Effectiveness of an Online Preceptor Development Program Across Health Professions.

Introduction: An online Preceptor Development Program (PDP) was developed to meet the needs of geographically dispersed preceptors across health professions. We aimed to measure the audience, their engagement, and effectiveness of an online PDP developed and implemented amid the COVID-19 pandemic. Methods: The mixed methods study included survey and attendance data for live and asynchronous formats. T-tests compared overall session perception to self-reported session impact. Objective alignment and self-reported measures of impact were analyzed around Kirkpatrick's levels of reaction, learning, and behavior. Results: Participants engaged in live and/or asynchronous PDP sessions from various professions, specialties, and geographical locations. Quantitative findings indicated significant associations between overall session perception and knowledge (session 2), competence (sessions 1, 2), and performance (session 1). Objectives were met, and key learning takeaways were reported. While most participants indicated no barriers to implementation, two barriers identified were a lack of time and uninterested students. Conclusion: Participants were afforded flexibility and choice and likely benefitted in the areas of knowledge, competence, and behavior. This online PDP effectively addressed preceptor needs in common areas, including feedback. Future program development may include advisory group input and interactive learning opportunities.

The effect of age on emotion processing in individuals with mood disorders and in healthy individuals.

Introduction: Emotion processing is an essential part of interpersonal relationships and social interactions. Changes in emotion processing have been found in both mood disorders and in aging, however, the interaction between such factors has yet to be examined in detail. This is of interest due to the contrary nature of the changes observed in existing research - a negativity bias in mood disorders versus a positivity effect with aging. It is also unclear how changes in non-emotional cognitive function with aging and in mood disorders, interact with these biases. Methods and results: In individuals with mood disorders and in healthy control participants, we examined emotional processing and its relationship to age in detail. Data sets from two studies examining facial expression recognition were pooled. In one study, 98 currently depressed individuals (either unipolar or bipolar) were compared with 61 healthy control participants, and in the other, 100 people with bipolar disorder (in various mood states) were tested on the same facial expression recognition task. Repeated measures analysis of variance was used to examine the effects of age and mood disorder diagnosis alongside interactions between individual emotion, age, and mood disorder diagnosis. A positivity effect was associated with increasing age which was evident irrespective of the presence of mood disorder or current mood episode. Discussion: Results suggest a positivity effect occurring at a relatively early age but with no evidence of a bias toward negative emotions in mood disorder or specifically, in depressed episodes. The positivity effect in emotional processing in aging appears to occur even within people with mood disorders. Further research is needed to understand how this fits with negative biases seen in previous studies in mood disorders.

Race and sex associations with tacrolimus pharmacokinetics in stable kidney transplant recipients.

STUDY OBJECTIVE: This study investigated race and sex differences in tacrolimus pharmacokinetics and pharmacodynamics in stable kidney transplant recipients. DESIGN AND SETTING: A cross-sectional, open-label, single center, 12-h pharmacokinetic-pharmacodynamic study was conducted. Tacrolimus pharmacokinetic parameters included area under the concentration-time curve (AUC0-12 ), AUC0-4 , 12-h troughs (C12 h ), maximum concentrations (Cmax ), oral clearance (Cl), with dose-normalized AUC0-12 , troughs, and Cmax with standardized adverse effect scores. Statistical models were used to analyze end points with individual covariate-adjustment including clinical factors, genotypic variants CYP3A5*3, CYP3A5*6, CYP3A5*7(CYP3A5*3*6*7) metabolic composite, and ATP binding cassette gene subfamily B member 1 (ABCB1) polymorphisms. PATIENTS: 65 stable, female and male, Black and White kidney transplant recipients receiving tacrolimus and mycophenolic acid ≥6 months post-transplant were evaluated. MEASUREMENTS AND MAIN RESULTS: Black recipients exhibited higher tacrolimus AUC0-12 (Race: p = 0.005), lower AUC* (Race: p < 0.001; Race × Sex: p = 0.068), and higher Cl (Race: p < 0.001; Sex: p = 0.066). Greater cumulative (Sex: p < 0.001; Race × Sex: p = 0.014), neurologic (Sex: p = 0.021; Race × Sex: p = 0.005), and aesthetic (Sex: p = 0.002) adverse effects were found in females, with highest scores in Black women. In 84.8% of Black and 68.8% of White patients, the target AUC0-12 was achieved (p = 0.027). In 31.3% of White and 9.1% of Black recipients, AUC0-12 was <100 ng‧h/ml despite tacrolimus troughs in the target range (p = 0.027). The novel CYP3A5*3*6*7 metabolic composite was the significant covariate accounting for 15%-19% of tacrolimus variability in dose (p = 0.002); AUC0-12 h * (p < 0.001), and Cl (p < 0.001). CONCLUSIONS: Tacrolimus pharmacokinetics and adverse effects were different among stable kidney transplant recipient groups based upon race and sex with interpatient variability associated with the CYP3A5*3*6*7 metabolic composite. More cumulative, neurologic, and aesthetic adverse effects were noted among females. Tacrolimus regimens that consider race and sex may reduce adverse effects and enhance allograft outcomes by facilitating more patients to achieve the targeted AUC0-12 h .

Emotion processing in depression and anxiety disorders in older adults: systematic review.

BACKGROUND: Emotional cognition and effective interpretation of affective information is an important factor in social interactions and everyday functioning, and difficulties in these areas may contribute to aetiology and maintenance of mental health conditions. In younger people with depression and anxiety, research suggests significant alterations in behavioural and brain activation aspects of emotion processing, with a tendency to appraise neutral stimuli as negative and attend preferentially to negative stimuli. However, in ageing, research suggests that emotion processing becomes subject to a 'positivity effect', whereby older people attend more to positive than negative stimuli. AIMS: This review examines data from studies of emotion processing in Late-Life Depression and Late-Life Anxiety to attempt to understand the significance of emotion processing variations in these conditions, and their interaction with changes in emotion processing that occur with ageing. METHOD: We conducted a systematic review following PRISMA guidelines. Articles that used an emotion-based processing task, examined older persons with depression or an anxiety disorder and included a healthy control group were included. RESULTS: In Late-Life Depression, there is little consistent behavioural evidence of impaired emotion processing, but there is evidence of altered brain circuitry during these processes. In Late-Life Anxiety and Post-Traumatic Stress disorder, there is evidence of interference with processing of negative or threat-related words. CONCLUSIONS: How these findings fit with the positivity bias of ageing is not clear. Future research is required in larger groups, further examining the interaction between illness and age and the significance of age at disease onset.

Elucidating the Molecular Determinants of Aß Aggregation with Deep Mutational Scanning.

Despite the importance of Aß aggregation in Alzheimer's disease etiology, our understanding of the sequence determinants of aggregation is sparse and largely derived from in vitro studies. For example, in vitro proline and alanine scanning mutagenesis of Aß40 proposed core regions important for aggregation. However, we lack even this limited mutagenesis data for the more disease-relevant Aß42 Thus, to better understand the molecular determinants of Aß42 aggregation in a cell-based system, we combined a yeast DHFR aggregation assay with deep mutational scanning. We measured the effect of 791 of the 798 possible single amino acid substitutions on the aggregation propensity of Aß42 We found that ∼75% of substitutions, largely to hydrophobic residues, maintained or increased aggregation. We identified 11 positions at which substitutions, particularly to hydrophilic and charged amino acids, disrupted Aß aggregation. These critical positions were similar but not identical to critical positions identified in previous Aß mutagenesis studies. Finally, we analyzed our large-scale mutagenesis data in the context of different Aß aggregate structural models, finding that the mutagenesis data agreed best with models derived from fibrils seeded using brain-derived Aß aggregates.

Multiplex assessment of protein variant abundance by massively parallel sequencing.

Determining the pathogenicity of genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes requires generalizable, scalable assays. We describe variant abundance by massively parallel sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance simultaneously. We apply VAMP-seq to quantify the abundance of 7,801 single-amino-acid variants of PTEN and TPMT, proteins in which functional variants are clinically actionable. We identify 1,138 PTEN and 777 TPMT variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and show that p.Pro38Ser, which accounts for ~10% of PTEN missense variants in melanoma, functions via a dominant-negative mechanism. Finally, we demonstrate that VAMP-seq is applicable to other genes, highlighting its generalizability.

Quantitative Missense Variant Effect Prediction Using Large-Scale Mutagenesis Data.

Large datasets describing the quantitative effects of mutations on protein function are becoming increasingly available. Here, we leverage these datasets to develop Envision, which predicts the magnitude of a missense variant's molecular effect. Envision combines 21,026 variant effect measurements from nine large-scale experimental mutagenesis datasets, a hitherto untapped training resource, with a supervised, stochastic gradient boosting learning algorithm. Envision outperforms other missense variant effect predictors both on large-scale mutagenesis data and on an independent test dataset comprising 2,312 TP53 variants whose effects were measured using a low-throughput approach. This dataset was never used for hyperparameter tuning or model training and thus serves as an independent validation set. Envision prediction accuracy is also more consistent across amino acids than other predictors. Finally, we demonstrate that Envision's performance improves as more large-scale mutagenesis data are incorporated. We precompute Envision predictions for every possible single amino acid variant in human, mouse, frog, zebrafish, fruit fly, worm, and yeast proteomes (https://envision.gs.washington.edu/).

Analysis of Large-Scale Mutagenesis Data To Assess the Impact of Single Amino Acid Substitutions.

Mutagenesis is a widely used method for identifying protein positions that are important for function or ligand binding. Advances in high-throughput DNA sequencing and mutagenesis techniques have enabled measurement of the effects of nearly all possible amino acid substitutions in many proteins. The resulting large-scale mutagenesis data sets offer a unique opportunity to draw general conclusions about the effects of different amino acid substitutions. Thus, we analyzed 34,373 mutations in 14 proteins whose effects were measured using large-scale mutagenesis approaches. Methionine was the most tolerated substitution, while proline was the least tolerated. We found that several substitutions, including histidine and asparagine, best recapitulated the effects of other substitutions, even when the identity of the wild-type amino acid was considered. The effects of histidine and asparagine substitutions also correlated best with the effects of other substitutions in different structural contexts. Furthermore, highly disruptive substitutions like aspartic and glutamic acid had the most discriminatory power for detecting ligand interface positions. Our work highlights the utility of large-scale mutagenesis data, and our conclusions can help guide future single substitution mutational scans.

Comparison of Clinical Outcomes and Adverse Events Associated With Glucose-Lowering Drugs in Patients With Type 2 Diabetes: A Meta-analysis.

IMPORTANCE: Numerous glucose-lowering drugs are used to treat type 2 diabetes. OBJECTIVE: To estimate the relative efficacy and safety associated with glucose-lowering drugs including insulin. DATA SOURCES: Cochrane Library Central Register of Controlled Trials, MEDLINE, and EMBASE databases through March 21, 2016. STUDY SELECTION: Randomized clinical trials of 24 weeks' or longer duration. DATA EXTRACTION AND SYNTHESIS: Random-effects network meta-analysis. MAIN OUTCOMES AND MEASURES: The primary outcome was cardiovascular mortality. Secondary outcomes included all-cause mortality, serious adverse events, myocardial infarction, stroke, hemoglobin A1c (HbA1C) level, treatment failure (rescue treatment or lack of efficacy), hypoglycemia, and body weight. RESULTS: A total of 301 clinical trials (1,417,367 patient-months) were included; 177 trials (56,598 patients) of drugs given as monotherapy; 109 trials (53,030 patients) of drugs added to metformin (dual therapy); and 29 trials (10,598 patients) of drugs added to metformin and sulfonylurea (triple therapy). There were no significant differences in associations between any drug class as monotherapy, dual therapy, or triple therapy with odds of cardiovascular or all-cause mortality. Compared with metformin, sulfonylurea (standardized mean difference [SMD], 0.18 [95% CI, 0.01 to 0.34]), thiazolidinedione (SMD, 0.16 [95% CI, 0.00 to 0.31]), DPP-4 inhibitor (SMD, 0.33 [95% CI, 0.13 to 0.52]), and α-glucosidase inhibitor (SMD, 0.35 [95% CI, 0.12 to 0.58]) monotherapy were associated with higher HbA1C levels. Sulfonylurea (odds ratio [OR], 3.13 [95% CI, 2.39 to 4.12]; risk difference [RD], 10% [95% CI, 7% to 13%]) and basal insulin (OR, 17.9 [95% CI, 1.97 to 162]; RD, 10% [95% CI, 0.08% to 20%]) were associated with greatest odds of hypoglycemia. When added to metformin, drugs were associated with similar HbA1C levels, while SGLT-2 inhibitors offered the lowest odds of hypoglycemia (OR, 0.12 [95% CI, 0.08 to 0.18]; RD, -22% [-27% to -18%]). When added to metformin and sulfonylurea, GLP-1 receptor agonists were associated with the lowest odds of hypoglycemia (OR, 0.60 [95% CI, 0.39 to 0.94]; RD, -10% [95% CI, -18% to -2%]). CONCLUSIONS AND RELEVANCE: Among adults with type 2 diabetes, there were no significant differences in the associations between any of 9 available classes of glucose-lowering drugs (alone or in combination) and the risk of cardiovascular or all-cause mortality. Metformin was associated with lower or no significant difference in HbA1C levels compared with any other drug classes. All drugs were estimated to be effective when added to metformin. These findings are consistent with American Diabetes Association recommendations for using metformin monotherapy as initial treatment for patients with type 2 diabetes and selection of additional therapies based on patient-specific considerations.

A Molecular Evolutionary Reference for the Human Variome.

Widespread sequencing efforts are revealing unprecedented amount of genomic variation in populations. Such information is routinely used to derive consensus reference sequences and to infer positions subject to natural selection. Here, we present a new molecular evolutionary method for estimating neutral evolutionary probabilities (EPs) of each amino acid, or nucleotide state at a genomic position without using intraspecific polymorphism data. Because EPs are derived independently of population-level information, they serve as null expectations that can be used to evaluate selective forces on alleles at both polymorphic and monomorphic positions in populations. We applied this method to coding sequences in the human genome and produced a comprehensive evolutionary variome reference for all human proteins. We found that EPs accurately predict neutral and disease-associated alleles. Through an analysis of discordance between allelic EPs and their observed population frequencies, we discovered thousands of novel candidate sites for nonneutral evolution in human proteins. Many of these were validated in a joint analysis of disease-associated variants and population data. The EP method is also directly applicable to the analysis of noncoding sequences and genomic analyses of nonmodel species.

Drug-, dose- and sex-dependent effects of chronic fluoxetine, reboxetine and venlafaxine on open-field behavior and spatial memory in rats.

In an effort to address the need to include both sexes in studies of effects of the SSRI fluoxetine, the NRI reboxetine and the SNRI venlafaxine on anxiety-related behavior and memory along with the use of chronic drug administration, male and female PVG/c rats were fed diets containing two doses of each drug for 21 days. The rats' anxiety level was then assessed in an open field. Short-term spatial memory for a brightness change in a Y maze was also measured. While there was little evidence of anxiolytic effects of any of the drugs, both fluoxetine and, to a lesser extent, venlafaxine appeared to be mainly anxiogenic in their action depending on both dose and sex. Reboxetine was relatively ineffective in this respect. Ability to locate the Y-maze arm that had changed (from white to black) seemed to be impaired for male (but not female) rats by both fluoxetine and venlafaxine and, to a much lesser extent, by reboxetine. Given the relative ineffectiveness of reboxetine in either test, it is possible that the effects of the other two drugs on both anxiety and memory were mainly due to their serotonin reuptake inhibiting properties. The differences that occurred between males and females in responsiveness to all three drugs supported the long-held view that both sexes should be investigated in studies of this sort, especially in view of reports of sex differences in effects of clinically prescribed antidepressants.

Influence of sex and race on mycophenolic acid pharmacokinetics in stable African American and Caucasian renal transplant recipients.

BACKGROUND AND OBJECTIVES: No evaluation of sex and race influences on mycophenolic acid (MPA) pharmacokinetics and adverse effects (AEs) during enteric-coated mycophenolate sodium (ECMPS) and tacrolimus immunosuppression are available. The primary objective of this study was to investigate the influence of sex and race on MPA and MPA glucuronide (MPAG) pharmacokinetics in stable renal transplant recipients receiving ECMPS and tacrolimus METHODS: The pharmacokinetics of MPA and MPAG and their associated gastrointestinal AEs were investigated in 67 stable renal transplant recipients: 22 African American males (AAMs), 13 African American females (AAFs), 16 Caucasian males (CMs), and 16 Caucasian females (CFs) receiving ECMPS and tacrolimus. A validated gastrointestinal AE rating included diarrhea, dyspepsia, vomiting, and acid-suppressive therapy was completed. Apparent clearance, clearance normalized to body mass index (BMI), area under the concentration-time curve from time zero to 12 h (AUC12) and dose-normalized AUC12 (AUC*) were determined using a statistical model that incorporated gastrointestinal AE and clinical covariates. RESULTS: Males had more rapid apparent MPA clearance (CMs 13.8 ± 6.27 L/h vs. AAMs 10.2 ± 3.73 L/h) than females (CFs 8.70 ± 3.33 L/h and AAFs 9.71 ± 3.94 L/h; p = 0.014) with a race-sex interaction (p = 0.043). Sex differences were observed in MPA clearance/BMI (p = 0.033) and AUC* (p = 0.033). MPA AUC12 was greater than 60 mg·h/L in 57 % of renal transplant recipients (RTR) with 71 % of patients demonstrating gastrointestinal AEs and a higher score noted in females. In all patients, females exhibited 1.40-fold increased gastrointestinal AE scores compared with males (p = 0.024). Race (p = 0.044) and sex (p = 0.005) differences were evident with greater MPAG AUC12 in AAFs and CFs. CONCLUSION: Sex and race differences were evident, with females having slower MPA clearance, higher MPAG AUC12, and more severe gastrointestinal AEs. These findings suggest sex and race should be considered during MPA immunosuppression.

Signatures of natural selection on mutations of residues with multiple posttranslational modifications.

Posttranslational modifications (PTMs) regulate molecular structures and functions of proteins by covalently binding to amino acids. Hundreds of thousands of PTMs have been reported for the human proteome, with multiple PTMs known to affect tens of thousands of lysine (K) residues. Our molecular evolutionary analyses show that K residues with multiple PTMs exhibit greater conservation than those with a single PTM, but the difference is rather small. In contrast, short-term evolutionary trends revealed in an analysis of human population variation exhibited a much larger difference. Lysine residues with three PTMs show 1.8-fold enrichment of Mendelian disease-associated variants when compared with K residues with two PTMs, with the latter showing 1.7-fold enrichment of these variants when compared with the K residues with one PTM. Rare polymorphisms in humans show a similar trend, which suggests much greater negative selection against mutations of K residues with multiple PTMs within population. Conversely, common polymorphisms are overabundant at unmodified K residues and at K residues with fewer PTMs. The observed difference between inter- and intraspecies patterns of purifying selection on residues with PTMs suggests extensive species-specific drifting of PTM positions. These results suggest that the functionality of a protein is likely conserved, without necessarily conserving the PTM positions over evolutionary time.

Race and drug formulation influence on mycophenolic acid pharmacokinetics in stable renal transplant recipients.

BACKGROUND: Limited mycophenolic acid (MPA) data are available comparing racial influence on mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS) pharmacokinetics. METHODS: Intrapatient MPA pharmacokinetics of MMF versus EC-MPS were compared in 13 male African American (AA) and 14 Caucasian (C) renal transplant recipients (RTRs). RTRs were switched to equivalent doses of the alternate formulation for at least 10 days prior to the second study. Mycophenolic acid clearance and dose-normalized area under the concentration-time curve(0-12) (AUC*) were determined. Mixed model statistics evaluated the main effects of race, drug formulation, and interaction of race and drug formulation (R × D) with albumin, cyclosporine trough, renal function, and diabetes and enterhepatic recirculation. RESULTS: Significant R × D was identified for MPA AUC* for EC-MPS (AA, 0.056 ± 0.029 [mg·h/L]/mg; C, 0.080 ± 0.044 [mg·h/L]/mg) compared with MMF (AA, 0.053 ± 0.019 [mg·h/L]/mg; C, 0.060 ± 0.025 [mg·h/L]/mg), P = .022. Significant R × D was identified with albumin in the model for MPA clearance for MMF (AA, 21.7 ± 8.9 L/h; C, 20.5 ± 10.8 L/h) compared with EC-MPS (AA, 22.2 ± 10.1 L/h; C, 16.2 ± 9.1 L/h), P = .032. CONCLUSIONS: Race influences MPA exposure between MMF and EC-MPS and may warrant therapeutic monitoring during formulation conversion.

The evolutionary history of amino acid variations mediating increased resistance of S. aureus identifies reversion mutations in metabolic regulators.

The evolution of resistance in Staphylococcus aureus occurs rapidly, and in response to all known antimicrobial treatments. Numerous studies of model species describe compensatory roles of mutations in mediating competitive fitness, and there is growing evidence that these mutation types also drive adaptation of S. aureus strains. However, few studies have tracked amino acid changes during the complete evolutionary trajectory of antibiotic adaptation or been able to predict their functional relevance. Here, we have assessed the efficacy of computational methods to predict biological resistance of a collection of clinically known Resistance Associated Mutations (RAMs). We have found that >90% of known RAMs are incorrectly predicted to be functionally neutral by at least one of the prediction methods used. By tracing the evolutionary histories of all of the false negative RAMs, we have discovered that a significant number are reversion mutations to ancestral alleles also carried in the MSSA476 methicillin-sensitive isolate. These genetic reversions are most prevalent in strains following daptomycin treatment and show a tendency to accumulate in biological pathway reactions that are distinct from those accumulating non-reversion mutations. Our studies therefore show that in addition to non-reversion mutations, reversion mutations arise in isolates exposed to new antibiotic treatments. It is possible that acquisition of reversion mutations in the genome may prevent substantial fitness costs during the progression of resistance. Our findings pose an interesting question to be addressed by further clinical studies regarding whether or not these reversion mutations lead to a renewed vulnerability of a vancomycin or daptomycin resistant strain to antibiotics administered at an earlier stage of infection.

Comparison of 12-hour creatinine clearance and estimated glomerular filtration rate in renal transplant recipients.

Glomerular filtration rate (GFR) is an essential clinical assessment of renal function post-renal transplantation. Creatinine clearance (CrCl) measured over 12 h and estimated GFR (e-GFR) (calculated by the Modification of diet in renal disease equation) were compared in 28 stable renal transplant recipients (RTRs). This single center study included 14 African American (AA) and 14 Caucasian (CC) recipients. The 12-h creatinine clearance (CrCl-12 h) was determined by monitored urine collection and by e-GFR on two occasions (two phases) separated by at least 2 weeks. Statistics included mixed model analysis of CrCl-12 h and e-GFR relative to race, phase, and difference between parameters. In the first phase, the e-GFR was higher in AA males (58.4 ± 14.8 mL/min) than the CC males (46.2 ± 10.2 mL/min) (p = 0.032), whereas the CrCl-12 h of AA males (70.8 ± 8.7 mL/min) and CC males (63.3 ± 21.7 mL/min) was not different (p = 0.740). During the second phase, the e-GFR in AA and CC RTRs was 55.4 ± 10.1 mL/min and 47.6 ± 10.7 mL/min (p = 0.117), respectively, whereas CrCl-12 h in AAs was 64.71 ± 17.9 mL/min and in CCs was 62.0 ± 14.9 mL/min (p = 1.000). The CrCl-12 h was higher than the e-GFR (p < 0.001) irrespective of race or phase. CrCl-12 h was not different on both occasions (p = 0.289) in all the patients. CrCl-12 h was consistently greater than e-GFR. The difference between these e-GFR estimates may have an importance in the care of RTRs.

Performance of computational tools in evaluating the functional impact of laboratory-induced amino acid mutations.

Site-directed mutagenesis is frequently used by scientists to investigate the functional impact of amino acid mutations in the laboratory. Over 10,000 such laboratory-induced mutations have been reported in the UniProt database along with the outcomes of functional assays. Here, we explore the performance of state-of-the-art computational tools (Condel, PolyPhen-2 and SIFT) in correctly annotating the function-altering potential of 10,913 laboratory-induced mutations from 2372 proteins. We find that computational tools are very successful in diagnosing laboratory-induced mutations that elicit significant functional change in the laboratory (up to 92% accuracy). But, these tools consistently fail in correctly annotating laboratory-induced mutations that show no functional impact in the laboratory assays. Therefore, the overall accuracy of computational tools for laboratory-induced mutations is much lower than that observed for the naturally occurring human variants. We tested and rejected the possibilities that the preponderance of changes to alanine and the presence of multiple base-pair mutations in the laboratory were the reasons for the observed discordance between the performance of computational tools for natural and laboratory mutations. Instead, we discover that the laboratory-induced mutations occur predominately at the highly conserved positions in proteins, where the computational tools have the lowest accuracy of correct prediction for variants that do not impact function (neutral). Therefore, the comparisons of experimental-profiling results with those from computational predictions need to be sensitive to the evolutionary conservation of the positions harboring the amino acid change.

Rampant purifying selection conserves positions with posttranslational modifications in human proteins.

Posttranslational modifications (PTMs) are chemical alterations that are critical to protein conformation and activation states. Despite their functional importance and reported involvement in many diseases, evolutionary analyses have produced enigmatic results because only weak or no selective pressures have been attributed to many types of PTMs. In a large-scale analysis of 16,836 PTM positions from 4,484 human proteins, we find that positions harboring PTMs show evidence of higher purifying selection in 70% of the phosphorylated and N-linked glycosylated proteins. The purifying selection is up to 42% more severe at PTM residues as compared with the corresponding unmodified amino acids. These results establish extensive selective pressures in the long-term history of positions that experience PTMs in the human proteins. Our findings will enhance our understanding of the historical function of PTMs over time and help in predicting PTM positions by using evolutionary comparisons.