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The Navajo Nation (NN), a sovereign indigenous tribal nation in the Southwestern United States, is home to 523 abandoned uranium mines (AUMs). Previous health studies have articulated numerous human health hazards associated with AUMs and multiple environmental mechanisms/pathways (e.g., air, water, and soil) for contaminant transport. Despite this evidence, the limited modeling of AUM contamination that exists relies solely on proximity to mines and only considers single rather than combined pathways from which the contamination is a product. In order to better understand the spatial dynamics of contaminant exposure across the NN, we adopted the following established geospatial and computational methods to develop a more sophisticated environmental risk map illustrating the potential for AUM contamination: GIS-based multi-criteria decision analysis (GIS-MCDA), fuzzy logic, and analytic hierarchy process (AHP). Eight criteria layers were selected for the GIS-MCDA model: proximity to AUMs, roadway proximity, drainage proximity, topographic landforms, wind index, topographic wind exposure, vegetation index, and groundwater contamination. Model sensitivity was evaluated using the one-at-a-time method, and statistical validation analysis was conducted using two separate environmental datasets. The sensitivity analysis indicated consistency and reliability of the model. Model results were strongly associated with environmental uranium concentrations. The model classifies 20.2% of the NN as high potential for AUM contamination while 65.7% and 14.1% of the region are at medium and low risk, respectively. This study is entirely a novel application and a crucial first step toward informing future epidemiologic studies and ongoing remediation efforts to reduce human exposure to AUM waste.

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (VSV) containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (VSVSARS- CoV-2) elicited by Apilimod and Vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants 1 – 4 . SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells 5 – 9 . However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes. COVID-19 booster immunizations aimed at spike protein from new SARS-CoV-2 variants induce robust germinal centre B cell responses against the original spike protein, as well as de novo B cell responses against the variant spike protein.

Rapidly emerging SARS-CoV-2 variants jeopardize antibody-based countermeasures. Although cell culture experiments have demonstrated a loss of potency of several anti-spike neutralizing antibodies against variant strains of SARS-CoV-2 1 – 3 , the in vivo importance of these results remains uncertain. Here we report the in vitro and in vivo activity of a panel of monoclonal antibodies (mAbs), which correspond to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron and Lilly, against SARS-CoV-2 variant viruses. Although some individual mAbs showed reduced or abrogated neutralizing activity in cell culture against B.1.351, B.1.1.28, B.1.617.1 and B.1.526 viruses with mutations at residue E484 of the spike protein, low prophylactic doses of mAb combinations protected against infection by many variants in K18-hACE2 transgenic mice, 129S2 immunocompetent mice and hamsters, without the emergence of resistance. Exceptions were LY-CoV555 monotherapy and LY-CoV555 and LY-CoV016 combination therapy, both of which lost all protective activity, and the combination of AbbVie 2B04 and 47D11, which showed a partial loss of activity. When administered after infection, higher doses of several mAb cocktails protected in vivo against viruses with a B.1.351 spike gene. Therefore, many—but not all—of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2. Experiments in mouse and hamster models show that monoclonal antibody combinations, using antibodies that correspond to products in clinical development, largely retain their efficacy in protecting against currently prevailing variant strains of SARS-CoV-2.

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.

Medium containing Fetal Bovine Serum (FBS) provides a supportive environment for isolation and expansion of mesenchymal stromal/stem cells (MSCs); however, the inherent variability of FBS may contribute to inconsistencies in cell growth and yield between batches of stem cell products. For this reason, we set out to develop a serum-free medium capable of supporting the in vitro expansion of MSCs. First a naïve serum-free medium was formulated by Sato's approach. Once it was established that the naïve serum-free medium supported the expansion of canine adipose-derived MSCs (Ad-MSCs), the serum-free medium was optimized by addition of growth factors. Combinations of growth factors were chosen and compared by their effect on cell proliferation and colony formation. Growth characteristics of canine adipose-derived MSCs cultured in the serum-free medium were comparable to those cultured in standard FBS containing medium. In addition, cell surface marker expression and differentiation potential of serum-free and FBS-based cultures were also comparable. However, a commercial serum-free medium developed for human MSC culture did not support growth of canine Ad-MSCs. In summary, canine Ad-MSCs isolated and cultured in serum-free medium retained the basic characteristics of MSCs cultured in FBS containing medium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein—catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.

Clostridium difficile toxin A (TcdA) is a major exotoxin contributing to disruption of the colonic epithelium during C. difficile infection. TcdA contains a carbohydrate-binding combined repetitive oligopeptides (CROPs) domain that mediates its attachment to cell surfaces, but recent data suggest the existence of CROPs-independent receptors. Here, we carried out genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)-mediated screens using a truncated TcdA lacking the CROPs, and identified sulfated glycosaminoglycans (sGAGs) and low-density lipoprotein receptor (LDLR) as host factors contributing to binding and entry of TcdA. TcdA recognizes the sulfation group in sGAGs. Blocking sulfation and glycosaminoglycan synthesis reduces TcdA binding and entry into cells. Binding of TcdA to the colonic epithelium can be reduced by surfen, a small molecule that masks sGAGs, by GM-1111, a sulfated heparan sulfate analogue, and by sulfated cyclodextrin, a sulfated small molecule. Cells lacking LDLR also show reduced sensitivity to TcdA, although binding between LDLR and TcdA are not detected, suggesting that LDLR may facilitate endocytosis of TcdA. Finally, GM-1111 reduces TcdA-induced fluid accumulation and tissue damage in the colon in a mouse model in which TcdA is injected into the caecum. These data demonstrate in vivo and pathological relevance of TcdA–sGAGs interactions, and reveal a potential therapeutic approach of protecting colonic tissues by blocking these interactions. Clostridium difficile toxin A utilizes host sulfated glycosaminoglycans and low-density lipoprotein receptor for host cell entry and intoxication.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to persist, demonstrating the risks posed by emerging infectious diseases to national security, public health, and the economy. Development of new vaccines and antibodies for emerging viral threats requires substantial resources and time, and traditional development platforms for vaccines and antibodies are often too slow to combat continuously evolving immunological escape variants, reducing their efficacy over time. Previously, we designed a next-generation synthetic humanized nanobody (Nb) phage display library and demonstrated that this library could be used to rapidly identify highly specific and potent neutralizing heavy chain-only antibodies (HCAbs) with prophylactic and therapeutic efficacy in vivo against the original SARS-CoV-2. In this study, we used a combination of high throughput screening and machine learning (ML) models to identify HCAbs with potent efficacy against SARS-CoV-2 viral variants of interest (VOIs) and concern (VOCs). To start, we screened our highly diverse Nb phage display library against several pre-Omicron VOI and VOC receptor binding domains (RBDs) to identify panels of cross-reactive HCAbs. Using HCAb affinity for SARS-CoV-2 VOI and VOCs (pre-Omicron variants) and model features from other published data, we were able to develop a ML model that successfully identified HCAbs with efficacy against Omicron variants, independent of our experimental biopanning workflow. This biopanning informed ML approach reduced the experimental screening burden by 78% to 90% for the Omicron BA.5 and Omicron BA.1 variants, respectively. The combined approach can be applied to other emerging viruses with pandemic potential to rapidly identify effective therapeutic antibodies against emerging variants.

Rocky Mountain Spotted Fever (RMSF) is a potentially fatal tick-borne disease historically prevalent in the eastern and southeastern U.S. Since the early 2000s, there has been a notable rise in RMSF cases in the south-western U.S. Despite the documented role of dogs in tick-borne disease transmission, research on the influence of other factors, such as veterinary care access, climatic conditions and landscape characteristics on RMSF incidence is limited. This study investigated the combined impact of these factors on RMSF using county-level temperature, relative humidity, precipitation, land cover, dog populations and veterinary care access in Arizona from 2006 to 2021. Employing a spatial negative binomial regression model, the study revealed significant associations between veterinary care access, precipitation, relative humidity, shrubland, and RMSF incidence across three models incorporating lagged effects (0-month, 1-month, and 2-month) for climatic variables. A key finding was that counties experiencing higher veterinary care access were more likely to report lower RMSF case counts (incidence rate ratio (IRR): 0.9237). The mean precipitation consistently showed the highest positive IRR (1.8137) across all models, indicating its strong influence. In contrast, relative humidity (IRR: 0.9413) and shrubland presence (IRR: 0.9265) demonstrated significant negative associations with RMSF incidence. These findings underscore the importance of veterinary care access, climatic factors, and land cover in shaping RMSF dynamics, particularly in regions with increasing incidence rates.