A Genetic Map of the Response to DNA Damage in Human Cells.

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

TLR7 gain-of-function genetic variation causes human lupus.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease1-7, evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA8,9 and binds to guanosine10-12. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP10-12, and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.

Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates.

Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.

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.

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.

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.

HELB Is a Feedback Inhibitor of DNA End Resection.

DNA double-strand break repair by homologous recombination is initiated by the formation of 3' single-stranded DNA (ssDNA) overhangs by a process termed end resection. Although much focus has been given to the decision to initiate resection, little is known of the mechanisms that regulate the ongoing formation of ssDNA tails. Here we report that DNA helicase B (HELB) underpins a feedback inhibition mechanism that curtails resection. HELB is recruited to ssDNA by interacting with RPA and uses its 5'-3' ssDNA translocase activity to inhibit EXO1 and BLM-DNA2, the nucleases catalyzing resection. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. Consistent with its role as a resection antagonist, loss of HELB results in PARP inhibitor resistance in BRCA1-deficient tumor cells. We conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB.

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.

An individual level infectious disease model in the presence of uncertainty from multiple, imperfect diagnostic tests.

Bayesian compartmental infectious disease models yield important inference on disease transmission by appropriately accounting for the dynamics and uncertainty of infection processes. In addition to estimating transition probabilities and reproductive numbers, these statistical models allow researchers to assess the probability of disease risk and quantify the effectiveness of interventions. These infectious disease models rely on data collected from all individuals classified as positive based on various diagnostic tests. In infectious disease testing, however, such procedures produce both false-positives and false-negatives at varying rates depending on the sensitivity and specificity of the diagnostic tests being used. We propose a novel Bayesian spatio-temporal infectious disease modeling framework that accounts for the additional uncertainty in the diagnostic testing and classification process that provides estimates of the important transmission dynamics of interest to researchers. The method is applied to data on the 2006 mumps epidemic in Iowa, in which over 6,000 suspected mumps cases were tested using a buccal or oral swab specimen, a urine specimen, and/or a blood specimen. Although all procedures are believed to have high specificities, the sensitivities can be low and vary depending on the timing of the test as well as the vaccination status of the individual being tested.

Bayesian multivariate longitudinal model for immune responses to Leishmania: A tick-borne co-infection study.

While many Bayesian state-space models for infectious disease processes focus on population infection dynamics (eg, compartmental models), in this work we examine the evolution of infection processes and the complexities of the immune responses within the host using these techniques. We present a joint Bayesian state-space model to better understand how the immune system contributes to the control of Leishmania infantum infections over the disease course. We use longitudinal molecular diagnostic and clinical data of a cohort of dogs to describe population progression rates and present evidence for important drivers of clinical disease. Among these results, we find evidence for the importance of co-infection in disease progression. We also show that as dogs progress through the infection, parasite load is influenced by their age, ectoparasiticide treatment status, and serology. Furthermore, we present evidence that pathogen load information from an earlier point in time influences its future value and that the size of this effect varies depending on the clinical stage of the dog. In addition to characterizing the processes driving disease progression, we predict individual and aggregate patterns of Canine Leishmaniasis progression. Both our findings and the application to individual-level predictions are of direct clinical relevance, presenting possible opportunities for application in veterinary practice and motivating lines of additional investigation to better understand and predict disease progression. Finally, as an important zoonotic human pathogen, these results may support future efforts to prevent and treat human Leishmaniosis.

VID22 counteracts G-quadruplex-induced genome instability.

Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism.

Incorporating infectious duration-dependent transmission into Bayesian epidemic models.

Compartmental models are commonly used to describe the spread of infectious diseases by estimating the probabilities of transitions between important disease states. A significant challenge in fitting Bayesian compartmental models lies in the need to estimate the duration of the infectious period, based on limited data providing only symptom onset date or another proxy for the start of infectiousness. Commonly, the exponential distribution is used to describe the infectious duration, an overly simplistic approach, which is not biologically plausible. More flexible distributions can be used, but parameter identifiability and computational cost can worsen for moderately sized or large epidemics. In this article, we present a novel approach, which considers a curve of transmissibility over a fixed infectious duration. The incorporation of infectious duration-dependent (IDD) transmissibility, which decays to zero during the infectious period, is biologically reasonable for many viral infections and fixing the length of the infectious period eases computational complexity in model fitting. Through simulation, we evaluate different functional forms of IDD transmissibility curves and show that the proposed approach offers improved estimation of the time-varying reproductive number. We illustrate the benefit of our approach through a new analysis of the 1995 outbreak of Ebola Virus Disease in the Democratic Republic of the Congo.

Joint Bayesian longitudinal models for mixed outcome types and associated model selection techniques.

Motivated by data measuring progression of leishmaniosis in a cohort of US dogs, we develop a Bayesian longitudinal model with autoregressive errors to jointly analyze ordinal and continuous outcomes. Multivariate methods can borrow strength across responses and may produce improved longitudinal forecasts of disease progression over univariate methods. We explore the performance of our proposed model under simulation, and demonstrate that it has improved prediction accuracy over traditional Bayesian hierarchical models. We further identify an appropriate model selection criterion. We show that our method holds promise for use in the clinical setting, particularly when ordinal outcomes are measured alongside other variables types that may aid clinical decision making. This approach is particularly applicable when multiple, imperfect measures of disease progression are available.

Mask Effectiveness for Preventing Secondary Cases of COVID-19, Johnson County, Iowa, USA.

In September of 2020, the Iowa Department of Public Health released guidance stating that persons exposed to someone with coronavirus disease (COVID-19) need not quarantine if the case-patient and the contact wore face masks at the time of exposure. This guidance differed from that issued by the Centers for Disease Control and Prevention. To determine the best action, we matched exposure information from COVID-19 case investigations with reported test results and calculated the secondary attack rates (SARs) after masked and unmasked exposures. Mask use by both parties reduced the SAR by half, from 25.6% to 12.5%. Longer exposure duration significantly increased SARs. Masks significantly reduced virus transmission when worn by both the case-patient and the contact, but SARs for each group were higher than anticipated. This finding suggests that quarantine after COVID-19 exposure is beneficial even if parties wore masks.

Exploratory decisions of Trinidadian guppies when uncertain about predation risk.

Animals can reduce their uncertainty of predation risk by gathering new information via exploration behaviour. However, a decision to explore may also be costly due to increased predator exposure. Here, we found contextual effects of predation risk on the exploratory activity of Trinidadian guppies Poecilia reticulata in a novel environment. First, guppies were exposed to a 3-day period of either high or low background predation risk in the form of repeated exposure to either injured conspecific cues (i.e. alarm cues) or control water, respectively. A day later, guppies were moved into a testing arena with limited visual information due to structural barriers and were then presented with an acute chemical stimulus, either alarm cues (a known and reliable indicator of risk), a novel odour (an ambiguous cue), or control water. In the presence of control water, guppies from high and low background risk showed a similar willingness to explore the arena. However, high-risk individuals significantly reduced their spatial evenness, although not their movement latency, in the presence of both the alarm and novel cues. When these high-risk individuals were a member of a shoal, they became willing to explore the environment more evenly in the presence of alarm cues while remaining cautious toward the novel cue, indicating an effect of the greater uncertainty associated with the novel cue. In contrast, low-risk guppies showed a willingness to explore the arena regardless of acute threat or social context. Such contextual effects of background risk and social context highlight the complexity of exploratory decisions when uncertain.

Gender differences in lifetime and current use of online support for recovery from alcohol use disorder.

BACKGROUND: Digital recovery support services (D-RSS) use technology to engage individuals seeking recovery from alcohol use disorder (AUD). Given sparse data on use of these emergent services as well as longstanding and stark gender disparities in use of traditional alcohol treatment services, we sought to quantify lifetime and current D-RSS use and to test associations with several recovery outcomes, with particular attention to gender differences. METHODS: We analyzed data obtained in fall 2020 in a national survey of adults with a resolved alcohol problem (n = 1487). We estimated lifetime and current D-RSS use, prevalence of various types of D-RSS, and related outcomes (e.g., recovery stability, relapse events, quality of life). Stratified logistic regression models identified correlates of D-RSS use for women and men, controlling for demographic and AUD characteristics. RESULTS: Overall, an estimated 14.9% of the population of adults with a resolved alcohol problem reported lifetime use of D-RSS, with no difference by gender. Current use was lower and was reported by more men than women (9.9% vs. 5.8%, respectively). Men had higher odds of D-RSS use if they had <1 year of recovery (adjusted odds ratio [aOR] 7.84), 1 to 5 years of recovery (aOR 2.17), and if never married (aOR 3.29). Among women, higher odds of D-RSS were associated with AUD symptom count (aOR 1.30), being unemployed (aOR 9.85), and having minor children in the household (aOR 3.58). Among women, there was no association between D-RSS use and recovery stability, relapse events, and quality of life. However, among men D-RSS use was associated with reporting that the COVID-19 pandemic had made it more difficult to resist alcohol or drugs and with lower self-reported quality of life. CONCLUSIONS: D-RSS are a promising technological approach to support recovery. There is room to increase their use, and gender-specific approaches may be needed given different correlates of use for women and men. In addition, further research is needed to explore whether D-RSS may confer benefits through similar mechanisms as in-person recovery services.

Development of a yeast whole-cell biocatalyst for MHET conversion into terephthalic acid and ethylene glycol.

BACKGROUND: Over the 70 years since the introduction of plastic into everyday items, plastic waste has become an increasing problem. With over 360 million tonnes of plastics produced every year, solutions for plastic recycling and plastic waste reduction are sorely needed. Recently, multiple enzymes capable of degrading PET (polyethylene terephthalate) plastic have been identified and engineered. In particular, the enzymes PETase and MHETase from Ideonella sakaiensis depolymerize PET into the two building blocks used for its synthesis, ethylene glycol (EG) and terephthalic acid (TPA). Importantly, EG and TPA can be re-used for PET synthesis allowing complete and sustainable PET recycling. RESULTS: In this study we used Saccharomyces cerevisiae, a species utilized widely in bioindustrial fermentation processes, as a platform to develop a whole-cell catalyst expressing the MHETase enzyme, which converts monohydroxyethyl terephthalate (MHET) into TPA and EG. We assessed six expression architectures and identified those resulting in efficient MHETase expression on the yeast cell surface. We show that the MHETase whole-cell catalyst has activity comparable to recombinant MHETase purified from Escherichia coli. Finally, we demonstrate that surface displayed MHETase is active across a range of pHs, temperatures, and for at least 12 days at room temperature. CONCLUSIONS: We demonstrate the feasibility of using S. cerevisiae as a platform for the expression and surface display of PET degrading enzymes and predict that the whole-cell catalyst will be a viable alternative to protein purification-based approaches for plastic degradation.

Post-replication repair: Rad5/HLTF regulation, activity on undamaged templates, and relationship to cancer.

The eukaryotic post-replication repair (PRR) pathway allows completion of DNA replication when replication forks encounter lesions on the DNA template and are mediated by post-translational ubiquitination of the DNA sliding clamp proliferating cell nuclear antigen (PCNA). Monoubiquitinated PCNA recruits translesion synthesis (TLS) polymerases to replicate past DNA lesions in an error-prone manner while addition of K63-linked polyubiquitin chains signals for error-free template switching to the sister chromatid. Central to both branches is the E3 ubiquitin ligase and DNA helicase Rad5/helicase-like transcription factor (HLTF). Mutations in PRR pathway components lead to genomic rearrangements, cancer predisposition, and cancer progression. Recent studies have challenged the notion that the PRR pathway is involved only in DNA lesion tolerance and have shed new light on its roles in cancer progression. Molecular details of Rad5/HLTF recruitment and function at replication forks have emerged. Mounting evidence indicates that PRR is required during lesion-less replication stress, leading to TLS polymerase activity on undamaged templates. Analysis of PRR mutation status in human cancers and PRR function in cancer models indicates that down regulation of PRR activity is a viable strategy to inhibit cancer cell growth and reduce chemoresistance. Here, we review these findings, discuss how they change our views of current PRR models, and look forward to targeting the PRR pathway in the clinic.

Early-life and parental predation risk shape fear acquisition in adult minnows.

Exposure to predation risk can induce a fearful baseline state, as well as fear reactions toward novel situations (i.e., neophobia). Some research indicates that risk exposure during sensitive periods makes adults more prone to acquiring long-term fearful phenotypes. However, chronic risk can also lead to ignoring threats in order to maintain other activities. We sought to assess how a relatively long period of low risk, experienced either early in life or by the previous generation, influences fear behaviour acquired from a short period of high risk as adults. We used fathead minnows as study subjects and simulated predation risk with repeated exposures to conspecific chemical alarm cues. The period of high risk experienced by adults induced typical fear behaviour (baseline freezing and neophobia), whereas the early-life low-risk period 1 year prior caused only a reduction in baseline foraging. We found no evidence that the early-life risk significantly altered the fear acquired from the adult-risk period. However, in a second experiment, a low-risk period during the parental generation interacted with a high-risk period experienced by the adult offspring. The combination of both risk periods heightened baseline freezing despite parental risk having little effect independently. Hence, our study provides evidence that parental risk exposure can lead to an additive intergenerational effect on fear acquisition in minnows.

Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiaeeco1-1 allele.

Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial, with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within ß7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored the function of the enzyme at elevated temperatures. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.