Adaptive Capacity of a DNA Polymerase Clamp-loader ATPase Complex.

The ability of mutations to facilitate adaptation is central to evolution. To understand how mutations can lead to functional adaptation in a complex molecular machine, we created a defective version of the T4 clamp-loader complex, which is essential for DNA replication. This variant, which is ∼5,000-fold less active than the wild type, was made by replacing the catalytic domains with those from another phage. A directed-evolution experiment revealed that multiple substitutions to a single negatively charged residue in the chimeric clamp loader-Asp 86-restore fitness to within ∼20-fold of wild type. These mutations remove an adventitious electrostatic repulsive interaction between Asp 86 and the sliding clamp. Thus, the fitness decrease of the chimeric clamp loader is caused by a reduction in affinity between the clamp loader and the clamp. Deep mutagenesis shows that the reduced fitness of the chimeric clamp loader is also compensated for by lysine and arginine substitutions of several DNA-proximal residues in the clamp loader or the sliding clamp. Our results demonstrate that there is a latent capacity for increasing the affinity of the clamp loader for DNA and the sliding clamp, such that even single-point mutations can readily compensate for the loss of function due to suboptimal interactions elsewhere.

Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM.

Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved 'DEAD-box' motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved.

Validation of a geospatial aggregation method for congressional districts and other US administrative geographies.

Stakeholders need data on health and drivers of health parsed to the boundaries of essential policy-relevant geographies. US Congressional Districts are an example of a policy-relevant geography which generally lack health data. One strategy to generate Congressional District heath data metric estimates is to aggregate estimates from other geographies, for example, from counties or census tracts to Congressional Districts. Doing so requires several methodological decisions. We refine a method to aggregate health metric estimates from one geography to another, using a population weighted approach. The method's accuracy is evaluated by comparing three aggregated metric estimates to metric estimates from the US Census American Community Survey for the same years: Broadband Access, High School Completion, and Unemployment. We then conducted four sensitivity analyses testing: the effect of aggregating counts vs. percentages; impacts of component geography size and data missingness; and extent of population overlap between component and target geographies. Aggregated estimates were very similar to estimates for identical metrics drawn directly from the data source. Sensitivity analyses suggest the following best practices for Congressional district-based metrics: utilizing smaller, more plentiful geographies like census tracts as opposed to larger, less plentiful geographies like counties, despite potential for less stable estimates in smaller geographies; favoring geographies with higher percentage population overlap.

Large language models generate functional protein sequences across diverse families.

Deep-learning language models have shown promise in various biotechnological applications, including protein design and engineering. Here we describe ProGen, a language model that can generate protein sequences with a predictable function across large protein families, akin to generating grammatically and semantically correct natural language sentences on diverse topics. The model was trained on 280 million protein sequences from >19,000 families and is augmented with control tags specifying protein properties. ProGen can be further fine-tuned to curated sequences and tags to improve controllable generation performance of proteins from families with sufficient homologous samples. Artificial proteins fine-tuned to five distinct lysozyme families showed similar catalytic efficiencies as natural lysozymes, with sequence identity to natural proteins as low as 31.4%. ProGen is readily adapted to diverse protein families, as we demonstrate with chorismate mutase and malate dehydrogenase.

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the prehairpin intermediate of the spike protein.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein. Significance Statement: SARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.

Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction.

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

Young Adults and Adverse Childhood Events: A Potent Measure of Cardiovascular Risk.

INTRODUCTION: While cardiovascular disease is the leading cause of death, its determinants include unhealthy behaviors and clinical risk factors and are recognized as the "actual causes" of death. Risk likely accumulates over the life course, and adverse childhood experiences may increase the risk of "actual causes" of death. The objectives of the study are to determine the prevalence and test the association of adverse childhood experiences among unhealthy behaviors and risk factors as a primordial risk factor among young adults. METHODS: Data were extracted from the 2009 and 2011 Behavioural Risk Factor Surveillance System. Individuals ages 18-99 years provided complete information on adverse childhood experiences, health behaviors, and risk factors. Adverse childhood experiences were categorized and evaluated as cumulative burden. Multivariable logistic models, including stratified analysis for young adults, tested the association of adverse childhood experiences burden with unhealthy behaviors and risk factors. RESULTS: Among 45,482 study participants, 52% report one adverse childhood experience and 25% report 2 adverse childhood experience categories. Among the total study population, 37% report violence/emotional abuse, 34% report neglect, and 12% report sexual abuse. Even one adverse childhood experience is strongly associated with hypertension, dyslipidemia, and diabetes, and while the association increases in a dose-response (P trend < .001) for all, it is especially more pronounced among the younger adults, with minimal attenuation of effects in the fully adjusted models. CONCLUSION: The prevalence of adverse childhood experiences in this study population is high. Even one adverse childhood experience is strongly and independently associated with cardiovascular risk factors, with implications for primordial prevention. Future studies are needed to develop screening and treatment strategies targeted to this high-risk group, especially among young adults.

A set of experimentally validated, mutually orthogonal primers for combinatorially specifying genetic components.

The design and synthesis of novel genes and deoxyribonucleic acid (DNA) sequences is a central technique in synthetic biology. Current methods of high throughput gene synthesis use pooled oligonucleotides obtained from custom-designed DNA microarray chips, and rely on orthogonal (non-interacting) polymerase chain reaction primers to specifically de-multiplex, by amplification, the precise subset of oligonucleotides necessary to assemble a full length gene. The availability of a large validated set of mutually orthogonal primers is therefore a crucial reagent for high-throughput gene synthesis. Here, we present a set of 166 20-nucleotide primers that are experimentally verified to be non-interacting, capable of specifying 13 695 unique genes. These primers represent a valuable resource to the synthetic biology community for specifying genetic components that can be assembled through a scalable and modular architecture.

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing.

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 ß-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to ß-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

Pathways from parental educational attainment to adolescent blood pressure.

OBJECTIVES: Lower parental education is associated with higher adolescent blood pressure (BP). We examined the contribution of modifiable risk factors from infancy to adolescence that could potentially explain the link between parental education and SBP and DBP in the offspring. METHODS: In a prospective Chinese birth cohort, 'Children of 1997' of 5604 adolescents (68% follow-up), we analyzed the relation between parental educational attainment and sex-specific, age-specific and height-specific BP z-scores at ∼13 years. Using mediation analysis, we examined the contribution of household income at birth (both absolute income and relative income deprivation), exposures during infancy (breastfeeding and early life second-hand smoking), lifestyles during childhood (diet, physical activity and screen-time), weight or BMI status during fetal, infancy, childhood and puberty, pubertal stage as well as parental BMI. RESULTS: We found that adolescent BMI, but not birth weight or infant growth or childhood BMI, mediated the inverse association of parental education with adolescent SBP (proportion mediated: 24%), followed by maternal BMI (proportion mediated: 18%). Factors explaining the link between parental education and DBP were less clear. Absolute income, breastfeeding, childhood diet and physical activity, pubertal stage and paternal BMI did not mediate the association between parental education and adolescent BP. CONCLUSION: Low parental education is a risk factor for high SBP and, to a lesser extent, DBP in adolescents. Important mediators of this relation include adolescent and maternal body weight.

Grandparental education, parental education and adolescent blood pressure.

BACKGROUND: Maternal and paternal education could affect childhood blood pressure differently. Grandparental education might also play a role. Disentangling their contribution to childhood blood pressure may shed light on the persistence of disparities and potential windows of intervention. METHODS: Using 5604 participants from a Chinese birth cohort born in 1997 and followed-up until ~13years (68% of follow-up), we examined the associations of parental education and grandparental education with age-, sex, and height-specific blood pressure z-scores or prehypertension status. RESULTS: Parental education was inversely associated with adolescent systolic (-0.11 z-score, equivalent to -1.17mmHg, 95% confidence interval (CI) -0.19 to -0.04 for grade ≥12 compared with grade ≤9) and diastolic blood pressure (-0.07 z-score, equivalent to -0.79mmHg, 95% CI -0.11 to -0.04). The magnitude of association was similar for maternal or paternal education. Grandparental education was not associated with adolescent blood pressure. No association with prehypertension was found. CONCLUSIONS: In an economically developed non-Western setting, both maternal and paternal, but not grandparental, education was associated with adolescent blood pressure. Blood pressure may be responsive to contemporary family socioeconomic conditions that may be scrutinized for suitable interventions.

Can social policy influence socioeconomic disparities? Korean War GI Bill eligibility and markers of depression.

PURPOSE: The Korean War GI Bill provided socioeconomic benefits to veterans; however, its association with health is unclear; we hypothesize GI Bill eligibility is associated with fewer depressive symptoms and smaller disparities. METHODS: Data from 246 Korean War GI Bill eligible veterans and 240 nonveterans from the Health and Retirement Study were matched on birth year, southern birth, race, height, and childhood health using coarsened exact matching. Number of depressive symptoms in 2010 (average age = 78 years) was assessed using a modified, validated Center for Epidemiologic Studies-Depression Scale, dichotomized to reflect elevated depressive symptoms. Regression analyses were stratified into low (at least one parent < 8 years schooling/missing data, n = 167) or high (both parents ≥ 8 years schooling, n = 319) childhood socioeconomic status (cSES) groups. RESULTS: Korean War GI Bill eligibility predicted fewer depressive symptoms among individuals from low cSES backgrounds [ß = -0.64, 95% confidence interval (CI) = (-1.18, -0.09), P = .022]. Socioeconomic disparities were smaller among veterans than nonveterans for number of depressive symptoms [ß = -0.76, 95% CI = (-1.33, -0.18), P = .010] and elevated depressive symptoms [ß = -11.7, 95% CI = (-8.2, -22.6), P = .035]. CONCLUSIONS: Korean War GI Bill eligibility predicted smaller socioeconomic disparities in depression markers.

Self-reports of medication side effects and pain-related activity interference in patients with chronic pain: a longitudinal cohort study.

The primary purpose of this study was to examine the association between self-reports of medication side effects and pain-related activity interference in patients with chronic pain. The potential moderators of the association between reports of side effects and pain-related activity interference were also examined. A total of 111 patients with chronic musculoskeletal pain were asked to provide, once a month for a period of 6 months, self-reports of medication use and the presence of any perceived side effects (eg, nausea, dizziness, headaches) associated with their medications. At each of these time points, patients were also asked to provide self-reports of pain intensity, negative affect, and pain-related activity interference. Multilevel modeling analyses revealed that month-to-month increases in perceived medication side effects were associated with heightened pain-related activity interference (P < 0.05). Importantly, multilevel models revealed that perceived medication side effects were associated with heightened pain-related activity interference even after controlling for the influence of patient demographics, pain intensity, and negative affect. This study provides preliminary evidence that reports of medication side effects are associated with heightened pain-related activity interference in patients with chronic pain beyond the influence of other pain-relevant variables. The implications of our findings for clinical practice and the management of patients with chronic pain conditions are discussed.

Elderly breast and colorectal cancer patients' clinical course: patient and contextual influences.

BACKGROUND: The social and medical environments that surround people are each independently associated with their cancer course. The extent to which these characteristics may together mediate patients' cancer care and outcomes is not known. METHODS: Using multilevel methods and data, we studied elderly breast and colorectal cancer patients (level I) within urban social (level II: ZIP code tabulation area) and health care (level III: hospital service area) contexts. We sought to determine (1) which, if any, observable social and medical contextual attributes were associated with patient cancer outcomes after controlling for observable patient attributes, and (2) the magnitude of residual variation in patient cancer outcomes at each level. RESULTS: Numerous patient attributes and social area attributes, including poverty, were associated with unfavorable patient cancer outcomes across the full clinical cancer continuum for both cancers. Health care area attributes were not associated with patient cancer outcomes. After controlling for observable covariates at all 3 levels, there was substantial residual variation in patient cancer outcomes at all levels. CONCLUSIONS: After controlling for patient attributes known to confer risk of poor cancer outcomes, we find that neighborhood socioeconomic disadvantage exerts an independent and deleterious effect on residents' cancer outcomes, but the area supply of the specific types of health care studied do not. Multilevel interventions targeted at cancer patients and their social areas may be useful. We also show substantial residual variation in patient outcomes across social and health care areas, a finding potentially relevant to traditional small area variation research methods.