealthy neighborhoods. Baseline outcomes did not differ between trial and non-trial clinics: trial clinics had 1.0 more patient-year of OUD treatment per 10,000 patients (95% CI: − 2.9, 5.0) and a 4% higher rate of days of acute care utilization than non-trial clinics (rate ratio: 1.04; 95% CI: 0.76, 1.42). Conclusions trial clinics and non-trial clinics were similar regarding most measured patient characteristics, and no differences were observed in baseline measures of trial primary and secondary outcomes. These findings suggest trial clinics were representative of comparably sized clinics within the same health systems. Although results do not reflect generalizability more broadly, this study illustrates an approach to assess representativeness of clinics in future pragmatic primary care trials.

Abstract Whole-sporozoite (WSp) malaria vaccines induce protective immune responses in animal malaria models and in humans. A recent clinical trial with a WSp vaccine comprising genetically attenuated parasites (GAP) which arrest growth early in the liver (PfSPZ-GA1), showed that GAPs can be safely administered to humans and immunogenicity is comparable to radiation-attenuated PfSPZ Vaccine. GAPs that arrest late in the liver stage (LA-GAP) have potential for increased potency as shown in rodent malaria models. Here we describe the generation of four putative P. falciparum LA-GAPs, generated by CRISPR/Cas9-mediated gene deletion. One out of four gene-deletion mutants produced sporozoites in sufficient numbers for further preclinical evaluation. This mutant, PfΔmei2, lacking the mei2-like RNA gene, showed late liver growth arrest in human liver-chimeric mice with human erythrocytes, absence of unwanted genetic alterations and sensitivity to antimalarial drugs. These features of PfΔmei2 make it a promising vaccine candidate, supporting further clinical evaluation. PfΔmei2 (GA2) has passed regulatory approval for safety and efficacy testing in humans based on the findings reported in this study.

Abstract Antibodies against the Plasmodium falciparum circumsporozoite protein (PfCSP) can block hepatocyte infection by sporozoites and protect against malaria. Needle-free vaccination strategies are desirable, yet most PfCSP-targeted vaccines like RTS,S require needle-based administration. Here, we evaluated the edible algae, Arthrospira platensis (commonly called ‘spirulina’) as a malaria vaccine platform. Spirulina were genetically engineered to express virus-like particles (VLPs) consisting of the woodchuck hepatitis B core capsid protein (WHcAg) displaying a (NANP)15 PfCSP antigen on its surface. PfCSP-spirulina administered to mice intranasally followed by oral PfCSP-spirulina boosters resulted in a strong, systemic anti-PfCSP immune response that was protective against subcutaneous challenge with PfCSP-expressing P. yoelii. Unlike male mice, female mice did not require Montanide adjuvant to reach high antibody titers or protection. The successful use of spirulina as a vaccine delivery system warrants further development of spirulina-based vaccines as a useful tool in addressing malaria and other diseases of global health importance.

Abstract Microglial morphology is used to measure neuroinflammation and pathology. For reliable inference, it is critical that microglial morphology is accurately quantified and that results can be easily interpreted and compared across studies and laboratories. The process through which microglial morphology is quantified is a key methodological choice and little is known about how this choice may bias conclusions. We applied five of the most commonly used ImageJ-based methods for quantifying the microglial morphological response to a stimulus to identical photomicrographs and individual microglial cells isolated from these photomicrographs, which allowed for direct comparisons of results generated using these approaches. We found a lack of comparability across methods that analyzed full photomicrographs, with significant discrepancies in results among the five methods. Quantitative methods to analyze microglial morphology should be selected based on several criteria, and combinations of these methods may give the most biologically accurate representation of microglial morphology.

Plasmodium vivax is a malaria-causing pathogen that establishes a dormant form in the liver (the hypnozoite), which can activate weeks, months, or years after the primary infection to cause a relapse, characterized by secondary blood-stage infection. These asymptomatic and undetectable latent liver infections present a significant obstacle to the goal of global malaria eradication. We use a human liver-chimeric mouse model (FRG huHep) to study P. vivax hypnozoite latency and activation in an in vivo model system. Functional activation of hypnozoites and formation of secondary schizonts is demonstrated by first eliminating primary liver schizonts using a schizont-specific antimalarial tool compound, and then measuring recurrence of secondary liver schizonts in the tissue and an increase in parasite RNA within the liver. We also reveal that, while primaquine does not immediately eliminate hypnozoites from the liver, it arrests developing schizonts and prevents activation of hypnozoites, consistent with its clinical activity in humans. Our findings demonstrate that the FRG huHep model can be used to study the biology of P. vivax infection and latency and assess the activity of anti-relapse drugs.

Abstract Background Plasmodium falciparum (Pf) sporozoite (SPZ) vaccines are the only candidate malaria vaccines that induce > 90% vaccine efficacy (VE) against controlled human malaria infection and the only malaria vaccines to have achieved reproducible VE against malaria in adults in Africa. The goal is to increase the impact and reduce the cost of PfSPZ vaccines by optimizing vaccine potency and manufacturing, which will benefit from identification of immunological responses contributing to protection in humans. Currently, there is no authentic animal challenge model for assessing P. falciparum malaria VE. Alternatively, Plasmodium knowlesi (Pk), which infects humans and non-human primates (NHPs) in nature, can be used to experimentally infect rhesus macaques (Macaca mulatta) to assess VE. Methods Sanaria has, therefore, produced purified, vialed, cryopreserved PkSPZ and conducted challenge studies in several naïve NHP cohorts. In the first cohort, groups of three rhesus macaques each received doses of 5 × 102, 2.5 × 103, 1.25 × 104 and 2.5 × 104 PkSPZ administered by direct venous inoculation. The infectivity of 1.5 × 103 PkSPZ cryopreserved with an altered method and of 1.5 × 103 PkSPZ cryopreserved for four years was tested in a second and third cohort of rhesus NHPs. The lastly, three pig-tailed macaques (Macaca nemestrina), a natural P. knowlesi host, were challenged with 2.5 × 103 PkSPZ cryopreserved six years earlier. Results In the first cohort, all 12 animals developed P. knowlesi parasitaemia by thick blood smear, and the time to positivity (prepatent period) followed a non-linear 4-parameter logistic sigmoidal model with a median of 11, 10, 8, and 7 days, respectively (r2 = 1). PkSPZ cryopreserved using a modified rapid-scalable method infected rhesus with a pre-patent period of 10 days, as did PkSPZ cryopreserved four years prior to infection, similar to the control group. Cryopreserved PkSPZ infected pig-tailed macaques with median time to positivity by thin smear, of 11 days. Conclusion This study establishes the capacity to consistently infect NHPs with purified, vialed, cryopreserved PkSPZ, providing a foundation for future studies to probe protective immunological mechanisms elicited by PfSPZ vaccines that cannot be established in humans.

Plasmid DNA offers extended transgene expression duration compared to mRNA technologies. Here, using a multi-step screening platform, the authors report the best performing nanoparticle formulations for liver-targeted plasmid DNA expression in vivo.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ injury including testicular inflammation, reduced testosterone, and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells, however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury could be initiated by direct virus infection or exposure to systemic inflammatory mediators or viral antigens. We characterized SARS-CoV-2 infection in different human testicular 2D and 3D culture systems including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not productively infect any testicular cell type. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma decreased cell viability and resulted in the death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 Envelope protein caused inflammatory response and cytopathic effects dependent on TLR2, while Spike 1 or Nucleocapsid proteins did not. A similar trend was observed in the K18-hACE2 transgenic mice which demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis that correlated with peak lung inflammation. Virus antigens including Spike 1 and Envelope proteins were also detected in the serum during the acute stage of the disease. Collectively, these data strongly suggest that testicular injury associated with SARS-CoV-2 infection is likely an indirect effect of exposure to systemic inflammation and/or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

Abstract Vaccine-induced sterilizing protection from infection by Plasmodium parasites, the pathogens that cause malaria, will be essential in the fight against malaria as it would prevent both malaria-related disease and transmission. Stopping the relatively small number of parasites injected by the mosquito before they can migrate from the skin to the liver is an attractive means to this goal. Antibody-eliciting vaccines have been used to pursue this objective by targeting the major parasite surface protein present during this stage, the circumsporozoite protein (CSP). While CSP-based vaccines have recently had encouraging success in disease reduction, this was only achieved with extremely high antibody titers and appeared less effective for a complete block of infection (i.e., sterile protection). While such disease reduction is important, these and other results indicate that strategies focusing on CSP alone may not achieve the high levels of sterile protection needed for malaria eradication. Here, we show that monoclonal antibodies (mAbs) recognizing another sporozoite protein, TRAP/SSP2, exhibit a range of inhibitory activity and that these mAbs may augment CSP-based protection despite conferring no sterile protection on their own. Therefore, pursuing a multivalent subunit vaccine immunization is a promising strategy for improving infection-blocking malaria vaccines.