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CD4+ T cells have orchestrated and regulated immunity since the introduction of jawed vertebrates, yet our understanding of CD4+ T cell evolution, development, and cellular physiology has only begun to be unearthed in the past few decades. Discoveries of genetic diseases that ablate this cellular population have provided insight into their critical functions while transcriptomics, proteomics, and high-resolution microscopy have recently revealed new insights into CD4+ T cell anatomy and physiology. This article compiles historical, microscopic, and multi-omics data that can be used as a reference atlas and index to dissect cellular physiology within these influential cells and further understand pathologies like HIV infection that inflict human CD4+ T cells.

AIM: To report 4 cases of Cryptococcus gattii (C. gattii) species complex infection with diverse ophthalmic manifestations, and to review the literature to examine pathobiology of disease, classical ophthalmic presentations and outcomes, and treatment modalities for this emerging pathogen. METHODS: Cases of C. gattii meningoencephalitis with ophthalmic manifestations were identified via chart review at two institutions in Australia and one institution in the mid-west region of the United States and are reported as a case series. Additionally, a MEDLINE literature review was conducted to identify all reported cases of C. gattii with ophthalmic manifestations from 1990-2020. Cases were reviewed and tabulated, together with our series of patients, in this report. RESULTS: Four cases of C. gattii with ophthalmic manifestations are presented; three from Australia and one from the USA. A literature review identified a total of 331 cases of C. gattii with visual sequelae. The majority of cases occurred in immunocompetent individuals. Blurred vision and diplopia were the most common presenting symptoms, with papilloedema the most common sign, reported in 10%-50% of cases. Visual loss was reported in 10%-53% of cases, as compared to rates of visual loss of 1%-9% in C. neoformans infection. Elevated intracranial pressure, cerebrospinal fluid (CSF) fungal burden, and abnormal neurological exam at presentation correlated with poor visual outcomes. The mainstays of treatment are anti-fungal agents and aggressive management of intracranial hypertension with serial lumbar punctures. CSF diversion procedures should be considered for refractory cases. Acetazolamide and mannitol are associated with high complication rates, and adjuvant corticosteroids have demonstrated higher mortality rates; these treatments should be avoided. CONCLUSION: Permanent visual loss represents a devastating yet potentially preventable sequelae of C. gattii infection. Intracranial hypertension needs to be recognised early and aggressively managed. Referral to an ophthalmologist/neuro-ophthalmologist in all cases of cryptococcal infection independent of visual symptoms at time of diagnosis is recommended.

Abstract Background Genome-wide association studies (GWAS) have identified multiple common breast cancer susceptibility variants. Many of these variants have differential associations by estrogen receptor (ER) status, but how these variants relate with other tumor features and intrinsic molecular subtypes is unclear. Methods Among 106,571 invasive breast cancer cases and 95,762 controls of European ancestry with data on 173 breast cancer variants identified in previous GWAS, we used novel two-stage polytomous logistic regression models to evaluate variants in relation to multiple tumor features (ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and grade) adjusting for each other, and to intrinsic-like subtypes. Results Eighty-five of 173 variants were associated with at least one tumor feature (false discovery rate

Dennis et al. investigate potential breast cancer associations with rare germline copy number variants (CNVs) by conducting a genome-wide analysis in a large breast cancer case-control dataset. The authors detected associations with exonic deletions in established breast cancer susceptibility genes and suggestive associations for a number of non-coding CNVs.

ABSTRACT Transposable elements (TEs) are mobile genomic sequences that encompass roughly 50% of the human genome. Class 1 TEs, or “retrotransposons,” mobilize through the production of an RNA intermediate that is then reverse transcribed to form complementary DNA (cDNA) molecules capable of genomic reinsertion. While TEs are traditionally silenced to maintain genomic integrity, the recognition of immunostimulatory cues, such as those provided by microorganisms, drastically alters host transcription to induce the differential expression of TEs. Emerging evidence demonstrates that the inducible production of TE cDNA is not an inert phenomenon but instead has been coopted by host immunity to facilitate cross talk between host and constituents of the microbiota by agonizing intrinsic antiviral receptors. Here, we demonstrate that immunostimulation of toll-like receptor 4 (TLR4) with lipopolysaccharide (LPS) and TLR5 with bacterial flagella (FLA) alters the expression of retrotransposons, such as human endogenous retroviruses (HERVs) and long interspersed nuclear elements (LINEs). Next, we demonstrate that reverse transcriptase inhibitor (RTi) delivery ameliorates the acute production of the proinflammatory cytokine “tumor necrosis factor alpha” (TNF-α) in response to FLA in a monocytic cell line (THP-1). Collectively, our findings demonstrate that TLR5-mediated cross talk between the host and microbiota is partially dependent on the reverse transcription (RT) of retrotransposons. IMPORTANCE The microbiota is a potent reservoir of immunostimulatory and immunosuppressive motifs that fundamentally shape host immunity. Despite broad associations between microbial composition and host immunity, the mechanisms underlying host microbiota-induced immunoregulation remain poorly defined. Here, we demonstrate a novel mechanism by which motifs overabundant during dysbiotic conditions influence host immunity through the upregulation of endogenous RT to produce motifs that agonize antiviral receptors.

Abstract Introduction Exposure to patient death places healthcare workers at increased risk for burnout and traumatic stress, yet limited data exist exploring exposure to death among emergency medical services (EMS) clinicians. Our objective was to describe changes in EMS encounters involving on‐scene death from 2018 to 2021. Methods We retrospectively analyzed deidentified EMS records for 9‐1‐1 responses from the ESO Data Collaborative from 2018 to 2021. We identified cases where patient dispositions of death on scene, with or without attempted resuscitation, and without EMS transport. A non‐parametric test of trend was used to assess for monotonic increase in agency‐level encounters involving on‐scene death and the proportion of EMS clinicians exposed to ≥1 on‐scene death. Results We analyzed records from 1109 EMS agencies. These agencies responded to 4,286,976 calls in 2018, 5,097,920 calls in 2019, 4,939,651 calls in 2020, and 5,347,340 calls in 2021.The total number of encounters with death on scene rose from 49,802 in 2018 to 60,542 in 2019 to 76,535 in 2020 and 80,388 in 2021. Agency‐level annual counts of encounters involving death on scene rose from a median of 14 (interquartile range [IQR], 4–40) in 2018 to 2023 (IQR, 6–63) in 2021 (P‐trend < 0.001). In 2018, 56% of EMS clinicians responded to a call with death on scene, and this number rose to 63% of EMS clinicians in 2021 (P‐trend < 0.001). Conclusion From 2018 to 2021, EMS clinicians were increasingly exposed to death. This trend may be driven by COVID‐19 and its effects on the healthcare system and reinforces the need for evidence‐based death notification training to support EMS clinicians.

Natamycin is an antifungal polyene macrolide that is used as a food preservative but also to treat fungal keratitis and other yeast infections. In contrast to other polyene antimycotics, natamycin does not form ion pores in the plasma membrane, but its mode of action is poorly understood. Using nuclear magnetic resonance (NMR) spectroscopy of deuterated sterols, we find that natamycin slows the mobility of ergosterol and cholesterol in liquid-ordered (Lo) membranes to a similar extent. This is supported by molecular dynamics (MD) simulations, which additionally reveal a strong impact of natamycin dimers on sterol dynamics and water permeability. Interference with sterol-dependent lipid packing is also reflected in a natamycin-mediated increase in membrane accessibility for dithionite, particularly in bilayers containing ergosterol. NMR experiments with deuterated sphingomyelin (SM) in sterol-containing membranes reveal that natamycin reduces phase separation and increases lipid exchange in bilayers with ergosterol. In ternary lipid mixtures containing monounsaturated phosphatidylcholine, saturated SM, and either ergosterol or cholesterol, natamycin interferes with phase separation into Lo and liquid-disordered (Ld) domains, as shown by NMR spectroscopy. Employing the intrinsic fluorescence of natamycin in ultraviolet-sensitive microscopy, we can visualize the binding of natamycin to giant unilamellar vesicles (GUVs) and find that it has the highest affinity for the Lo phase in GUVs containing ergosterol. Our results suggest that natamycin specifically interacts with the sterol-induced ordered phase, in which it disrupts lipid packing and increases solvent accessibility. This property is particularly pronounced in ergosterol containing membranes, which could underlie the selective antifungal activity of natamycin.

The coastal waters of southern British Columbia, Canada, encompass habitat of international conservation significance to coastal and marine birds, including sizeable areas designated in the early 1900s as Migratory Bird Sanctuaries (MBS) to protect overwintering waterfowl from hunting near urban centres. Two of these, Shoal Harbour (SHMBS) and Victoria Harbour (VHMBS), have seen significant marine infrastructure development in recent decades and experience considerable vessel traffic. Vessel-related stressors are known to affect waterbirds, but traffic characteristics in coastal urban areas are poorly understood for the smaller vessels not tracked by Automatic Identification Systems (AIS). We conducted a pilot study using shore-based observers to develop small-vessel baselines for the winter months, when regional waterbird numbers are highest. During our surveys we recorded considerable inter-site variability in vessel traffic characteristics, with one site (SHMBS) a source of nearly twice as many vessel transits as the other (VHMBS). Most recorded vessels were small watercraft (mean length 26 ± 17', mode 18'), and vessels at the high-traffic site were both shorter and faster on average. One in six vessels were classified as 'noisy', of interest given that noise is an important component of vessel disturbance of waterbirds and other marine animals. Few vessels (7% of all recorded) were of the type required to carry AIS transponders, which highlights the monitoring gap created by using AIS-based approaches alone in nearshore waters, and allows for correction of AIS-derived vessel counts. Waterbird community composition also varied by locality, with one site dominated by gulls (Laridae), cormorants (Phalacrocoracidae), and seaducks (Tribe Mergini), and the other by gulls, cormorants, and alcids (Alcidae). Our results demonstrate that fine-scale local variability must be taken into account when managing for vessel traffic disturbance of waterbirds, particularly at sites of high human population density and increasing coastal development.

Abstract Background Mutations in certain genes are known to increase breast cancer risk. We study the relevance of rare protein-truncating variants (PTVs) that may result in loss-of-function in breast cancer susceptibility genes on tumor characteristics and survival in 8852 breast cancer patients of Asian descent. Methods Gene panel sequencing was performed for 34 known or suspected breast cancer predisposition genes, of which nine genes (ATM, BRCA1, BRCA2, CHEK2, PALB2, BARD1, RAD51C, RAD51D, and TP53) were associated with breast cancer risk. Associations between PTV carriership in one or more genes and tumor characteristics were examined using multinomial logistic regression. Ten-year overall survival was estimated using Cox regression models in 6477 breast cancer patients after excluding older patients (≥75years) and stage 0 and IV disease. Results PTV9genes carriership (n = 690) was significantly associated (p < 0.001) with more aggressive tumor characteristics including high grade (poorly vs well-differentiated, odds ratio [95% confidence interval] 3.48 [2.35–5.17], moderately vs well-differentiated 2.33 [1.56–3.49]), as well as luminal B [HER−] and triple-negative subtypes (vs luminal A 2.15 [1.58–2.92] and 2.85 [2.17–3.73], respectively), adjusted for age at diagnosis, study, and ethnicity. Associations with grade and luminal B [HER2−] subtype remained significant after excluding BRCA1/2 carriers. PTV25genes carriership (n = 289, excluding carriers of the nine genes associated with breast cancer) was not associated with tumor characteristics. However, PTV25genes carriership, but not PTV9genes carriership, was suggested to be associated with worse 10-year overall survival (hazard ratio [CI] 1.63 [1.16–2.28]). Conclusions PTV9genes carriership is associated with more aggressive tumors. Variants in other genes might be associated with the survival of breast cancer patients. The finding that PTV carriership is not just associated with higher breast cancer risk, but also more severe and fatal forms of the disease, suggests that genetic testing has the potential to provide additional health information and help healthy individuals make screening decisions.

Hedgehog-Interacting Protein (HHIP) is the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling. Here, the authors report structures of the HHIP N- and C-terminal domains, both in complexes with glycosaminoglycans, providing insights into the molecular basis for SHH sequestration and inhibition.

Enteroaggregative Escherichia coli (EAEC) is a common diarrhoeagenic human pathogen, isolated from patients in both developing and industrialized countries, that is becoming increasingly resistant to many frontline antibiotics. In this study, we screened 50 E. coli strains from children presenting with diarrhea at the outpatients clinic of Assiut University Children’s Hospital, Egypt. We show that all of these isolates were resistant to multiple classes of antibiotics and identified two as being typical EAEC strains. Using whole genome sequencing, we determined that both isolates carried, amongst others, blaCTX-M and blaTEM antibiotic resistance genes, as well as many classical EAEC virulence determinants, including the transcriptional regulator, AggR. We demonstrate that the expression of these virulence determinants is dependent on AggR, including aar, which encodes for a repressor of AggR, Aar. Since biofilm formation is the hallmark of EAEC infection, we examined the effect of Aar overexpression on both biofilm formation and AggR-dependent gene expression. We show that whilst Aar has a minimal effect on AggR-dependent transcription it is able to completely disrupt biofilm formation, suggesting that Aar affects these two processes differently. Taken together, our results suggest a model for the induction of virulence gene expression in EAEC that may explain the ubiquity of EAEC in both sick and healthy individuals.

The claim is sometimes made (and also contested) that, following contributions of Karl Barth and Karl Rahner, there has been a renewal of interest in the doctrine of the Trinity among Christian theologians. This essay further develops the metaphysically circumspect or ‘low-flying’ trinitarian theology I present in A Theology for the Twenty-First Century. Drawing on the work of Donald Baillie, B. A. Gerrish, James M. Gustafson, Hans Küng, H. Richard Niebuhr, Friedrich Schleiermacher, Paul Tillich and others, I argue that such a theology better accords with the New Testament than many realise, and that it has a broad and robust significance for theological ethics. Whether my theology contributes to the recent renewal of interest, however, depends on whether one judges that explicitly revisionary critics of orthodoxy’s metaphysical heights qualify as trinitarian theologians. Intradisciplinary and/or interdisciplinary implications: The essay indicates that traditional assumptions about trinitarian claims in the New Testament are too narrow, that it is important to attend to the fuller range of trinitarian reflections in the field of Church History and that a pragmatic, metaphysically circumspect trinitarian theology has profound implications for Christian life and faithfulness.

Abstract Lithium is a first-line treatment for bipolar disorder, where it acts as a mood-stabilizing agent. Although its precise mechanism remains unclear, neuroimaging studies have shown that lithium accumulates in the hippocampus and that chronic use amongst bipolar disorder patients is associated with larger hippocampal volumes. Here, we tested the chronic effects of low (0.75 mM) and high (2.25 mM) doses of lithium on human hippocampal progenitor cells and used immunocytochemistry to investigate the effects of lithium on cell parameters implicated in neurogenesis. Corresponding RNA-sequencing and gene-set enrichment analyses were used to evaluate whether genes affected by lithium in our model overlap with those regulating the volume of specific layers of the dentate gyrus. We observed that high-dose lithium treatment in human hippocampal progenitors increased the generation of neuroblasts (P ≤ 0.01), neurons (P ≤ 0.01), and glia (P ≤ 0.001), alongside the expression of genes, which regulate the volume of the molecular layer of the dentate gyrus. This study provides empirical support that adult hippocampal neurogenesis and gliogenesis are mechanisms that could contribute to the effects of lithium on human hippocampal volume.

Abstract Breast cancer metastasis accounts for most of the deaths from breast cancer. Identification of germline variants associated with survival in aggressive types of breast cancer may inform understanding of breast cancer progression and assist treatment. In this analysis, we studied the associations between germline variants and breast cancer survival for patients with distant metastases at primary breast cancer diagnosis. We used data from the Breast Cancer Association Consortium (BCAC) including 1062 women of European ancestry with metastatic breast cancer, 606 of whom died of breast cancer. We identified two germline variants on chromosome 1, rs138569520 and rs146023652, significantly associated with breast cancer-specific survival (P = 3.19 × 10−8 and 4.42 × 10−8). In silico analysis suggested a potential regulatory effect of the variants on the nearby target genes SDE2 and H3F3A. However, the variants showed no evidence of association in a smaller replication dataset. The validation dataset was obtained from the SNPs to Risk of Metastasis (StoRM) study and included 293 patients with metastatic primary breast cancer at diagnosis. Ultimately, larger replication studies are needed to confirm the identified associations.

Abstract Background Vaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited. Methods We used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19. Results Out of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2’s main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model. Conclusions Small clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.

Objective: To describe variables used by Saudi pediatric intensivists to make antibiotic-related decisions for children with suspected severe bacterial infections. Methods: We conducted a cross-sectional survey, which was developed using a multi-step methodological approach. The survey included 4 clinical scenarios of the most relevant bacterial infections in pediatric critical care (pneumonia, sepsis, meningitis and intra-abdominal infection). The potential determinants of antibiotic treatment duration addressed in all scenarios included clinical variables (patient characteristics, disease severity), laboratory infection markers, radiologic findings, and pathogens. Results: The response rate was 65% (55/85). Eight variables (immunodeficiency, 3 months of age, 2 or more organ dysfunctions, Pediatric Risk of Mortality III score >10, leukocytosis, elevated C-reactive protein [CRP], elevated erythrocyte sedimentation rate [ESR], and elevated procalcitonin [PCT]) were associated with prolonging antibiotic treatment duration for all 4 clinical scenarios, with a median increase ranging from 3.0 days (95% confidence interval [CI] 0.5, 3.5, leukocytosis) to 8.8 days (95% CI 5.5, 10.5, immunodeficiency). There were no variables that were consistently associated with shortening antibiotic duration across all scenarios. Lastly, the proportion of physicians who would continue antibiotics for ≥5 days despite a positive viral polymerase chain reaction test result was 67% for pneumonia, 85% for sepsis, 63% for meningitis, and 95% for intra-abdominal infections. Conclusion: Antibiotic-related decisions for critically ill patients are complex and depend on several factors. Saudi pediatric intensivists will use prolonged courses of antibiotics for younger patients, patients with severe clinical picture, and patients with persistently elevated laboratory markers and hospital acquired infections, even when current literature and guidelines do not suggest such practices. Antimicrobial stewardship programs should include interventions to address these misconceptions to ensure the rational use of antibiotics in pediatric intensive care units.

Abstract The ongoing global COVID-19 pandemic has thrown into sharp relief the gap between modern biology’s ability to investigate and respond to a novel pathogen and modern medicine’s ability to marshal effective front-line interventions to limit its immediate health impact. While we have witnessed the rapid development of innovative vaccines against SARS-CoV-2 using novel molecular platforms, these have yet to alter the pandemic’s long-term trajectory in all but a handful of high-income countries. Health workers at the clinical front lines have little more in their clinical armamentarium than was available a century ago—chiefly oxygen and steroids—and yet advances in modern immunology and immunotherapeutics suggest an underuse of extant and effective, if unorthodox, therapies, which we now call “Extreme Immunotherapies for Pandemics (EIPs).”

Abstract Structural variation in the complement 4 gene (C4) confers genetic risk for schizophrenia. The variation includes numbers of the increased C4A copy number, which predicts increased C4A mRNA expression. C4-anaphylatoxin (C4-ana) is a C4 protein fragment released upon C4 protein activation that has the potential to change the blood–brain barrier (BBB). We hypothesized that elevated plasma levels of C4-ana occur in individuals with schizophrenia (iSCZ). Blood was collected from 15 iSCZ with illness duration < 5 years and from 14 healthy controls (HC). Plasma C4-ana was measured by radioimmunoassay. Other complement activation products C3-ana, C5-ana, and terminal complement complex (TCC) were also measured. Digital-droplet PCR was used to determine C4 gene structural variation state. Recombinant C4-ana was added to primary brain endothelial cells (BEC) and permeability was measured in vitro. C4-ana concentration was elevated in plasma from iSCZ compared to HC (mean = 654 ± 16 ng/mL, 557 ± 94 respectively, p = 0.01). The patients also carried more copies of the C4AL gene and demonstrated a positive correlation between plasma C4-ana concentrations and C4A gene copy number. Furthermore, C4-ana increased the permeability of a monolayer of BEC in vitro. Our findings are consistent with a specific role for C4A protein in schizophrenia and raise the possibility that its activation product, C4-ana, increases BBB permeability. Exploratory analyses suggest the novel hypothesis that the relationship between C4-ana levels and C4A gene copy number could also be altered in iSCZ, suggesting an interaction with unknown genetic and/or environmental risk factors.

Abstract Background Advancements in cancer therapeutics have resulted in increases in cancer-related survival; however, there is a growing clinical dilemma. The current balancing of survival benefits and future cardiotoxic harms of oncotherapies has resulted in an increased burden of cardiovascular disease in breast cancer survivors. Risk stratification may help address this clinical dilemma. This study is the first to assess the association between a coronary artery disease-specific polygenic risk score and incident coronary artery events in female breast cancer survivors. Methods We utilized the Studies in Epidemiology and Research in Cancer Heredity prospective cohort involving 12,413 women with breast cancer with genotype information and without a baseline history of cardiovascular disease. Cause-specific hazard ratios for association of the polygenic risk score and incident coronary artery disease (CAD) were obtained using left-truncated Cox regression adjusting for age, genotype array, conventional risk factors such as smoking and body mass index, as well as other sociodemographic, lifestyle, and medical variables. Results Over a median follow-up of 10.3 years (IQR: 16.8) years, 750 incident fatal or non-fatal coronary artery events were recorded. A 1 standard deviation higher polygenic risk score was associated with an adjusted hazard ratio of 1.33 (95% CI 1.20, 1.47) for incident CAD. Conclusions This study provides evidence that a coronary artery disease-specific polygenic risk score can risk-stratify breast cancer survivors independently of other established cardiovascular risk factors.

Human Immunodeficiency virus type 1 (HIV-1) relies on host cell metabolism for all aspects of viral replication. Efficient HIV-1 entry, reverse transcription, and integration occurs in activated T cells because HIV-1 proteins co-opt host metabolic pathways to fuel the anabolic requirements of virion production. The HIV-1 viral life cycle is especially dependent on mTOR, which drives signaling and metabolic pathways required for viral entry, replication, and latency. As a central regulator of host cell metabolism, mTOR and its downstream effectors help to regulate the expression of enzymes within the glycolytic and pentose phosphate pathways along with other metabolic pathways regulating amino acid uptake, lipid metabolism, and autophagy. In HIV-1 pathogenesis, mTOR, in addition to HIF-1α and Myc signaling pathways, alter host cell metabolism to create an optimal environment for viral replication. Increased glycolysis and pentose phosphate pathway activity are required in the early stages of the viral life cycle, such as providing sufficient dNTPs for reverse transcription. In later stages, fatty acid synthesis is required for creating cholesterol and membrane lipids required for viral budding. Epigenetics of the provirus fueled by metabolism and mTOR signaling likewise controls active and latent infection. Acetyl-CoA and methyl group abundance, supplied by the TCA cycle and amino acid uptake respectively, may regulate latent infection and reactivation. Thus, understanding and exploring new connections between cellular metabolism and HIV-1 pathogenesis may yield new insights into the latent viral reservoirs and fuel novel treatments and cure strategies.

ABSTRACT Coccidioidomycosis, also called valley fever (VF), is a fungal infection with endemicity in desert regions of the western United States as well as certain arid regions of Central and South America. Laboratory-based diagnosis of VF often relies on the composite results from three serologic-based diagnostics, complement fixation, immunodiffusion, and enzyme immunoassay (EIA). EIA is commonly performed in clinical laboratories because results can be obtained in a few hours. Two commercially available EIAs, IMMY clarus Coccidioides antibody and Meridian Premier Coccidioides, look for the presence of anticoccidioidal IgG and IgM in patient sera that are diluted 1:441. Per regulatory requirements, this dilution step must be verified with a dilution step control despite not being provided as a reagent in either FDA-approved EIA kit. Therefore, clinical laboratories collect and reuse patient sera in subsequent tests that had a positive result in a previous test. This is a nonstandard process, reinforcing the need for a consistent and reliable dilution control. Here, we evaluate the performance of a humanized IgG and IgM antibody as a dilution control in both EIA kits. Both humanized IgG and IgM work well in each EIA and meet the appropriate threshold for positivity. IMPORTANCE In southwestern and western regions of the United States, at least half a million diagnostic tests for coccidioidomycosis (valley fever) are run annually. Enzyme immunoassays (EIAs) are blood tests which require precise dilution of patient serum prior to testing. To ensure patient serum is properly diluted, there is a regulatory requirement to ensure the dilution step is accurate. Two FDA-approved EIAs used to aid in the diagnosis of coccidioidomycosis do not contain controls for this dilution step, leaving clinical laboratories with the only option of using previously positive patient sera, which may not react in a reliable or predictable manner. Here, we evaluate a humanized monoclonal antibody against a coccidioidal antigen and its utility as a dilution control in both available commercial EIAs. The use of a humanized monoclonal antibody provides a standardized and well-characterized dilution control for use in serological assays that aid in diagnosis of coccidioidomycosis.

Summary: The host genetic factors conferring protection against HIV type 1 (HIV-1) acquisition remain elusive, and in particular the contributions of common genetic variants. Here, we performed the largest genome-wide association meta-analysis of HIV-1 acquisition, which included 7,303 HIV-1-positive individuals and 587,343 population controls. We identified 25 independent genetic loci with suggestive association, of which one was genome-wide significant within the major histocompatibility complex (MHC) locus. After exclusion of the MHC signal, linkage disequilibrium score regression analyses revealed a SNP heritability of 21% and genetic correlations with behavioral factors. A transcriptome-wide association study identified 15 susceptibility genes, including HERC1, UEVLD, and HIST1H4K. Convergent evidence from conditional analyses and fine-mapping identified HERC1 downregulation in immune cells as a robust mechanism associated with HIV-1 acquisition. Functional studies on HERC1 and other identified candidates, as well as larger genetic studies, have the potential to further our understanding of the host mechanisms associated with protection against HIV-1.

Monoclonal antibodies (mAbs) are important proteins used in many life science applications, from diagnostics to therapeutics. High demand for mAbs for different applications urges the development of rapid and reliable recombinant production platforms. Plants provide a quick and inexpensive system for producing recombinant mAbs. Moreover, when paired with an established platform for mAb discovery, plants can easily be tailored to produce mAbs of different isotypes against the same target. Here, we demonstrate that a hybridoma-generated mouse mAb against chitinase 1 (CTS1), an antigen from Coccidioides spp., can be biologically engineered for use with serologic diagnostic test kits for coccidioidomycosis (Valley Fever) using plant expression. The original mouse IgG was modified and recombinantly produced in glycoengineered Nicotiana benthamiana plants via transient expression as IgG and IgM isotypes with human kappa, gamma, and mu constant regions. The two mAb isotypes produced in plants were shown to maintain target antigen recognition to CTS1 using similar reagents as the Food and Drug Administration (FDA)-approved Valley Fever diagnostic kits. As none of the currently approved kits provide antibody dilution controls, humanization of antibodies that bind to CTS1, a major component of the diagnostic antigen preparation, may provide a solution to the lack of consistently reactive antibody controls for Valley Fever diagnosis. Furthermore, our work provides a foundation for reproducible and consistent production of recombinant mAbs engineered to have a specific isotype for use in diagnostic assays.

Abstract Background Given the high heterogeneity among breast tumors, associations between common germline genetic variants and survival that may exist within specific subgroups could go undetected in an unstratified set of breast cancer patients. Methods We performed genome-wide association analyses within 15 subgroups of breast cancer patients based on prognostic factors, including hormone receptors, tumor grade, age, and type of systemic treatment. Analyses were based on 91,686 female patients of European ancestry from the Breast Cancer Association Consortium, including 7531 breast cancer-specific deaths over a median follow-up of 8.1 years. Cox regression was used to assess associations of common germline variants with 15-year and 5-year breast cancer-specific survival. We assessed the probability of these associations being true positives via the Bayesian false discovery probability (BFDP

GWAS have enhanced our understanding for the genetic basis of breast cancer, but the majority of them were performed for European ancestry populations. Here, the authors use a cross-ancestry approach and report seven new variants associated with breast cancer risk among women of African ancestry.

The aim of this paper is to demonstrate how Bandura's Social Cognitive Theory (1986) influenced the development of several complementary models of the construct of Self-Regulation. Building on the foundation of Self-Determination Theory, SDT (2000), and Zimmerman's Self-Regulation Theory, SR (2001), with their assumptions, contributions, goddesses, and limitations, we come to the Self- vs. External Regulatory Theory, SR-ER (2021). Finally, we integrate recent evidence demonstrating the explanatory adequacy of the SR vs. ER model for different psychological constructions in different settings related to education, health, clinical practice and social work. Complementary, a new theoretical and empirical research agenda is presented, to continue testing the adequacy of SR vs. ER assumptions, and to better understand the behavioral variability of the different constructs studied.

Abstract While vaccines traditionally have been designed and used for protection against infection or disease caused by one specific pathogen, there are known off-target effects from vaccines that can impact infection from unrelated pathogens. The best-known non-specific effects from an unrelated or heterologous vaccine are from the use of the Bacillus Calmette-Guérin (BCG) vaccine, mediated partly through trained immunity. Other vaccines have similar heterologous effects. This review covers molecular mechanisms behind the heterologous effects, and the potential use of heterologous vaccination in the current COVID-19 pandemic. We then discuss novel pandemic response strategies based on rapidly deployed, widespread heterologous vaccination to boost population-level immunity for initial, partial protection against infection and/or clinical disease, while specific vaccines are developed.

Abstract New glass compositions containing high concentrations of Tb3+ ions were developed aiming at the production of magneto-optical (MO) fibers. This work reports on the structural and MO properties of a new glass composition based on (100 − x)(41GeO2–25B2O3–4Al2O3–10Na2O–20BaO) − xTb4O7. Morphological analysis (HR-TEM) of the sample with the highest concentration of Tb3+ ions confirmed the homogeneous distribution of Tb3+ ions and the absence of nanoclusters. All the samples presented excellent thermal stability against crystallization (ΔT > 100 °C). An optical fiber was manufactured by a fiber drawing process. The UV–Vis spectra of the glasses showed Tb3+ electronic transitions and optical windows varying from 0.4 to 1.6 μm. The magneto-optical properties and the paramagnetic behaviors of the glasses were investigated using Faraday rotation experiments. The Verdet constant (VB) values were calculated at 500, 650, 880, 1050, 1330, and 1550 nm. The maximum VB values obtained at 650 and 1550 nm for the glass with x = 18 mol% were -128 and − 17.6 rad T−1 m−1, respectively. The VB values at 500 and 1550 nm for the optical fiber containing 8 mol% of Tb4O7 were − 110.2 and − 9.5 rad T−1 m−1, respectively, while the optical loss at around 880 nm was 6.4 dB m−1.

Abstract Bi-allelic loss-of-function (LoF) variants in the base excision repair (BER) gene NTHL1 cause a high-risk hereditary multi-tumor syndrome that includes breast cancer, but the contribution of heterozygous variants to hereditary breast cancer is unknown. An analysis of 4985 women with breast cancer, enriched for familial features, and 4786 cancer-free women revealed significant enrichment for NTHL1 LoF variants. Immunohistochemistry confirmed reduced NTHL1 expression in tumors from heterozygous carriers but the NTHL1 bi-allelic loss characteristic mutational signature (SBS 30) was not present. The analysis was extended to 27,421 breast cancer cases and 19,759 controls from 10 international studies revealing 138 cases and 93 controls with a heterozygous LoF variant (OR 1.06, 95% CI: 0.82–1.39) and 316 cases and 179 controls with a missense variant (OR 1.31, 95% CI: 1.09–1.57). Missense variants selected for deleterious features by a number of in silico bioinformatic prediction tools or located within the endonuclease III functional domain showed a stronger association with breast cancer. Somatic sequencing of breast cancers from carriers indicated that the risk associated with NTHL1 appears to operate through haploinsufficiency, consistent with other described low-penetrance breast cancer genes. Data from this very large international multicenter study suggests that heterozygous pathogenic germline coding variants in NTHL1 may be associated with low- to moderate- increased risk of breast cancer.

Abstract Allogeneic mesenchymal stromal cells (MSCs) have been used clinically for decades, without cross‐matching, on the assumption that they are immune‐privileged. In the equine model, we demonstrate innate and adaptive immune responses after repeated intra‐articular injection with major histocompatibility complex (MHC) mismatched allogeneic MSCs, but not MHC matched allogeneic or autologous MSCs. We document increased peri‐articular edema and synovial effusion, increased synovial cytokine and chemokine concentrations, and development of donor‐specific antibodies in mismatched recipients compared with recipients receiving matched allogeneic or autologous MSCs. Importantly, in matched allogeneic and autologous recipients, but not mismatched allogeneic recipients, there was increased stromal derived factor‐1 along with increased MSC concentrations in synovial fluid. Until immune recognition of MSCs can be avoided, repeated clinical use of MSCs should be limited to autologous or cross‐matched allogeneic MSCs. When non–cross‐matched allogeneic MSCs are used in single MSC dose applications, presensitization against donor MHC should be assessed.

Abstract Mitochondria receive cholesterol from late endosomes and lysosomes (LE/LYSs) or from the plasma membrane for production of oxysterols and steroid hormones. This process depends on the endo-lysosomal sterol transfer protein Niemann Pick C2 (NPC2). Using the intrinsically fluorescent cholesterol analog, cholestatrienol, we directly observe sterol transport to mitochondria in fibroblasts upon treating NPC2 deficient human fibroblasts with NPC2 protein. Soft X-ray tomography reveals the ultrastructure of mitochondria and discloses close contact to endosome-like organelles. Using fluorescence microscopy, we localize endo-lysosomes containing NPC2 relative to mitochondria based on the Euclidian distance transform and use statistical inference to show that about 30% of such LE/LYSs are in contact to mitochondria in human fibroblasts. Using Markov Chain Monte Carlo image simulations, we show that interaction between both organelle types, a defining feature of membrane contact sites (MCSs) can give rise to the observed spatial organelle distribution. We devise a protocol to determine the surface fraction of endo-lysosomes in contact with mitochondria and show that this fraction does not depend on functional NPC1 or NPC2 proteins. Finally, we localize MCSs between LE/LYSs containing NPC2 and mitochondria in time-lapse image sequences and show that they either form transiently or remain stable for tens of seconds. Lasting MCSs between endo-lysosomes containing NPC2 and mitochondria move by slow anomalous sub-diffusion, providing location and time for sterol transport between both organelles. Our quantitative imaging strategy will be of high value for characterizing the dynamics and function of MCSs between various organelles in living cells.

Abstract Rare protein-truncating variants (PTVs) in PALB2 confer increased risk to breast cancer, but relatively few studies have reported the characteristics of tumours with PALB2 PTVs. In this study, we describe molecular characteristics of tumours with either germline or somatic alterations in PALB2. DNA from fresh frozen tumour tissues and matched peripheral blood lymphocytes for 560 breast cancer patients was subjected for whole-exome sequencing (WES), and RNA from tumour tissues was subjected to RNA sequencing (RNA-seq). We found six cases with germline and three with somatic protein-truncating variants in PALB2. The characteristics of tumours in patients with PALB2 PTVs were similar to those with BRCA1 and BRCA2 PTVs, having significantly more somatic alterations, and a high proportion of the mutational signature and genomic scar scores characteristic of deficiencies in homologous recombination (HR), compared to tumours arising in non-carriers. Unlike tumours arising in patients with BRCA1 and BRCA2 PTVs, PALB2 tumours did not have high prevalence of TP53 somatic alterations or an enriched immune microenvironment. In summary, PALB2 tumours show the homologous recombination deficiencies characteristic of BRCA1 and BRCA2 tumours, and highlight the potential clinical relevance of PALB2 mutational status in guiding therapeutic choices.

New magnetic glass compositions, based on system (100-x) (60GeO2 -25B2O3 –4Al2O3 -10Na2O–1PbO) – xTb4O7 were prepared by the melt-quenching technique. The materials were systematically characterized with respect to their structural (XRD, EXAFS, XANES and Raman), thermal (DSC), morphological, optical, magnetic and magneto-optical (MO) properties. Morphological and magnetic measurements show the homogeneous distribution of the Tb3+ ions as the magnetic susceptibility increases proportionally to the rare earth content. The optical window covers the range from 0.5 up to 1.6 μm, limited by absorption due to 4f8 -> 4f7 5 d 1and 4f8 -> 4f8 transitions of Tb3+. XANES and EXAFS measurements at the Ge–K edge show that the germanium atoms are always tetrahedrally coordinated, while results at the Tb-LIII edge shows terbium atoms to be eight-coordinated and exclusively present in the oxidation state 3+ independently of the glass composition. MO measurements show that the Verdet constant (VB) increases linearly with the Tb3+ concentration reaching the value of −83.9 rad T−1m−1 at 650 nm for glass containing 8 mol% of Tb4O7. Good thermal stability (ΔT = 200 °C) allowed obtaining an optical fiber of a glass containing 4 mol% of Tb4O7 featuring VB values of −37.9, −23.6 and −5.89 rad T−1 m−1, at 500, 650 and 1550 nm, respectively.

It is important to develop quantitative criteria to identify RNA-binding proteins and target RNAs. Here, the authors present a method to quantify cross-linking and apply it to recurrent cancer mutations in RNA-binding proteins.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.

Breast cancer risk for BRCA1/BRCA2 mutation carriers varies depending on other genetic factors. Here, the authors perform a case-only genome-wide association study and highlight novel loci associated with breast cancer risk for BRCA1/BRCA2 mutation carriers.

While antiretroviral therapy (ART) can completely suppress viremia, it is not a cure for HIV. HIV persists as a latent reservoir of infected cells, able to evade host immunity and re-seed infection following cessation of ART. Two promising immunotherapeutic strategies to eliminate both productively infected cells and reactivated cells of the reservoir are the adoptive transfer of potent HIV-specific T cells and the passive administration of HIV-specific broadly neutralizing antibodies also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). The simultaneous use of both as the basis of a single therapeutic has never been explored. We therefore sought to modify HIV-specific T cells from HIV-naive donors (to allow their use in the context of allotransplant, a promising platform for sterilizing cures) so they are able to secrete a broadly neutralizing antibody (bNAb) directed against the HIV envelope to elicit ADCC. We designed an antibody construct comprising bNAb 10-1074 heavy and light chains, fused to IgG3 Fc to elicit ADCC, with truncated cluster of differentiation 19 (CD19) as a selectable marker. HIV-specific T cells were expanded from HIV-naive donors by priming with antigen-presenting cells expressing overlapping HIV antigens in the presence of cytokines. T cells retained specificity against Gag, Nef, and Pol peptides (218.55 ± 300.14 interferon γ [IFNγ] spot-forming cells [SFC]/1 × 105) following transduction (38.92 ± 25.30) with the 10-1074 antibody constructs. These cells secreted 10-1074 antibodies (139.04 ± 114.42 ng/mL). The HIV-specific T cells maintained T cell function following transduction, and the secreted 10-1074 antibody bound HIV envelope (28.13% ± 19.42%) and displayed ADCC activity (10.47% ± 4.11%). Most critically, the 10-1074 antibody-secreting HIV-specific T cells displayed superior in vitro suppression of HIV replication. In summary, HIV-specific T cells can be engineered to produce antibodies mediating ADCC against HIV envelope-expressing cells. This combined innate/adaptive approach allows for synergy between the two immune arms, broadens the target range of the immune therapy, and provides further insight into what defines an effective anti-HIV response.

Uveal melanoma (UM) is a unique disease in that patients with primary UM are well stratified based on their risk of developing metastasis, yet there are limited effective treatments once metastases occur. There is an urgent need to better understand the distinct molecular pathogenesis of UM and the characteristics of patients at high risk for metastasis to identify neoantigenic targets that can be used in immunotherapy and to develop novel therapeutic strategies that may effectively target this lethal transition. An important and overlooked area of molecular pathogenesis and neoantigenic targets in UM comes from human endogenous retroviruses (HERVs). We investigated the HERV expression landscape in primary UM and found that tumors were stratified into 4 HERV-based subsets that provide clear delineation of risk outcome and support subtypes identified by other molecular indicators. Specific HERV loci are associated with the risk of uveal melanoma metastasis and may offer mechanistic insights into this process, including dysregulation of HERVs on chromosomes 3 and 8. A HERV signature composed of 17 loci was sufficient to classify tumors according to subtype with greater than 95% accuracy, including at least 1 intergenic HERV with coding potential (HERVE_Xp11.23) that could represent a potential HERV E target for immunotherapy.

We recently reported that a neurosteroid analogue with T-channel-blocking properties (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), induced hypnosis in rat pups without triggering neuronal apoptosis. Furthermore, we found that the inhibition of the CaV3.1 isoform of T-channels contributes to the hypnotic properties of 3β-OH in adult mice. However, the specific mechanisms underlying the role of other subtypes of voltage-gated calcium channels in thalamocortical excitability and oscillations in vivo during 3β-OH-induced hypnosis are largely unknown. Here, we used patch-clamp recordings from acute brain slices, in vivo electroencephalogram (EEG) recordings, and mouse genetics with wild-type (WT) and CaV2.3 knock-out (KO) mice to further investigate the molecular mechanisms of neurosteroid-induced hypnosis. Our voltage-clamp recordings showed that 3β-OH inhibited recombinant CaV2.3 currents. In subsequent current-clamp recordings in thalamic slices ex vivo, we found that selective CaV2.3 channel blocker (SNX-482) inhibited stimulated tonic firing and increased the threshold for rebound burst firing in WT animals. Additionally, in thalamic slices we found that 3β-OH inhibited spike-firing more profoundly in WT than in mutant mice. Furthermore, 3β-OH reduced bursting frequencies in WT but not mutant animals. In ensuing in vivo experiments, we found that intra-peritoneal injections of 3β-OH were less effective in inducing LORR in the mutant mice than in the WT mice, with expected sex differences. Furthermore, the reduction in total α, β, and low γ EEG power was more profound in WT than in CaV2.3 KO females over time, while at 60 min after injections of 3β-OH, the increase in relative β power was higher in mutant females. In addition, 3β-OH depressed EEG power more strongly in the male WT than in the mutant mice and significantly increased the relative δ power oscillations in WT male mice in comparison to the mutant male animals. Our results demonstrate for the first time the importance of the CaV2.3 subtype of voltage-gated calcium channels in thalamocortical excitability and the oscillations that underlie neurosteroid-induced hypnosis.

In an ex vivo rat ocular hypertension (OHT) model, the neurosteroid allopregnanolone (AlloP) exerts neuroprotective effects via enhancement of both GABAA receptors and autophagy. We now examine whether its enantiomer (ent-AlloP), which is largely inactive at GABA receptors, offers similar neuroprotection in ex vivo and in vivo rat OHT models. Ex vivo rat retinal preparations were incubated in a hyperbaric condition (10 and 75 mmHg) for 24 h. An in vivo ocular hypertension (OHT) model was induced by intracameral injection of polystyrene microbeads. We examined pharmacological effects of AlloP, ent-AlloP, picrotoxin (a GABAA receptor antagonist), and 3-MA (an autophagy inhibitor) histologically and biochemically. We found that both AlloP and ent-AlloP have marked neuroprotective effects in the retina, but effects of the unnatural enantiomer are independent of GABAA receptors. Electron microscopic analyses show that pressure elevation significantly increased autophagosomes (APs) in the nerve fiber layer and addition of AlloP also increased APs and degenerative autophagic vacuoles (AVds). ent-AlloP markedly increased APs and AVds compared to AlloP. Examination of LC3B-II and SQSTM1 protein levels using immunoblotting revealed that AlloP increased LC3B-II, and ent-AlloP further enhanced LC3B-II and suppressed SQSTM1, indicating that autophagy is a major mechanism underlying neuroprotection by ent-AlloP. In an rat in vivo OHT model, single intravitreal ent-AlloP injection prevented apoptotic cell death of retinal ganglion cells similar to AlloP. However, even in this model, ent-AlloP was more effective in activating autophagy than AlloP. We conclude that ent-AlloP may be a prototype of potential therapeutic for treatment of glaucoma as an autophagy enhancer without affecting GABA receptors.

Background Questions regarding the extent to which post-traumatic stress disorder (PTSD) is comorbid with alcohol and drug use are particularly germane in an era when the American College of Surgeons Committee on Trauma (ACS-COT) is considering policy requiring screening, intervention and/or referral services for patients presenting with psychological sequalae of traumatic injury. Literature review revealed few multisite trauma-center-based investigations that have assessed the association between PTSD symptoms and alcohol and drug use comorbidities in injured patients.Methods This investigation was a secondary analysis of baseline data collected prior to randomization in a 25-site trauma center pragmatic clinical trial. All 635 patients included in the investigation had elevated PTSD symptom levels at the time of trauma center admission. Self-report questionnaire screening, laboratory toxicology results, and electronic health record data were combined to assess the frequencies of alcohol, stimulant (i.e., amphetamine and cocaine), opioid and marijuana use comorbidities for injured patients. Logistic regression was used to assess the associations between demographic and injury characteristics and alcohol and drug use comorbidity.Results The frequency of patients with one or more alcohol or substance use comorbidity was between 62% and 79%. Over 50% of patients were positive for one or more alcohol or cannabis comorbidity. Approximately 26% of patients were positive for stimulants and 10% for opioid comorbidity.Discussion This multisite investigation suggests that between 62% and 79% of hospitalized injury survivors with elevated PTSD symptoms have one or more alcohol or drug use comorbidity. Orchestrated ACS-COT policy and trauma center service delivery development should incorporate the key finding that a substantial majority of patients with high levels of psychological distress (eg, elevated PTSD symptoms) may have alcohol and drug use comorbidities.Level of evidence Level II (epidemiological investigation of untreated controls from a multisite randomized clinical trial)Trial registration number NCT02655354.

Bacillus Calmette-Guérin (BCG) vaccine is known to have “bystander benefits” in protecting against heterologous infections; interim analysis of the “ACTIVATE” trial shows protection against respiratory infections in the elderly population. Epidemiologic studies suggest a potential benefit of BCG vaccination on COVID-19 outcomes. Differential past BCG vaccination policies between the former East and West German states provides a unique natural experiment to assess the potential effect of prior BCG vaccination on COVID-19. We estimated a 5% heterologous vaccine efficacy in the highly vaccinated former East Germany using the COVID-19 International Modeling (CoMo) Consortium model. A comparable BCG vaccination campaign undertaken prior to the pandemic in former West Germany, instituted along with known country-wide transmission reduction measures, is associated with a 37% decrease in projected mortality by mid-summer, 2020. These findings support a combined heterologous vaccine and non-pharmaceutical interventions (HVI+NPI) approach to mitigate the SARS-CoV-2 pandemic until SARS-CoV-2 specific vaccines are widely distributed.