A Ratiometric Catalog of Protein Isoform Shifts in the Cardiac Fetal Gene Program.

The fetal genetic program orchestrates cardiac development and the re-expression of fetal genes is thought to underlie cardiac disease and adaptation. Here, a proteomics ratio test using mass spectrometry is applied to find protein isoforms with statistically significant usage differences in the fetal vs. postnatal mouse heart. Changes in isoform usage ratios are pervasive at the protein level, with 104 significant events observed, including 88 paralog-derived isoform switching events and 16 splicing-derived isoform switching events between fetal and postnatal hearts. The ratiometric proteomic comparisons rediscovered hallmark fetal gene signatures including a postnatal switch from fetal ß (MYH7) toward ɑ (MYH6) myosin heavy chains and from slow skeletal muscle (TNNI1) toward cardiac (TNNI3) troponin I. Altered usages in metabolic proteins are prominent, including a platelet to muscle phosphofructokinase (PFKP - PFKM), enolase 1 to 3 (ENO1 - ENO3), and alternative splicing of pyruvate kinase M2 toward M1 (PKM2 - PKM1) isoforms in glycolysis. The data also revealed a parallel change in mitochondrial proteins in cardiac development, suggesting the shift toward aerobic respiration involves also a remodeling of the mitochondrial protein isoform proportion. Finally, a number of glycolytic protein isoforms revert toward their fetal forms in adult hearts under pathological cardiac hypertrophy, suggesting their functional roles in adaptive or maladaptive response, but this reversal is partial. In summary, this work presents a catalog of ratiometric protein markers of the fetal genetic program of the mouse heart, including previously unreported splice isoform markers.

Tissue Usage Preference and Intrinsically Disordered Region Remodeling of Alternative Splicing Derived Proteoforms in the Heart.

A computational analysis of mass spectrometry data was performed to uncover alternative splicing derived protein variants across chambers of the human heart. Evidence for 216 non-canonical isoforms was apparent in the atrium and the ventricle, including 52 isoforms not documented on SwissProt and recovered using an RNA sequencing derived database. Among non-canonical isoforms, 29 show signs of regulation based on statistically significant preferences in tissue usage, including a ventricular enriched protein isoform of tensin-1 (TNS1) and an atrium-enriched PDZ and LIM Domain 3 (PDLIM3) isoform 2 (PDLIM3-2/ALP-H). Examined variant regions that differ between alternative and canonical isoforms are highly enriched with intrinsically disordered regions. Moreover, over two-thirds of such regions are predicted to function in protein binding and RNA binding. The analysis here lends further credence to the notion that alternative splicing diversifies the proteome by rewiring intrinsically disordered regions, which are increasingly recognized to play important roles in the generation of biological function from protein sequences.

Changes in medical student attendance and its impact on student educational outcomes: a systematic review protocol.

INTRODUCTION: The COVID-19 pandemic has had a significant impact on medical education, with many institutions shifting to online learning to ensure the safety of students and staff. However, there has been a decline in in-person attendance at medical schools across the UK and worldwide following the relaxation of social distancing rules and the reinstation of in-person teaching. Importantly, this trend has been observed prior to the pandemic. While reflected within the literature, there is currently no systematic review describing these changes. We aim to find out how medical students' attendance is changing as documented within the literature and its impact on their educational outcomes. METHODS AND ANALYSIS: This systematic review will follow the guidelines of the Centre of Research and Dissemination, Meta-analyses of Observational Studies in Epidemiology and Preferred Reporting Items for Systematic Reviews and Meta-Analyses. We will search the major databases of Medline via Ovid, Embase via Ovid, Scopus, Web of Science, British Education Index via EBSCOhost and ERIC via EBSCOhost.Two reviewers will independently screen each paper and extract data, with a third reviewer for dispute resolution. All studies reporting on medical students from various universities, both graduate and undergraduate and describing changes in attendance and/or students' educational outcomes will be included. Risk of bias in individual studies will be assessed using the Newcastle-Ottawa Scale and confidence in cumulative evidence will be evaluated using the Grading of Recommendations, Assessment, Development and Evaluation-Confidence in the Evidence from Reviews of Qualitative Research approach. A narrative synthesis of the findings from all included studies will be reported. ETHICS AND DISSEMINATION: Ethical approval is not required for this systematic review of existing publicly available literature. We will subsequently aim to publish the results of this systematic review in a peer-reviewed journal.

Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.

The spatial and temporal distributions of proteins are critical to protein function, but cannot be directly assessed by measuring protein bundance. Here we describe a mass spectrometry-based proteomics strategy, Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently protein turnover rates and subcellular localization in the same experiment. Applying the method, we find that unfolded protein response (UPR) has different effects on protein turnover dependent on their subcellular location in human AC16 cells, with proteome-wide slowdown but acceleration among stress response proteins in the ER and Golgi. In parallel, UPR triggers broad differential localization of proteins including RNA-binding proteins and amino acid transporters. Moreover, we observe newly synthesized proteins including EGFR that show a differential localization under stress than the existing protein pools, reminiscent of protein trafficking disruptions. We next applied SPLAT to an induced pluripotent stem cell derived cardiomyocyte (iPSC-CM) model of cancer drug cardiotoxicity upon treatment with the proteasome inhibitor carfilzomib. Paradoxically, carfilzomib has little effect on global average protein half-life, but may instead selectively disrupt sarcomere protein homeostasis. This study provides a view into the interactions of protein spatial and temporal dynamics and demonstrates a method to examine protein homeostasis regulations in stress and drug response.

Proteomics applications in next generation induced pluripotent stem cell models.

INTRODUCTION: Induced pluripotent stem (iPS) cell technology has transformed biomedical research. New opportunities now exist to create new organoids, microtissues, and body-on-a-chip systems for basic biology investigations and clinical translations. AREAS COVERED: We discuss the utility of proteomics for attaining an unbiased view into protein expression changes during iPS cell differentiation, cell maturation, and tissue generation. The ability to discover cell-type specific protein markers during the differentiation and maturation of iPS-derived cells has led to new strategies to improve cell production yield and fidelity. In parallel, proteomic characterization of iPS-derived organoids is helping to realize the goal of bridging in vitro and in vivo systems. EXPERT OPINIONS: We discuss some current challenges of proteomics in iPS cell research and future directions, including the integration of proteomic and transcriptomic data for systems-level analysis.

Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.

The functions of proteins depend on their spatial and temporal distributions, which are not directly measured by static protein abundance. Under endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) pathway remediates proteostasis in part by altering the turnover kinetics and spatial distribution of proteins. A global view of these spatiotemporal changes has yet to emerge and it is unknown how they affect different cellular compartments and pathways. Here we describe a mass spectrometry-based proteomics strategy and data analysis pipeline, termed Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently the changes in protein turnover and subcellular distribution in the same experiment. Investigating two common UPR models of thapsigargin and tunicamycin challenge in human AC16 cells, we find that the changes in protein turnover kinetics during UPR varies across subcellular localizations, with overall slowdown but an acceleration in endoplasmic reticulum and Golgi proteins involved in stress response. In parallel, the spatial proteomics component of the experiment revealed an externalization of amino acid transporters and ion channels under UPR, as well as the migration of RNA-binding proteins toward an endosome co-sedimenting compartment. The SPLAT experimental design classifies heavy and light SILAC labeled proteins separately, allowing the observation of differential localization of new and old protein pools and capturing a partition of newly synthesized EGFR and ITGAV to the ER under stress that suggests protein trafficking disruptions. Finally, application of SPLAT toward human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) exposed to the cancer drug carfilzomib, identified a selective disruption of proteostasis in sarcomeric proteins as a potential mechanism of carfilzomib-mediated cardiotoxicity. Taken together, this study provides a global view into the spatiotemporal dynamics of human cardiac cells and demonstrates a method for inferring the coordinations between spatial and temporal proteome regulations in stress and drug response.

Prevalence of and Risk Factors for Drug-Related Readmissions in Older Adults: A Systematic Review and Meta-Analysis.

BACKGROUND: Older adults are at an increased risk of drug-related problems, especially following discharge from hospital. Drug-related readmissions place a large burden on the patient and the healthcare system. However, previous studies report inconsistent results on the prevalence and associated risk factors for drug-related hospital readmissions in older adults. OBJECTIVES: We aimed to assess the prevalence of drug-related readmissions in older adults aged 65 years and older and investigate the drug classes, preventability and risk factors most associated with these readmissions. METHODS: A systematic review and meta-analysis were undertaken to answer our objectives. A search of four databases (MEDLINE, Embase, CINAHL and Scopus) was conducted. Three authors independently performed title and abstract screening, full-text screening and data extraction of all included studies. A meta-analysis was conducted to calculate the pooled prevalence of drug-related readmissions across all studies, and a subgroup analysis was performed to explore heterogeneity among studies reporting on adverse drug reaction-related readmissions. RESULTS: A total of 1978 studies were identified in the initial search, of which four studies were included in the final synthesis. Three studies focused on readmissions due to adverse drug reactions and one study focused on readmissions due to drug-related problems. A pooled prevalence of 9% (95% confidence interval 2-18) was found for drug-related readmissions across all studies, and a pooled prevalence of 6% (95% confidence interval 4-10) was found for adverse drug reaction-related readmissions. Three studies explored the preventability of readmissions and 15.4-22.2% of cases were deemed preventable. The drug classes most associated with adverse drug reaction readmissions included anticoagulants, antibiotics, psychotropics and chemotherapy agents. Polypharmacy (the use of five or more medications) and several comorbidities such as cancer, liver disease, ischaemic heart disease and peptic ulcer disease were identified as risk factors for drug-related readmissions. CONCLUSIONS: Almost one in ten older adults discharged from hospital experienced a drug-related hospital readmission, with one fifth of these deemed preventable. Several comorbidities and the use of polypharmacy and high-risk drugs were identified as prominent risk factors for readmission. Further research is needed to explore possible causes of drug-related readmissions in older adults for a more guided approach to the development of effective medication management interventions.

Tissue Usage Preference and Intrinsically Disordered Region Remodeling of Alternative Splicing Derived Proteoforms in the Heart.

A computational analysis of mass spectrometry data was performed to uncover alternative splicing derived protein variants across chambers of the human heart. Evidence for 216 non-canonical isoforms was apparent in the atrium and the ventricle, including 52 isoforms not documented on SwissProt and recovered using an RNA sequencing derived database. Among non-canonical isoforms, 29 show signs of regulation based on statistically significant preferences in tissue usage, including a ventricular enriched protein isoform of tensin-1 (TNS1) and an atrium-enriched PDZ and LIM Domain 3 (PDLIM3) isoform 2 (PDLIM3-2/ALP-H). Examined variant regions that differ between alternative and canonical isoforms are highly enriched in intrinsically disordered regions, and over two-thirds of such regions are predicted to function in protein binding and/or RNA binding. The analysis here lends further credence to the notion that alternative splicing diversifies the proteome by rewiring intrinsically disordered regions, which are increasingly recognized to play important roles in the generation of biological function from protein sequences.

Poisonings in Older People with Dementia: A Systematic Scoping Review and Meta-Analysis.

BACKGROUND: Older people with dementia are at a particularly high risk of poisonings and their subsequent harms. OBJECTIVE: This review aimed to describe the key agents, incidence, risk factors, and disposition of poisonings in people with dementia reported in the literature. METHODS: Medline, Embase, CINAHL, and PsycINFO databases were searched from 1 September 2001 to 1 September 2021. Terms for dementia, poisonings, and older adults formed the search concepts. Quantitative studies published in English, describing poisonings in older people with dementia, including Alzheimer's disease, were included. Two investigators independently assessed articles for eligibility and extracted relevant data. A meta-analysis of the incidence of poisonings in people with dementia across studies was performed. RESULTS: Of 4,579 articles, 18 were included for final synthesis. Nervous system medications were implicated in over half of all medicinal poisonings, with anti-dementia agents, benzodiazepines, and opioids the most common classes. The non-medicinal agents frequently associated with poisonings were personal care and household products. The yearly incidence of poisoning varied across definitions of poisoning from 3% for International Classification of Disease-defined poisonings to 43% for adverse drug event-defined poisonings. Several risk factors were identified, including multimorbidity, psychotropic medication use, and living in residential care. Where described, up to one in five poisonings resulted in hospitalisation and in death. CONCLUSIONS: Poisonings are common in people with dementia, involving commonly prescribed medications or easily accessible substances. Given the significant outcomes associated, further research is required to better understand these poisonings and improve public health strategies to reduce the occurrence of this preventable harm.

Senescence in human AC16 cardiac cells is associated with thymidine kinase induction and histone loss.

AC16 cells are a transformed human cardiac cell line commonly used to study cardiomyocyte biology. We show that reduced proliferation and senescence markers can be robustly induced in AC16 cells cultured in low serum condition and treated with (i) low-dose doxorubicin, (ii) UV 254 nm, or (iii) H 2 O 2 exposure for up to 48 hours. Increased p21 (CDKN1A) and H2A.X variant histone (H2AX) levels serve as reliable molecular markers upon all three treatment conditions, but the up-regulation of another common senescence marker, p16 (CDKN2A) was not observed. A proteomics screen further shows that the loss of histones and the increased expression of thymidine kinases (TK1) are prominent features of AC16 cells under doxorubicin induced senescence.

Co-culture with normal and senescent AC16 cardiomyocytes modulates fibrotic response in primary human ventricular fibroblasts.

Transwell co-culture with human AC16 cardiomyocyte-like cells modifies the response of primary human ventricular fibroblasts to TGF-ß stimulation. Fibrotic response markers including collagen I (COL1A1) and ɑ-smooth muscle actin (ACTA2) are amplified in the presence of AC16 cells, whereas others including periostin (POSTN) and fibronectin (FN1) are suppressed. Similar modulation is observed when the ventricular fibroblasts are co-cultured with AC16 cells under baseline and induced senescence conditions. Given that the response to TGF-ß stimulation is commonly measured to study fibrotic signaling and drug treatments in vitro, the results here suggest that the effect of cellular crosstalk should be more broadly considered.

Daratumumab, bortezomib and dexamethasone at first relapse for patients with multiple myeloma: A real-world multicentre UK retrospective analysis.

Daratumumab, bortezomib and dexamethasone (DVd) is approved for patients with relapsed multiple myeloma following the CASTOR phase 3 clinical trial. This retrospective multicentre analysis assesses the overall response rate (ORR) and progression-free survival (PFS) in routine clinical practice for patients at first relapse treated with DVd incorporating weekly bortezomib. Data were collected from 296 sequential patients treated across 15 UK centres. After a median follow-up of 21 months, the ORR was 82% (26% partial response, 56% very good partial response or better) and the median PFS was 16 months [95% confidence interval (CI) 12-20 months]. Results were similar regardless of prior lenalidomide exposure. The median time to next treatment was 20 months (95% CI 15-25 months) and the estimated overall survival at two years was 74%. Patients with high-risk features (by cytogenetics, International Staging System or extramedullary disease) and those treated within 18 months of initiation of progression-free treatment, or within 12 months of autologous stem cell transplant, had significantly inferior outcomes. The grade 2 and 3 peripheral neuropathy rate was 7%. DVd with weekly bortezomib was effective in a heterogenous real-world population at first relapse with a low rate of peripheral neuropathy. However, high-risk patients had inferior outcomes and should be considered for alternative treatments.

Protein prediction models support widespread post-transcriptional regulation of protein abundance by interacting partners.

Protein and mRNA levels correlate only moderately. The availability of proteogenomics data sets with protein and transcript measurements from matching samples is providing new opportunities to assess the degree to which protein levels in a system can be predicted from mRNA information. Here we examined the contributions of input features in protein abundance prediction models. Using large proteogenomics data from 8 cancer types within the Clinical Proteomic Tumor Analysis Consortium (CPTAC) data set, we trained models to predict the abundance of over 13,000 proteins using matching transcriptome data from up to 958 tumor or normal adjacent tissue samples each, and compared predictive performances across algorithms, data set sizes, and input features. Over one-third of proteins (4,648) showed relatively poor predictability (elastic net r ≤ 0.3) from their cognate transcripts. Moreover, we found widespread occurrences where the abundance of a protein is considerably less well explained by its own cognate transcript level than that of one or more trans locus transcripts. The incorporation of additional trans-locus transcript abundance data as input features increasingly improved the ability to predict sample protein abundance. Transcripts that contribute to non-cognate protein abundance primarily involve those encoding known or predicted interaction partners of the protein of interest, including not only large multi-protein complexes as previously shown, but also small stable complexes in the proteome with only one or few stable interacting partners. Network analysis further shows a complex proteome-wide interdependency of protein abundance on the transcript levels of multiple interacting partners. The predictive model analysis here therefore supports that protein-protein interaction including in small protein complexes exert post-transcriptional influence on proteome compositions more broadly than previously recognized. Moreover, the results suggest mRNA and protein co-expression analysis may have utility for finding gene interactions and predicting expression changes in biological systems.

Sex-specific responses to slow progressive pressure overload in a large animal model of HFpEF.

Approximately 50% of all heart failure (HF) diagnoses can be classified as HF with preserved ejection fraction (HFpEF). HFpEF is more prevalent in females compared with males, but the underlying mechanisms are unknown. We previously showed that pressure overload (PO) in male felines induces a cardiopulmonary phenotype with essential features of human HFpEF. The goal of this study was to determine if slow progressive PO induces distinct cardiopulmonary phenotypes in females and males in the absence of other pathological stressors. Female and male felines underwent aortic constriction (banding) or sham surgery after baseline echocardiography, pulmonary function testing, and blood sampling. These assessments were repeated at 2 and 4 mo postsurgery to document the effects of slow progressive pressure overload. At 4 mo, invasive hemodynamic studies were also performed. Left ventricle (LV) tissue was collected for histology, myofibril mechanics, extracellular matrix (ECM) mass spectrometry, and single-nucleus RNA sequencing (snRNAseq). The induced pressure overload (PO) was not different between sexes. PO also induced comparable changes in LV wall thickness and myocyte cross-sectional area in both sexes. Both sexes had preserved ejection fraction, but males had a slightly more robust phenotype in hemodynamic and pulmonary parameters. There was no difference in LV fibrosis and ECM composition between banded male and female animals. LV snRNAseq revealed changes in gene programs of individual cell types unique to males and females after PO. Based on these results, both sexes develop cardiopulmonary dysfunction but the phenotype is somewhat less advanced in females.NEW & NOTEWORTHY We performed a comprehensive assessment to evaluate the effects of slow progressive pressure overload on cardiopulmonary function in a large animal model of heart failure with preserved ejection fraction (HFpEF) in males and females. Functional and structural assessments were performed at the organ, tissue, cellular, protein, and transcriptional levels. This is the first study to compare snRNAseq and ECM mass spectrometry of HFpEF myocardium from males and females. The results broaden our understanding of the pathophysiological response of both sexes to pressure overload. Both sexes developed a robust cardiopulmonary phenotype, but the phenotype was equal or a bit less robust in females.

Proteogenomics reveals sex-biased aging genes and coordinated splicing in cardiac aging.

The risks of heart diseases are significantly modulated by age and sex, but how these factors influence baseline cardiac gene expression remains incompletely understood. Here, we used RNA sequencing and mass spectrometry to compare gene expression in female and male young adult (4 mo) and early aging (20 mo) mouse hearts, identifying thousands of age- and sex-dependent gene expression signatures. Sexually dimorphic cardiac genes are broadly distributed, functioning in mitochondrial metabolism, translation, and other processes. In parallel, we found over 800 genes with differential aging response between male and female, including genes in cAMP and PKA signaling. Analysis of the sex-adjusted aging cardiac transcriptome revealed a widespread remodeling of exon usage patterns that is largely independent from differential gene expression, concomitant with upstream changes in RNA-binding protein and splice factor transcripts. To evaluate the impact of the splicing events on cardiac proteoform composition, we applied an RNA-guided proteomics computational pipeline to analyze the mass spectrometry data and detected hundreds of putative splice variant proteins that have the potential to rewire the cardiac proteome. Taken together, the results here suggest that cardiac aging is associated with 1) widespread sex-biased aging genes and 2) a rewiring of RNA splicing programs, including sex- and age-dependent changes in exon usages and splice patterns that have the potential to influence cardiac protein structure and function. These changes contribute to the emerging evidence for considerable sexual dimorphism in the cardiac aging process that should be considered in the search for disease mechanisms.NEW & NOTEWORTHY Han et al. used proteogenomics to compare male and female mouse hearts at 4 and 20 mo. Sex-biased cardiac genes function in mitochondrial metabolism, translation, autophagy, and other processes. Hundreds of cardiac genes show sex-by-age interactions, that is, sex-biased aging genes. Cardiac aging is accompanied with a remodeling of exon usage in functionally coordinated genes, concomitant with differential expression of RNA-binding proteins and splice factors. These features represent an underinvestigated aspect of cardiac aging that may be relevant to the search for disease mechanisms.

Defining the Roles of Cardiokines in Human Aging and Age-Associated Diseases.

In recent years an expanding collection of heart-secreted signaling proteins have been discovered that play cellular communication roles in diverse pathophysiological processes. This minireview briefly discusses current evidence for the roles of cardiokines in systemic regulation of aging and age-associated diseases. An analysis of human transcriptome and secretome data suggests the possibility that many other cardiokines remain to be discovered that may function in long-range physiological regulations. We discuss the ongoing challenges and emerging technologies for elucidating the identity and function of cardiokines in endocrine regulations.

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals.

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope-labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [13C6]lysine or [2H2]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.