The localization, mass, and dynamics of microtubules are important in many processes. Cells may actively monitor the state of their microtubules and respond to perturbation, but how this occurs ...outside mitosis is poorly understood. We used gene-expression analysis in quiescent cells to analyze responses to subtle and strong perturbation of microtubules. Genes encoding α-, β, and γ-tubulins (TUBAs, TUBBs, and TUBGs), but not δ- or ε-tubulins (TUBDs or TUBEs), exhibited the strongest differential expression response to microtubule-stabilizing versus destabilizing drugs. Quantitative PCR of exon versus intron sequences confirmed that these changes were caused by regulation of tubulin mRNA stability and not transcription. Using tubulin mRNA stability as a signature to query the Gene Expression Omnibus (GEO) database, we find that tubulin genes respond to toxins known to damage microtubules. Importantly, we find many other experimental perturbations, including multiple signaling and metabolic inputs that trigger tubulin differential expression, suggesting their novel, to our knowledge, role in the regulation of the microtubule cytoskeleton. Mechanistic follow-up confirms that one important physiological signal, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activity, indeed regulates tubulin mRNA stability via changes in microtubule dynamics. We propose that tubulin gene expression is regulated as part of many coordinated biological responses, with wide implications in physiology and toxicology. Furthermore, we present a new way to discover microtubule regulation using transcriptomics.
Clinical trials of novel therapeutics for Alzheimer's Disease (AD) have consumed a large amount of time and resources with largely negative results. Repurposing drugs already approved by the Food and ...Drug Administration (FDA) for another indication is a more rapid and less expensive option. We present DRIAD (Drug Repurposing In AD), a machine learning framework that quantifies potential associations between the pathology of AD severity (the Braak stage) and molecular mechanisms as encoded in lists of gene names. DRIAD is applied to lists of genes arising from perturbations in differentiated human neural cell cultures by 80 FDA-approved and clinically tested drugs, producing a ranked list of possible repurposing candidates. Top-scoring drugs are inspected for common trends among their targets. We propose that the DRIAD method can be used to nominate drugs that, after additional validation and identification of relevant pharmacodynamic biomarker(s), could be readily evaluated in a clinical trial.
Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require ...expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.
Treatment of BRAF‐mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient ...benefit. We use live‐cell imaging, single‐cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug. Drug‐adapted cells up‐regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly. This phenotype is transiently stable, reverting to the drug‐naïve state within 9 days of drug withdrawal. Transcriptional profiling of cell lines and human tumors implicates a c‐Jun/ECM/FAK/Src cascade in de‐differentiation in about one‐third of cell lines studied; drug‐induced changes in c‐Jun and NGFR levels are also observed in xenograft and human tumors. Drugs targeting the c‐Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (Emax) of RAF/MEK kinase inhibitors by promoting cell killing. Thus, analysis of reversible drug resistance at a single‐cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.
Synopsis
Responses of BRAFV600E melanoma cells to vemurafenib were studied at the single‐cell level using live‐cell imaging and by transcriptional and biochemical profiling to uncover a slowly dividing, de‐differentiated cell state associated with drug resistance but inhibitable by drug combinations.
Cell‐to‐cell variability in BRAFV600E melanomas generates drug‐tolerant subpopulations.
The drug‐tolerant, slowly dividing NFGRHigh state is transiently heritable.
Drugs against a proposed c‐Jun/ECM/FAK/Src cascade block acquisition of this phenotype.
The NGFRHigh drug‐tolerant state is also blocked by BET inhibitors in vitro and in vivo.
Drugs that block adaptation by cell subpopulations increase cell killing by RAF/MEK inhibitors.
LINCS‐compliant data and methods are freely available to enhance reproducibility.
Responses of BRAFV600E melanoma cells to vemurafenib were studied at the single‐cell level using live‐cell imaging and by transcriptional and biochemical profiling to uncover a slowly dividing, de‐differentiated cell state associated with drug resistance but inhibitable by drug combinations.
Tyrosine kinase inhibitors (TKIs) are anti-cancer therapeutics often prescribed for long-term treatment. Many of these treatments cause cardiotoxicity with limited cure. We aim to clarify molecular ...mechanisms of TKI-induced cardiotoxicity so as to find potential targets for treating the adverse cardiac complications.
Eight TKIs with different levels of cardiotoxicity reported are selected. Phenotypic and transcriptomic responses of human cardiomyocytes to TKIs at varying doses and times are profiled and analyzed. Stress responses and signaling pathways that modulate cardiotoxicity induced by three TKIs are validated in cardiomyocytes and rat hearts.
Toxicity rank of the eight TKIs determined by measuring their effects on cell viability, contractility, and respiration is largely consistent with that derived from database or literature, indicating that human cardiomyocytes are a good cellular model for studying cardiotoxicity. When transcriptomes are measured for selected TKI treatments with different levels of toxicity in human cardiomyocytes, the data are classified into 7 clusters with mainly single-drug clusters. Drug-specific effects on the transcriptome dominate over dose-, time- or toxicity-dependent effects. Two clusters with three TKIs (afatinib, ponatinib, and sorafenib) have the top enriched pathway as the endoplasmic reticulum stress (ERS). All three TKIs induce ERS in rat primary cardiomyocytes and ponatinib activates the IRE1α-XBP1s axis downstream of ERS in the hearts of rats underwent a 7-day course of drug treatment. To look for potential triggers of ERS, we find that the three TKIs induce transient reactive oxygen species followed by lipid peroxidation. Inhibiting either PERK or IRE1α downstream of ERS blocks TKI-induced cardiac damages, represented by the induction of cardiac fetal and pro-inflammatory genes without causing more cell death.
Our data contain rich information about phenotypic and transcriptional responses of human cardiomyocytes to eight TKIs, uncovering potential molecular mechanisms in modulating cardiotoxicity. ER stress is activated by multiple TKIs and leads to cardiotoxicity through promoting expression of pro-inflammatory factors and cardiac fetal genes. ER stress-induced inflammation is a promising therapeutic target to mitigate ponatinib- and sorafenib-induced cardiotoxicity.
Quantitative diagnostics that are rapid, inexpensive, sensitive, robust, and field-deployable are needed to contain the spread of infectious diseases and inform treatment strategies. While current ...gold-standard techniques are highly sensitive and quantitative, they are slow and require expensive equipment. Conversely, current rapid field-deployable assays available provide essentially binary information about the presence of the target analyte, not a quantitative measure of concentration. Here, we report the development of a molecular diagnostic test quantitative recombinase polymerase amplification (qRPA) that utilizes competitive amplification during a recombinase polymerase amplification (RPA) assay to provide semi-quantitative information on a target nucleic acid. We demonstrate that qRPA can quantify DNA, RNA, and viral titers in HIV and COVID-19 patient samples and that it is more robust to environmental perturbations than traditional RPA. These features make qRPA potentially useful for at-home testing to monitor the progress of viral infections or other diseases.
Deubiquitinating enzymes (DUBs), ~100 of which are found in human cells, are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. They are involved in a wide ...range of cellular activities and are emerging therapeutic targets for cancer and other diseases. Drugs targeting USP1 and USP30 are in clinical development for cancer and kidney disease respectively. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and multiple protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper are browsable online in a newly developed
DUB Portal
and promise to improve understanding of DUBs as a family as well as the activities of incompletely characterized DUBs (e.g. USPL1 and USP32) and those already targeted with investigational cancer therapeutics (e.g. USP14, UCHL5, and USP7).
Background
The electronic health record (EHR) presents new opportunities for the timely identification of patients at high risk of critical illness and the implementation of preventive strategies. ...This study aims to externally validate an EHR‐based Elders Risk Assessment (ERA) score to identify older patients at high risk of future critical illness during a primary care visit.
Methods
This historical cohort study included patients aged ≥65 years who had primary care visits at Mayo Clinic Rochester, MN, between July 2019 and December 2021. The ERA score at the time of the primary care visit was used to predict critical illness, defined as death or ICU admission within 1 year of the visit.
Results
A total of 12,885 patients were included in the analysis. The median age at the time of the primary care visit was 75 years, with 44.6% being male. 93.7% of participants were White, and 64.2% were married. The median (25th, 75th percentile) ERA score was 4 (0, 9). 11.3% of study participants were admitted to the ICU or died within 1 year of the visit. The ERA score predicted critical illness within 1 year of a primary care visit with an area under the receiver operating characteristic curve of 0.84 (95% CI 0.83–0.85), which indicates good discrimination. An ERA score of 9 was identified as optimal for implementing and testing potential preventive strategies, with the odds ratio of having the primary outcome in patients with ERA score ≥9 being 11.33 (95%CI 9.98–12.87).
Conclusions
This simple EHR‐based risk assessment model can predict critical illness within 1 year of primary care visits in older patients. The findings of this study can serve as a basis for testing and implementation of preventive strategies to promote the well‐being of older adults at risk of critical illness and its consequences.
See related article by Haimovich and Deardorff.
•We developed an automation and accession compatible clinical swab.•The Rhinostic swab preforms as well as standard CLIA use approved swabs.•Samples are stable on Rhinostic swabs with both wet and ...dry transport.•Clinically, the Rhinostic swabs were 100 % concordant with other tested swabs.
The COVID-19 pandemic has resulted in an unparalleled need for viral testing capacity across the world and is a critical requirement for successful re-opening of economies. The logistical barriers to near-universal testing are considerable. We have designed an injection molded polypropylene anterior nares swab, the Rhinostic, with a screw cap integrated into the swab handle that is compatible with fully automated sample accessioning and processing. The ability to collect and release both human and viral material is comparable to that of several commonly used swabs on the market. SARS-CoV-2 is stable on dry Rhinostic swabs for at least 3 days, even at 42 °C, and elution can be achieved with small volumes. To test the performance of the Rhinostic in patients, 119 samples were collected with Rhinostic and the positive and negative determinations were 100 % concordant with samples collected using Clinical Laboratory Improvement Amendments (CLIA) use approved nasal swabs at a clinical lab. The Rhinostic swab and barcoded tube set can be produced, sterilized, and packaged cost effectively and is designed to be adopted by clinical laboratories using automation to increase throughput and dramatically reduce the cost of a standard SARS-CoV-2 detection pipeline.
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