Explore the vast range of titles available.

The millions of protein sequences generated by genomics are expected to transform protein engineering and personalized medicine. To achieve these goals, tools for predicting outcomes of amino acid changes must be improved. Currently, advances are hampered by insufficient experimental data about nonconserved amino acid positions. Since the property \"nonconserved\" is identified using a sequence alignment, we designed experiments to recapitulate that context: Mutagenesis and functional characterization was carried out in 15 LacI/GalR homologs (rows) at 12 nonconserved positions (columns). Multiple substitutions were made at each position, to reveal how various amino acids of a nonconserved column were tolerated in each protein row. Results showed that amino acid preferences of nonconserved positions were highly context-dependent, had few correlations with physico-chemical similarities, and were not predictable from their occurrence in natural LacI/GalR sequences. Further, unlike the \"toggle switch\" behaviors of conserved positions, substitutions at nonconserved positions could be rank-ordered to show a \"rheostatic\", progressive effect on function that spanned several orders of magnitude. Comparisons to various sequence analyses suggested that conserved and strongly co-evolving positions act as functional toggles, whereas other important, nonconserved positions serve as rheostats for modifying protein function. Both the presence of rheostat positions and the sequence analysis strategy appear to be generalizable to other protein families and should be considered when engineering protein modifications or predicting the impact of protein polymorphisms.

Protein families might evolve paralogous functions on their common tertiary scaffold in two ways. First, the locations of functionally-important sites might be \"hard-wired\" into the structure, with novel functions evolved by altering the amino acid (e.g. Ala vs Ser) at these positions. Alternatively, the tertiary scaffold might be adaptable, accommodating a unique set of functionally important sites for each paralogous function. To discriminate between these possibilities, we compared the set of functionally important sites in the six largest paralogous subfamilies of the LacI/GalR transcription repressor family. LacI/GalR paralogs share a common tertiary structure, but have low sequence identity (≤ 30%), and regulate a variety of metabolic processes. Functionally important positions were identified by conservation and co-evolutionary sequence analyses. Results showed that conserved positions use a mixture of the \"hard-wired\" and \"accommodating\" scaffold frameworks, but that the co-evolution networks were highly dissimilar between any pair of subfamilies. Therefore, the tertiary structure can accommodate multiple networks of functionally important positions. This possibility should be included when designing and interpreting sequence analyses of other protein families. Software implementing conservation and co-evolution analyses is available at https://sourceforge.net/projects/coevolutils/.

BackgroundAlthough social factors influence uptake of preventive services, the association between social needs and influenza vaccination has not been comprehensively evaluated for adults seeking primary care in the USA.ObjectiveTo determine the association between unmet social needs and influenza vaccination.DesignRetrospective, cross-sectional, multivariable logistic regression.ParticipantsPersons completing ambulatory visits in a primary care department at a midwestern, urban, multispecialty, academic medical center between July 2017 and July 2019 (N = 7955 individuals included).Main MeasuresCompletion of influenza vaccination in the 2018–2019 influenza season (primary outcome) or any year (secondary outcome) against 11 essential social needs (childcare, companionship, food security, health literacy, home safety, neighborhood safety, housing, health care provider costs, prescription costs, transportation, and utilities). Demographics, diabetic status, COPD, smoking status, office visit frequency, and hierarchical condition category risk scores were included as covariates.Key ResultsIndividuals with transportation vulnerability were less likely to be vaccinated against influenza (current-year aOR 0.65, 95% CI: 0.53–0.78, p < 0.001; any-year aOR 0.58, 95% CI: 0.47–0.71, p < 0.001). Poor health literacy promoted any-year, but not current-year, influenza vaccination (any-year aOR 1.30, 95% CI: 1.01–1.69, p = 0.043). Older age, female sex, diabetes, more comorbidities, and more frequent primary care visits were associated with greater influenza vaccination. Persons with Black or other/multiple race and current smokers were less frequently vaccinated.ConclusionsTransportation vulnerability, health literacy, smoking, age, sex, race, comorbidity, and office visit frequency are associated with influenza vaccination. Primary care–led interventions should consider these factors when designing outreach interventions.Trial RegistrationNot applicable

Introduction: Rural and under-resourced urban communities face unique challenges in addressing patients’ social determinants of health needs (SDoH). Community health workers (CHWs) can support patients experiencing social needs, yet little is known about how rural and under-resourced primary care clinics are screening for SDoH or utilizing CHWs. Methods: Interviews were conducted with primary care clinic providers and managers across a geographically large and predominately rural state to assess screening practices for SDoH and related community resources, and perspectives on using CHWs to address SDoH. Interviews were conducted by phone, recorded, and transcribed. Data were analyzed using thematic analysis. We completed interviews with 27 respondents (12 providers and 15 clinic managers) at 26 clinics. Results: Twelve (46.1%) clinics had a standardized process for capturing SDoH, but this was primarily limited to Medicare wellness visits. Staffing and time were identified as barriers to proper SDoH screening. Lack of transportation and affordable medication were the most cited SDoH. While respondents were all aware of CHWs, only 8 (30.8%) included a CHW on their care team. Perceived barriers to engaging CHWs included cost, space, and availability of qualified CHWs. Perceived benefits of engaging CHWs in their practice were: assisting patients with navigating resources and programs, relieving clinical staff of non-medical tasks, and bridging language barriers. Conclusions: Rural and under-resourced primary care clinics need help in identifying and addressing SDoH. CHWs could play an important part in addressing social needs and promoting preventive care if financial constraints could be addressed and local CHWs could be trained.

The Communities Organizing to Promote Equity (COPE) Project was implemented in 20 counties across Kansas to build capacity to address health equity by forming local health equity action teams (LHEATS), hiring and training community health workers, facilitating state-wide learning collaboratives, and tailoring communication strategies. We conducted interviews and focus groups with project stakeholders who identified pragmatic recommendations related to LHEAT formation and leadership, establishing trust, nurturing autonomy, and optimizing impact. Insights can improve future community-based health equity efforts. ( Am J Public Health. 2024;114(S7):S570–S574. https://doi.org/10.2105/AJPH.2024.307802 )

Abstract Amino acid variation at “rheostat” positions provides opportunity to modulate various aspects of protein function – such as binding affinity or allosteric coupling – across a wide range. Previously a subclass of “multiplex” rheostat positions was identified at which substitutions simultaneously modulated more than one functional parameter. Using the Miller laboratory’s dataset of ∼4000 variants of lactose repressor protein (LacI), we compared the structural properties of multiplex rheostat positions with (i) “single” rheostat positions that modulate only one functional parameter, (ii) “toggle” positions that follow textbook substitution rules, and (iii) “neutral” positions that tolerate any substitution without changing function. The combined rheostat classes comprised >40% of LacI positions, more than either toggle or neutral positions. Single rheostat positions were broadly distributed over the structure. Multiplex rheostat positions structurally overlapped with positions involved in allosteric regulation. When their phenotypic outcomes were interpreted within a thermodynamic framework, functional changes at multiplex positions were uncorrelated. This suggests that substitutions lead to complex changes in the underlying molecular biophysics. Bivariable and multivariable analyses of evolutionary signals within multiple sequence alignments could not differentiate single and multiplex rheostat positions. Phylogenetic analyses – such as ConSurf – could distinguish rheostats from toggle and neutral positions. Multivariable analyses could also identify a subset of neutral positions with high probability. Taken together, these results suggest that detailed understanding of the underlying molecular biophysics, likely including protein dynamics, will be required to discriminate single and multiplex rheostat positions from each other and to predict substitution outcomes at these sites. Competing Interest Statement The authors have declared no competing interest.

Revolutionary advances in sequencing technology have dramatically expanded the set of known, naturally-occurring protein sequences. Protein sequences arise from an evolutionary process and during evolution proteins experience pressure to maintain and diversity their functions via mutation. Some mutations arise merely from neutral drift, but other changes enable organisms to adapt to their unique niche. Positions that are important for structure or function are expected to be mutationally constrained during evolution. To that end, many algorithms have been devised to identify mutational constraints in the evolutionary record in order to predict the location of functionally important sites. Accurate prediction of functionally important positions would have important practical implications. For example, individual humans each carry about 10,000 exomic sequence polymorphisms. Which of these are functionally and/or clinically significant? Similarly, protein engineers may target such sites for mutagenesis to derive variant functions. To detect these constraints, homologous proteins must first be sorted into protein families, based on sequence similarity, which typically indicates structural and functional similarity. Protein family multiple sequence alignments (MSAs) can then be computationally analyzed to understand the family in light of its evolutionary history. MSA analyses have detect various evolutionary patterns that are thought to confer functional significance. For example, positions that are absolutely conserved across a family are commonly inferred to play important structural or functional roles and, consequently, be intolerant to mutation. Other analyses attempt to identify important non-conserved positions, some of which must be functionally significant for the family to evolve functional variations. One important example is “co-evolutionary” analyses, which seek pairs of positions that vary in a coordinated manner across evolution. MSA analyses make a number of simplifying assumptions to abstract away the full complexity of real proteins. Here, we have (1) assessed the validity of some of these assumptions, and (2) investigated strategies to maximize the usefulness of existing tools in identifying functionally important positions, in light of their limitations, and (3) evaluated the ability of existing tools to identify known-significant positions. To that end, we have applied MSA analyses to the LacI/GalR bacterial transcription regulator family as our primary model system. Our studies have proceeded in three phases. First, preceding work indicated that published predictions based on a small LacI/GalR MSA fail to identify several functionally-significant positions in the 18-amino acid linker of LacI/GalR proteins. We have investigated whether making better use of these tools – by expanding the set of sequence in the LacI/GalR MSA and sorting the family based on external experimental knowledge – can improve predictive accuracy. Interestingly, comparison of existing predictions to all available experimental data also suggests that – contrary to a common assumption – functionally neutral positions may be much more rare than previously thought. Second, LacI/GalR proteins exhibit substantial functional diversity, even though their structures are extremely similar. One question is: how can a common structure support high levels of functional diversity? We have used conservation and co-evolutionary analyses to determine whether (a) functionally significant positions are dictated by the tertiary structure – an assumption of most MSA analyses – or (b) whether the structure serves as an accommodating scaffold, by permitting multiple subfamily-specific networks of functionally significant positions. Finally, alternative co-evolutionary algorithms disagree about which pairs of positions are evolutionarily-linked. However, we have analyzed alternative co-evolution networks using graph theory and have observed that the eigenvector network centrality (a) improves agreement between diverse analyses, and (b) can identify functionally significant positions in protein families. Thus, eigenvector centrality may be a useful framework for interpreting co-evolution analyses. Taken together, our studies provide tools to make best use of existing MSA analyses and indicate that future tools should avoid making several common assumptions.

Understanding how each residue position contributes to protein function has been a long-standing goal in protein science. Substitution studies have historically focused on conserved protein positions. However, substitutions of nonconserved positions can also modify function. Indeed, we recently identified nonconserved positions that have large substitution effects in human liver pyruvate kinase (hLPYK), including altered allosteric coupling. To facilitate a comparison of which characteristics determine when a nonconserved position does vs. does not contribute to function, the goal of the current work was to identify neutral positions in hLPYK. However, existing hLPYK data showed that three features commonly associated with neutral positions – high sequence entropy, high surface exposure, and alanine scanning – lacked the sensitivity needed to guide experimental studies. We used multiple evolutionary patterns identified in a sequence alignment of the PYK family to identify which positions were least patterned, reasoning that these were most likely to be neutral. Nine positions were tested with a total of 117 amino acid substitutions. Although exploring all potential functions is not feasible for any protein, five parameters associated with substrate/effector affinities and allosteric coupling were measured for hLPYK variants. For each position, the aggregate functional outcomes of all variants were used to quantify a “neutrality” score. Three positions showed perfect neutral scores for all five parameters. Furthermore, the nine positions showed larger neutral scores than 17 positions located near allosteric binding sites. Thus, our strategy successfully enriched the dataset for positions with neutral and modest substitutions.

In the goal of interpreting human exomes, when predictive programs assign functional importance to some positions, they implicitly assume the existence of non-important positions: those that accommodate many side chain chemistries without altering function (neutral). However, very few (if any) experimental studies have demonstrated the existence of neutral positions. We sought experimental evidence for neutral positions using human liver pyruvate kinase (hLPYK) as a model system. To that end, we used multiple evolutionary criteria to identify 20 possibly neutral positions. Nine positions were further tested with a total of 117 amino acid substitutions. Although all potential hLPYK functions can never be explored, we measured effects on 5 parameters associated with substrate/ligand affinities and allosteric coupling. At each position, the aggregate outcomes of multiple variants were used to quantify neutrality scores. Three of the nine positions showed perfect neutral scores in all 5 parameters; a fourth position had high neutral scores. Although our strategy for predicting positions had low predictive power for the identification of neutral positions, all positions had neutral scores that were much higher than positions in functional sites. Given this evidence for the existence of neutral positions, similar studies should be carried out for other proteins to generate a database of well characterized neutral positions that can then be available to benchmark and validate predictions about amino acid substitutions.