Mapping the Evolutionary Space of SARS-CoV-2 Variants to Anticipate Emergence of Subvariants Resistant to COVID-19 Therapeutics.

New sublineages of SARS-CoV-2 variants-of-concern (VOCs) continuously emerge with mutations in the spike glycoprotein. In most cases, the sublineage-defining mutations vary between the VOCs. It is unclear whether these differences reflect lineage-specific likelihoods for mutations at each spike position or the stochastic nature of their appearance. Here we show that SARS-CoV-2 lineages have distinct evolutionary spaces (a probabilistic definition of the sequence states that can be occupied by expanding virus subpopulations). This space can be accurately inferred from the patterns of amino acid variability at the whole-protein level. Robust networks of co-variable sites identify the highest-likelihood mutations in new VOC sublineages and predict remarkably well the emergence of subvariants with resistance mutations to COVID-19 therapeutics. Our studies reveal the contribution of low frequency variant patterns at heterologous sites across the protein to accurate prediction of the changes at each position of interest.

Predicting dental caries outcomes in young adults using machine learning approach.

OBJECTIVES: To predict the dental caries outcomes in young adults from a set of longitudinally-obtained predictor variables and identify the most important predictors using machine learning techniques. METHODS: This study was conducted using the Iowa Fluoride Study dataset. The predictor variables - sex, mother's education, family income, composite socio-economic status (SES), caries experience at ages 9, 13, and 17, and the cumulative estimates of risk and protective factors, including fluoride, dietary, and behavioral variables from ages 5-9, 9-13, 13-17, and 17-23 were used to predict the age 23 D2+MFS count. The following machine learning models (LASSO regression, generalized boosting machines (GBM), negative binomial (NegGLM), and extreme gradient boosting models (XGBOOST)) were compared under 5-fold cross validation with nested resampling techniques. RESULTS: The prevalence of cavitated level caries experience at age 23 (mean D2+MFS count) was 4.75. The predictive analysis found LASSO to be the best performing model (compared to GBM, NegGLM, and XGBOOST), with a root mean square error (RMSE) of 0.70, and coefficient of determination (R2) of 0.44. After dichotomization of the predicted and observed values of the LASSO regression, the classification results showed accuracy, precision, recall, and ROC AUC of 83.7%, 85.9%, 93.1%, 68.2%, respectively. Previous caries experience at age 13 and age 17 and sugar-sweetened beverages intakes at age 13 and age 17 were found to be the four most important predictors of cavitated caries count at age 23. CONCLUSION: Our machine learning model showed high accuracy and precision in the prediction of caries in young adults from a longitudinally-obtained predictor variables. Our model could, in the future, after further development and validation with other diverse population data, be used by public health specialists and policy-makers as a screening tool to identify the risk of caries in young adults and apply more targeted interventions. However, data from a more diverse population are needed to improve the quality and generalizability of caries prediction.

Predictive Modeling for One-Year Lower Extremity Endovascular Revascularization Failure in Black Persons.

INTRODUCTION: Black persons bear a disproportionate burden of peripheral artery disease (PAD) and experience higher rates of endovascular revascularization failure (ERF) when compared with non-Hispanic White persons. We aimed to identify predictors of ERF in Black persons using predictive modeling. METHODS: This retrospective study included all persons identifying as Black who underwent an initial endovascular revascularization procedure for PAD between 2011 and 2018 at a midwestern tertiary care center. Three predictive models were developed using (1) logistic regression, (2) penalized logistic regression (least absolute shrinkage and selection operator [LASSO]), and (3) random forest (RF). Predictive performance was evaluated under repeated cross-validation. RESULTS: Of the 163 individuals included in the study, 113 (63.1%) experienced ERF at 1 y. Those with ERF had significant differences in symptom status (P < 0.001), lesion location (P < 0.001), diabetes status (P = 0.037), and annual procedural volume of the attending surgeon (P < 0.001). Logistic regression and LASSO models identified tissue loss, smoking, femoro-popliteal lesion location, and diabetes control as risk factors for ERF. The RF model identified annual procedural volume, age, PAD symptoms, number of comorbidities, and lesion location as most predictive variables. LASSO and RF models were more sensitive than logistic regression but less specific, although all three methods had an overall accuracy of ≥75%. CONCLUSIONS: Black persons undergoing endovascular revascularization for PAD are at high risk of ERF, necessitating need for targeted intervention. Predictive models may be clinically useful for identifying high-risk patients, although individual predictors of ERF varied by model. Further exploration into these models may improve limb salvage for this population.

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

Cytidine deaminases APOBEC3C and APOBEC3D promote DNA replication stress resistance in pancreatic cancer cells.

Gemcitabine is a potent inhibitor of DNA replication and is a mainstay therapeutic for diverse cancers, particularly pancreatic ductal adenocarcinoma (PDAC). However, most tumors remain refractory to gemcitabine therapies. Here, to define the cancer cell response to gemcitabine, we performed genome-scale CRISPR-Cas9 chemical-genetic screens in PDAC cells and found selective loss of cell fitness upon disruption of the cytidine deaminases APOBEC3C and APOBEC3D. Following gemcitabine treatment, APOBEC3C and APOBEC3D promote DNA replication stress resistance and cell survival by deaminating cytidines in the nuclear genome to ensure DNA replication fork restart and repair in PDAC cells. We provide evidence that the chemical-genetic interaction between APOBEC3C or APOBEC3D and gemcitabine is absent in nontransformed cells but is recapitulated across different PDAC cell lines, in PDAC organoids and in PDAC xenografts. Thus, we uncover roles for APOBEC3C and APOBEC3D in DNA replication stress resistance and offer plausible targets for improving gemcitabine-based therapies for PDAC.

Rare SH2B3 coding variants in lupus patients impair B cell tolerance and predispose to autoimmunity.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them, SH2B3-a negative regulator of cytokine and growth factor receptor signaling-harbors rare coding variants in over 5% of SLE patients. Here, we show that unlike the variant found exclusively in healthy controls, SH2B3 rare variants found in lupus patients are predominantly hypomorphic alleles, failing to suppress IFNGR signaling via JAK2-STAT1. The generation of two mouse lines carrying patients' variants revealed that SH2B3 is important in limiting the number of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair the negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role for SH2B3 in human B cell tolerance and lupus risk.

Within-host bayesian joint modeling of longitudinal and time-to-event data of Leishmania infection.

The host immune system plays a significant role in managing and clearing pathogen material during an infection, but this complex process presents numerous challenges from a modeling perspective. There are many mathematical and statistical models for these kinds of processes that take into account a wide range of events that happen within the host. In this work, we present a Bayesian joint model of longitudinal and time-to-event data of Leishmania infection that considers the interplay between key drivers of the disease process: pathogen load, antibody level, and disease. The longitudinal model also considers approximate inflammatory and regulatory immune factors. In addition to measuring antibody levels produced by the immune system, we adapt data from CD4+ and CD8+ T cell proliferation, and expression of interleukin 10, interferon-gamma, and programmed cell death 1 as inflammatory or regulatory factors mediating the disease process. The model is developed using data collected from a cohort of dogs naturally exposed to Leishmania infantum. The cohort was chosen to start with healthy infected animals, and this is the majority of the data. The model also characterizes the relationship features of the longitudinal outcomes and time-to-death due to progressive Leishmania infection. In addition to describing the mechanisms causing disease progression and impacting the risk of death, we also present the model's ability to predict individual trajectories of Canine Leishmaniosis (CanL) progression. The within-host model structure we present here provides a way forward to address vital research questions regarding the understanding of the progression of complex chronic diseases such as Visceral Leishmaniasis, a parasitic disease causing significant morbidity worldwide.

Genetic screens in Saccharomyces cerevisiae identify a role for 40S ribosome recycling factors Tma20 and Tma22 in nonsense-mediated decay.

The decay of messenger RNA with a premature termination codon by nonsense-mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD in Saccharomyces cerevisiae. In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2, and UPF3), as well as NMD4 and EBS1, we identify factors known to function in posttermination recycling and characterize their contribution to NMD. These observations in S. cerevisiae expand on data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD by showing that perturbations in factors implicated in 40S recycling also correlate with a loss of NMD.

Comparison of virus aerosol concentrations across a face shield worn on a healthcare personnel during a simulated patient cough.

BACKGROUND: Patients diagnosed with coronavirus disease 2019 (COVID-19) aerosolize severe acute respiratory coronavirus virus 2 (SARS-CoV-2) via respiratory efforts, expose, and possibly infect healthcare personnel (HCP). To prevent transmission of SARS-CoV-2 HCP have been required to wear personal protective equipment (PPE) during patient care. Early in the COVID-19 pandemic, face shields were used as an approach to control HCP exposure to SARS-CoV-2, including eye protection. METHODS: An MS2 bacteriophage was used as a surrogate for SARS-CoV-2 and was aerosolized using a coughing machine. A simulated HCP wearing a disposable plastic face shield was placed 0.41 m (16 inches) away from the coughing machine. The aerosolized virus was sampled using SKC biosamplers on the inside (near the mouth of the simulated HCP) and the outside of the face shield. The aerosolized virus collected by the SKC Biosampler was analyzed using a viability assay. Optical particle counters (OPCs) were placed next to the biosamplers to measure the particle concentration. RESULTS: There was a statistically significant reduction (P < .0006) in viable virus concentration on the inside of the face shield compared to the outside of the face shield. The particle concentration was significantly lower on the inside of the face shield compared to the outside of the face shield for 12 of the 16 particle sizes measured (P < .05). CONCLUSIONS: Reductions in virus and particle concentrations were observed on the inside of the face shield; however, viable virus was measured on the inside of the face shield, in the breathing zone of the HCP. Therefore, other exposure control methods need to be used to prevent transmission from virus aerosol.

Uncertainty about predation risk: a conceptual review.

Uncertainty has long been of interest to economists and psychologists and has more recently gained attention among ecologists. In the ecological world, animals must regularly make decisions related to finding resources and avoiding threats. Here, we describe uncertainty as a perceptual phenomenon of decision-makers, and we focus specifically on the functional ecology of such uncertainty regarding predation risk. Like all uncertainty, uncertainty about predation risk reflects informational limitations. When cues are available, they may be novel (i.e. unknown information), incomplete, unreliable, overly abundant and complex, or conflicting. We review recent studies that have used these informational limitations to induce uncertainty of predation risk. These studies have typically used either over-responses to novelty (i.e. neophobia) or memory attenuation as proxies for measuring uncertainty. Because changes in the environment, particularly unpredictable changes, drive informational limitations, we describe studies assessing unpredictable variance in spatio-temporal predation risk, intensity of predation risk, predator encounter rate, and predator diversity. We also highlight anthropogenic changes within habitats that are likely to have dramatic impacts on information availability and thus uncertainty in antipredator decisions in the modern world.

DECTIN-1: A modifier protein in CTLA-4 haploinsufficiency.

Autosomal dominant loss-of-function (LoF) variants in cytotoxic T-lymphocyte associated protein 4 (CTLA4) cause immune dysregulation with autoimmunity, immunodeficiency and lymphoproliferation (IDAIL). Incomplete penetrance and variable expressivity are characteristic of IDAIL caused by CTLA-4 haploinsufficiency (CTLA-4h), pointing to a role for genetic modifiers. Here, we describe an IDAIL proband carrying a maternally inherited pathogenic CTLA4 variant and a paternally inherited rare LoF missense variant in CLEC7A, which encodes for the ß-glucan pattern recognition receptor DECTIN-1. The CLEC7A variant led to a loss of DECTIN-1 dimerization and surface expression. Notably, DECTIN-1 stimulation promoted human and mouse regulatory T cell (Treg) differentiation from naïve αß and γδ T cells, even in the absence of transforming growth factor-ß. Consistent with DECTIN-1's Treg-boosting ability, partial DECTIN-1 deficiency exacerbated the Treg defect conferred by CTL4-4h. DECTIN-1/CLEC7A emerges as a modifier gene in CTLA-4h, increasing expressivity of CTLA4 variants and acting in functional epistasis with CTLA-4 to maintain immune homeostasis and tolerance.

FBXW7-loss Sensitizes Cells to ATR Inhibition Through Induced Mitotic Catastrophe.

FBXW7 is a commonly mutated tumor suppressor gene that functions to regulate numerous oncogenes involved in cell-cycle regulation. Genome-wide CRISPR fitness screens identified a signature of DNA repair and DNA damage response genes as required for the growth of FBXW7-knockout cells. Guided by these findings, we show that FBXW7-mutant cells have high levels of replication stress, which results in a genotype-specific vulnerability to inhibition of the ATR signaling pathway, as these mutant cells become heavily reliant on a robust S-G2 checkpoint. ATR inhibition induces an accelerated S-phase, leading to mitotic catastrophe and cell death caused by the high replication stress present in FBXW7-/- cells. In addition, we provide evidence in cell and organoid studies, and mining of publicly available high-throughput drug screening efforts, that this genotype-specific vulnerability extends to multiple types of cancer, providing a rational means of identifying responsive patients for targeted therapy. SIGNIFICANCE: We have elucidated the synthetic lethal interactions between FBXW7 mutation and DNA damage response genes, and highlighted the potential of ATR inhibitors as targeted therapies for cancers harboring FBXW7 alterations.

Olfaction and reaction: The role of olfactory and hypothalamic investment in the antipredator responses to chemical alarm cues by northern redbelly dace.

Neuroplasticity enables teleosts to promote or downregulate the growth of their brains regionally. To compensate for the effects of predation pressure, teleosts may alter their brain morphology and behavioral responses to mitigate its impact on individual fitness. High-predation environments often promote specific patterns of brain growth and produce bolder and more proactive populations. Owing to the expense of maintaining neural tissue, relative size indicates the regions most relied upon. In northern redbelly dace Chrosomus eos, as little as 2 weeks of elevated predation pressure, resulted in increased investment in their olfactory bulbs and optic tecta, while the imposition of captivity produced smaller, less symmetric hypothalami. Taken together, these results suggest that an individual could potentially become better able to detect a threat, and simultaneously less inclined to react to it, making the impact of either change in isolation is difficult to discern. Here, we compared interindividual variation in gross brain morphology, risk-taking tactics in a novel arena (shy-bold personality), and responding to olfactory cues (proactive/reactive stress-coping style). We hypothesized that olfactory investment would positively correlate with response intensity to predator cue concentration and respond across a wider range of cue concentrations, while hypothalamus size would correlate with shyness and reactivity. Exposure to heightened risk produced more bold/proactive individuals, with larger olfactory bulbs and smaller hypothalami. However, the direction of the correlation between hypothalamus size and behavior varied by treatment, and olfactory investment only corresponded with response intensity amongst proactive individuals. Our findings illustrate the potential pitfalls of relating gross brain morphology to complex behavior and suggest that stress-coping style is a relevant consideration in future studies.

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells.

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

Evaluation of face shields used during aerosol generating procedures.

Transnasal flexible laryngoscopy is considered an aerosol generating procedure. A negative pressure face shield (NPFS) was developed to control aerosol from the patient during laryngoscopy. The purpose of this study was to determine the effectiveness of the NPFS at controlling virus aerosol compared to a standard disposable plastic face shield. The face shields were placed on a simulated patient coughing machine. MS2 bacteriophage was used as a surrogate for SARS-CoV-2 and was aerosolized using the coughing machine. The aerosolized virus was sampled on the inside and outside of the face shields. The virus aerosol concentration was not significantly different between the inside and outside of the traditional plastic face shield (p = 0.12). However, the particle concentrations across all particle sizes measured were significantly decreased outside the face shield. The virus and particle concentrations were significantly decreased (p < 0.01) outside the NPFS operating at a flow rate of 38.6 L per minute (LPM). When the NPFS was operated at 10 LPM, virus concentrations were not significantly different (p = 0.09) across the face shield. However, the number particle concentrations across all particle sizes measured were significantly different (p < 0.05).

Dental caries incidence and associated factors in young adults.

OBJECTIVES: To assess the caries incidence from late adolescence to early adulthood and to identify the factors associated with caries incidence. METHODS: This is a secondary analysis of longitudinal caries data of young adults aged 17-23 from the Iowa Fluoride Study cohort. The inclusion criteria required completion of dental exams at both ages 17 and 23 and having cumulative exposure (AUC) variables data for at least 8 out of the 11 time periods between ages 17 and 23. Mean imputation was used to handle the missing explanatory variable data. Multiple linear regressions were conducted using a generalized linear model to assess the effects of sociodemographic and behavioral/dietary variables on the age 17-23 adjusted cavitated caries (D2+ MFS) increment (AdjCI17-23 ). Multicollinearity was assessed using the variance inflation factor (VIF) and the final model was selected based on the Akaike Information Criterion (AIC) using backward selection and the net effects calculated. RESULTS: The mean AdjCI17-23 was 2.08 (SD = 4.02). The net effects (main effect plus interactions) of higher composite socioeconomic status, higher combined daily fluoride intake, higher frequency of milk intake, lower amount of sugar-sweetened beverages intake, and lower age 17 dental caries counts were associated with lower mean AdjCI17-23 . CONCLUSION: The incidence of caries from age 17 to 23 in this study was low. This study suggests and reinforces the need to continue to advocate for caries preventive strategies such as fluoride use, encouraging milk intake, and reducing sugar-sweetened beverage intakes.

Within-Host Bayesian Joint Modeling of Longitudinal and Time-to-Event Data of Leishmania Infection.

The host immune system plays a significant role in managing and clearing pathogen material during an infection, but this complex process presents numerous challenges from a modeling perspective. There are many mathematical and statistical models for these kinds of processes that take into account a wide range of events that happen within the host. In this work, we present a Bayesian joint model of longitudinal and time-to-event data of Leishmania infection that considers the interplay between key drivers of the disease process: pathogen load, antibody level, and disease. The longitudinal model also considers approximate inflammatory and regulatory immune factors. In addition to measuring antibody levels produced by the immune system, we adapt data from CD4+ and CD8+ T cell proliferation, and expression of interleukin 10, interferon-gamma, and programmed cell death 1 as inflammatory or regulatory factors mediating the disease process. The model is developed using data collected from a cohort of dogs naturally exposed to Leishmania infantum. The cohort was chosen to start with healthy infected animals, and this is the majority of the data. The model also characterizes the relationship features of the longitudinal outcomes and time of death due to progressive Leishmania infection. In addition to describing the mechanisms causing disease progression and impacting the risk of death, we also present the model's ability to predict individual trajectories of Canine Leishmaniosis (CanL) progression. The within-host model structure we present here provides a way forward to address vital research questions regarding the understanding progression of complex chronic diseases such as Visceral Leishmaniasis, a parasitic disease causing significant morbidity worldwide.

Microhabitat conditions drive uncertainty of risk and shape neophobic responses in Trinidadian guppies, Poecilia reticulata.

In response to uncertain risks, prey may rely on neophobic phenotypes to reduce the costs associated with the lack of information regarding local conditions. Neophobia has been shown to be driven by information reliability, ambient risk and predator diversity, all of which shape uncertainty of risk. We similarly expect environmental conditions to shape uncertainty by interfering with information availability. In order to test how environmental variables might shape neophobic responses in Trinidadian guppies (Poecilia reticulata), we conducted an in situ field experiment of two high-predation risk guppy populations designed to determine how the 'average' and 'variance' of several environmental factors might influence the neophobic response to novel predator models and/or novel foraging patches. Our results suggest neophobia is shaped by water velocity, microhabitat complexity, pool width and depth, as well as substrate diversity and heterogeneity. Moreover, we found differential effects of the 'average' and 'variance' environmental variables on food- and predator-related neophobia. Our study highlights that assessment of neophobic drivers should consider predation risk, various microhabitat conditions and neophobia being tested. Neophobic phenotypes are expected to increase the probability of prey survival and reproductive success (i.e. fitness), and are therefore likely linked to population health and species survival. Understanding the drivers and consequences of uncertainty of risk is an increasingly pressing issue, as ecological uncertainty increases with the combined effects of climate change, anthropogenic disturbances and invasive species.

Two independent DNA repair pathways cause mutagenesis in template switching deficient Saccharomyces cerevisiae.

Upon DNA replication stress, cells utilize the postreplication repair pathway to repair single-stranded DNA and maintain genome integrity. Postreplication repair is divided into 2 branches: error-prone translesion synthesis, signaled by proliferating cell nuclear antigen (PCNA) monoubiquitination, and error-free template switching, signaled by PCNA polyubiquitination. In Saccharomyces cerevisiae, Rad5 is involved in both branches of repair during DNA replication stress. When the PCNA polyubiquitination function of Rad5 s disrupted, Rad5 recruits translesion synthesis polymerases to stalled replication forks, resulting in mutagenic repair. Details of how mutagenic repair is carried out, as well as the relationship between Rad5-mediated mutagenic repair and the canonical PCNA-mediated mutagenic repair, remain to be understood. We find that Rad5-mediated mutagenic repair requires the translesion synthesis polymerase ζ but does not require other yeast translesion polymerase activities. Furthermore, we show that Rad5-mediated mutagenic repair is independent of PCNA binding by Rev1 and so is separable from canonical mutagenic repair. In the absence of error-free template switching, both modes of mutagenic repair contribute additively to replication stress response in a replication timing-independent manner. Cellular contexts where error-free template switching is compromised are not simply laboratory phenomena, as we find that a natural variant in RAD5 is defective in PCNA polyubiquitination and therefore defective in error-free repair, resulting in Rad5- and PCNA-mediated mutagenic repair. Our results highlight the importance of Rad5 in regulating spontaneous mutagenesis and genetic diversity in S. cerevisiae through different modes of postreplication repair.