Amelioration of Anxiety Associated with Opioid Withdrawal by Activation of Spinal Mechanoreceptors Via Novel Heterodyned Whole Body Vibration.

Objectives: Opioid use disorder (OUD)-associated overdose deaths have reached epidemic proportions worldwide. An important driving force for relapse is anxiety associated with opioid withdrawal. We hypothesized that our new technology, termed heterodyned whole-body vibration (HWBV) would ameliorate anxiety associated with OUD. Methods: Using a randomized, placebo (sham)-controlled, double-blind study design in an NIH-sponsored Phase 1 trial, we evaluated 60 male and 26 female participants diagnosed with OUD and undergoing treatment at pain and rehabilitation clinics. We utilized the Hamilton Anxiety Scale (HAM-A) and a daily visual analog scale anxiety rating (1-10) to evaluate anxiety. Subjects were treated for 10 min 5X/week for 4 weeks with either sham vibration (no interferential beat or harmonics) or HWBV (beats and harmonics). The participants also completed a neuropsychological test battery at intake and discharge. Results: In OUD subjects with moderate anxiety, there was a significant improvement in daily anxiety scores in the HWBV group compared to the sham treatment group (p=3.41 × 10-7). HAM-A scores in OUD participants at intake showed moderate levels of anxiety in OUD participants (HWBV group: 15.9 ± 1.6; Sham group: 17.8 ± 1.6) and progressively improved in both groups at discharge, but improvement was greater in the HWBV group (p=1.37 × 10-3). Furthermore, three indices of neuropsychological testing (mental rotations, spatial planning, and response inhibition) were significantly improved by HWBV treatment. Conclusions: These findings support HWBV as a novel, non-invasive, non-pharmacological treatment for anxiety associated with OUD.

Chronic Migraine May Be Associated With Postprandial Hypoglycemia in Adult Men: A Case Series.

Migraine is a common neurological disorder that significantly impacts patients around the world. In the United States, one in six individuals suffers from a migraine disorder. Despite its high prevalence, the etiology of migraine is not well understood. Multiple factors likely contribute to the development of both acute and chronic migraine, making the consensus as to the cause and treatment difficult. Presented here are three case studies involving adult males suffering from chronic migraine. Each subject provided a medical history and underwent physical, psychological, and neurological examinations. In addition, relevant bloodwork and cervical spine X-rays were obtained. Physical examination, laboratory studies, imaging, and psychological metrics were unremarkable with the notable exception of the three-hour oral glucose tolerance tests. All three patients displayed hypoglycemia at three hours. Furthermore, their symptoms markedly improved with the initiation of a ketogenic diet. These data are suggestive of a potential link between postprandial hypoglycemia and chronic migraine. Despite the small sample size, we feel that this report presents possible evidence for a connection between postprandial hypoglycemia and chronic migraine. Furthermore, properly controlled studies of larger sample sizes are required, but we suggest that clinicians consider screening patients for this easily overlooked metabolic disturbance, especially in the absence of other options.

Toward robust clinical genome interpretation: Developing a consistent terminology to characterize Mendelian disease-gene relationships-allelic requirement, inheritance modes, and disease mechanisms.

PURPOSE: The terminology used for gene-disease curation and variant annotation to describe inheritance, allelic requirement, and both sequence and functional consequences of a variant is currently not standardized. There is considerable discrepancy in the literature and across clinical variant reporting in the derivation and application of terms. Here, we standardize the terminology for the characterization of disease-gene relationships to facilitate harmonized global curation and to support variant classification within the ACMG/AMP framework. METHODS: Terminology for inheritance, allelic requirement, and both structural and functional consequences of a variant used by Gene Curation Coalition members and partner organizations was collated and reviewed. Harmonized terminology with definitions and use examples was created, reviewed, and validated. RESULTS: We present a standardized terminology to describe gene-disease relationships, and to support variant annotation. We demonstrate application of the terminology for classification of variation in the ACMG SF 2.0 genes recommended for reporting of secondary findings. Consensus terms were agreed and formalized in both Sequence Ontology (SO) and Human Phenotype Ontology (HPO) ontologies. Gene Curation Coalition member groups intend to use or map to these terms in their respective resources. CONCLUSION: The terminology standardization presented here will improve harmonization, facilitate the pooling of curation datasets across international curation efforts and, in turn, improve consistency in variant classification and genetic test interpretation.

An Overview of Machine Learning Applications in Sports Injury Prediction.

Use injuries, i.e., injuries caused by repetitive strain on the body, represent a serious problem in athletics that has traditionally relied on historic datasets and human experience for prevention. Existing methodologies have been frustratingly slow at developing higher precision prevention practices. Technological advancements have permitted the emergence of artificial intelligence and machine learning (ML) as promising toolsets to enhance both injury mitigation and rehabilitation protocols. This article provides a comprehensive overview of recent advances in ML techniques as they have been applied to sports injury prediction and prevention. A comprehensive literature review was conducted searching PubMed/Medline, Institute of Electrical and Electronics Engineers (IEEE)/Institute of Engineering and Technology (IET), and ScienceDirect. Ovid Discovery and Google Scholar were used to provide additional aggregate results and a grey literature search. A focus was placed on papers published from 2017 to 2022. Algorithms of interest were limited to K-Nearest Neighbor (KNN), K-means, decision tree, random forest, gradient boosting and AdaBoost, and neural networks. A total of 42 original research papers were included, and their results were summarized. We conclude that given the current lack of open source, uniform data sets, as well as a reliance on dated regression models, no strong conclusions about the real-world efficacy of ML as it applies to sports injury prediction can be made. However, it is suggested that addressing these two issues will allow powerful, novel ML architectures to be deployed, thus rapidly advancing the state of this field, and providing validated clinical tools.

LSTM-based recurrent neural network provides effective short term flu forecasting.

BACKGROUND: Influenza virus is responsible for a yearly epidemic in much of the world. To better predict short-term, seasonal variations in flu infection rates and possible mechanisms of yearly infection variation, we trained a Long Short-Term Memory (LSTM)-based deep neural network on historical Influenza-Like-Illness (ILI), climate, and population data. METHODS: Data were collected from the Centers for Disease Control and Prevention (CDC), the National Center for Environmental Information (NCEI), and the United States Census Bureau. The model was initially built in Python using the Keras API and tuned manually. We explored the roles of temperature, precipitation, local wind speed, population size, vaccination rate, and vaccination efficacy. The model was validated using K-fold cross validation as well as forward chaining cross validation and compared to several standard algorithms. Finally, simulation data was generated in R and used for further exploration of the model. RESULTS: We found that temperature is the strongest predictor of ILI rates, but also found that precipitation increased the predictive power of the network. Additionally, the proposed model achieved a +1 week prediction mean absolute error (MAE) of 0.1973. This is less than half of the MAE achieved by the next best performing algorithm. Additionally, the model accurately predicted simulation data. To test the role of temperature in the network, we phase-shifted temperature in time and found a predictable reduction in prediction accuracy. CONCLUSIONS: The results of this study suggest that short term flu forecasting may be effectively accomplished using architectures traditionally reserved for time series analysis. The proposed LSTM-based model was able to outperform comparison models at the +1 week time point. Additionally, this model provided insight into the week-to-week effects of climatic and biotic factors and revealed potential patterns in data series. Specifically, we found that temperature is the strongest predictor of seasonal flu infection rates. This information may prove to be especially important for flu forecasting given the uncertain long-term impact of the SARS-CoV-2 pandemic on seasonal influenza.

Whole-Body Vibration Prevents Neuronal, Neurochemical, and Behavioral Effects of Morphine Withdrawal in a Rat Model.

Peripheral mechanoreceptor-based treatments such as acupuncture and chiropractic manipulation have shown success in modulating the mesolimbic dopamine (DA) system originating in the ventral tegmental area (VTA) of the midbrain and projecting to the nucleus accumbens (NAc) of the striatum. We have previously shown that mechanoreceptor activation via whole-body vibration (WBV) ameliorates neuronal and behavioral effects of chronic ethanol exposure. In this study, we employ a similar paradigm to assess the efficacy of WBV as a preventative measure of neuronal and behavioral effects of morphine withdrawal in a Wistar rat model. We demonstrate that concurrent administration of WBV at 80 Hz with morphine over a 5-day period significantly reduced adaptations in VTA GABA neuronal activity and NAc DA release and modulated expression of δ-opioid receptors (DORs) on NAc cholinergic interneurons (CINs) during withdrawal. We also observed a reduction in behavior typically associated with opioid withdrawal. WBV represents a promising adjunct to current intervention for opioid use disorder (OUD) and should be examined translationally in humans.

Towards robust clinical genome interpretation: developing a consistent terminology to characterize disease-gene relationships - allelic requirement, inheritance modes and disease mechanisms.

PURPOSE: The terminology used for gene-disease curation and variant annotation to describe inheritance, allelic requirement, and both sequence and functional consequences of a variant is currently not standardized. There is considerable discrepancy in the literature and across clinical variant reporting in the derivation and application of terms. Here we standardize the terminology for the characterization of disease-gene relationships to facilitate harmonized global curation, and to support variant classification within the ACMG/AMP framework. METHODS: Terminology for inheritance, allelic requirement, and both structural and functional consequences of a variant used by Gene Curation Coalition (GenCC) members and partner organizations was collated and reviewed. Harmonized terminology with definitions and use examples was created, reviewed, and validated. RESULTS: We present a standardized terminology to describe gene-disease relationships, and to support variant annotation. We demonstrate application of the terminology for classification of variation in the ACMG SF 2.0 genes recommended for reporting of secondary findings. Consensus terms were agreed and formalized in both sequence ontology (SO) and human phenotype ontology (HPO) ontologies. GenCC member groups intend to use or map to these terms in their respective resources. CONCLUSION: The terminology standardization presented here will improve harmonization, facilitate the pooling of curation datasets across international curation efforts and, in turn, improve consistency in variant classification and genetic test interpretation.

Long-Term Health Outcomes of Infantile Spasms Following Prednisolone vs. Adrenocorticotropic Hormone Treatment Characterized Using Phenome-Wide Association Study.

Objective: To determine differences in long-term health and neurological outcomes following infantile spasms (IS) in patients treated with adrenocorticotropic hormone (ACTH) vs. prednisolone/prednisone (PRED). Methods: A retrospective, case-control study of patients with an International Classification of Diseases, Ninth Revision, Clinical Modifications (ICD-9) diagnosis of IS, identified over a 10-year period from a national administrative database, was conducted. IS patients treated with ACTH or PRED were determined and cohorts established by propensity score matching. Outcomes, defined by hospital discharge ICD codes, were followed for each patient for 5 years. Related ICD codes were analyzed jointly as phenotype codes (phecodes). Analysis of phecodes between cohorts was performed including phenome-wide association analysis. Results: A total of 5,955 IS patients were identified, and analyses were subsequently performed for 493 propensity score matched patients, each in the ACTH and PRED cohorts. Following Bonferroni correction, no phecode was more common in either cohort (p < 0.001). However, assuming an a priori difference, one phecode, abnormal findings on study of brain or nervous system (a category of abnormal neurodiagnostic tests), was more common in the PRED cohort (p <0.05), and was robust to sensitivity analysis. Variability in outcomes was noted between hospitals. Significance: We found that long-term outcomes for IS patients following ACTH or PRED treatment were very similar, including for both neurological and non-neurological outcomes. In the PRED-treated cohort there was a higher incidence of abnormal neurodiagnostic tests, assuming an a priori statistical model. Future studies can evaluate whether variability in outcomes between hospitals may be affected by post-treatment differences in care models.

The gene regulation knowledge commons: the action area of GREEKC.

As computational modeling becomes more essential to analyze and understand biological regulatory mechanisms, governance of the many databases and knowledge bases that support this domain is crucial to guarantee reliability and interoperability of resources. To address this, the COST Action Gene Regulation Ensemble Effort for the Knowledge Commons (GREEKC, CA15205, www.greekc.org) organized nine workshops in a four-year period, starting September 2016. The workshops brought together a wide range of experts from all over the world working on various steps in the knowledge management process that focuses on understanding gene regulatory mechanisms. The discussions between ontologists, curators, text miners, biologists, bioinformaticians, philosophers and computational scientists spawned a host of activities aimed to standardize and update existing knowledge management workflows and involve end-users in the process of designing the Gene Regulation Knowledge Commons (GRKC). Here the GREEKC consortium describes its main achievements in improving this GRKC.

Sequence Ontology terminology for gene regulation.

The Sequence Ontology (SO) is a structured, controlled vocabulary that provides terms and definitions for genomic annotation. The Gene Regulation Ensemble Effort for the Knowledge Commons (GREEKC) initiative has gathered input from many groups of researchers, including the SO, the Gene Ontology (GO), and gene regulation experts, with the goal of curating information about how gene expression is regulated at the molecular level. Here we discuss recent updates to the SO reflecting current knowledge. We have developed more accurate human-readable terms (also known as classes), including new definitions, and relationships related to the expression of genes. New findings continue to give us insight into the biology of gene regulation, including the order of events, and participants in those events. These updates to the SO support logical reasoning with the current understanding of gene expression regulation at the molecular level.

Vitamin C sensitizes triple negative breast cancer to PI3K inhibition therapy.

Rationale: The clinical use of PI3K inhibitors, such as buparlisib, has been plagued with toxicity at effective doses. The aim of this study is to determine if vitamin C, a potent epigenetic regulator, can improve the therapeutic outcome and reduce the dose of buparlisib in treating PIK3CA-mutated triple negative breast cancer (TNBC). Methods: The response of TNBC cells to buparlisib was assessed by EC50 measurements, apoptosis assay, clonogenic assay, and xenograft assay in mice. Molecular approaches including Western blot, immunofluorescence, RNA sequencing, and gene silencing were utilized as experimental tools. Results: Treatment with buparlisib at lower doses, along with vitamin C, induced apoptosis and inhibited the growth of TNBC cells in vitro. Vitamin C via oral delivery rendered a sub-therapeutic dose of buparlisib able to inhibit TNBC xenograft growth and to markedly block metastasis in mice. We discovered that buparlisib and vitamin C coordinately reduced histone H3K4 methylation by enhancing the nuclear translocation of demethylase, KDM5, and by serving as a cofactor to promote KDM5-mediated H3K4 demethylation. The expression of genes in the PI3K pathway, such as AKT2 and mTOR, was suppressed by vitamin C in a KDM5-dependent manner. Vitamin C and buparlisib cooperatively blocked AKT phosphorylation. Inhibition of KDM5 largely abolished the effect of vitamin C on the response of TNBC cells to buparlisib. Additionally, vitamin C and buparlisib co-treatment changed the expression of genes, including PCNA and FILIP1L, which are critical to cancer growth and metastasis. Conclusion: Vitamin C can be used to reduce the dosage of buparlisib needed to produce a therapeutic effect, which could potentially ease the dose-dependent side effects in patients.

Methods and feasibility study for exome sequencing as a universal second-tier test in newborn screening.

PURPOSE: Newborn screening disorders increasingly require genetic variant analysis as part of second-tier or confirmatory testing. Sanger sequencing and gene-specific next-generation sequencing (NGS)-based tests, the current methods of choice, are costly and lack scalability when expanding to new conditions. We describe a scalable, exome sequencing-based NGS pipeline with a priori analysis restriction that can be universally applied to any NBS disorder. METHODS: De-identified abnormal newborn screening specimens representing severe combined immune deficiency (SCID), cystic fibrosis (CF), VLCAD deficiency, metachromatic leukodystrophy (MLD), and in silico sequence read data sets were used to validate the pipeline. To support interpretation and clinical decision-making within the bioinformatics pipeline, variants from multiple databases were curated and validated. RESULTS: CFTR variant panel analysis correctly identified all variants. Concordance compared with diagnostic testing results for targeted gene analysis was between 78.6% and 100%. Validation of the bioinformatics pipeline with in silico data sets revealed a 100% detection rate. Varying degrees of overlap were observed between ClinVar and other databases ranging from 3% to 65%. Data normalization revealed that 11% of variants across the databases required manual curation. CONCLUSION: This pipeline allows for restriction of analysis to variants within a single gene or multiple genes, and can be readily expanded to full exome analysis if clinically indicated and parental consent is granted.

Vitamin C regulates Schwann cell myelination by promoting DNA demethylation of pro-myelinating genes.

Ascorbic acid (vitamin C) is critical for Schwann cells to myelinate peripheral nerve axons during development and remyelination after injury. However, its exact mechanism remains elusive. Vitamin C is a dietary nutrient that was recently discovered to promote active DNA demethylation. Schwann cell myelination is characterized by global DNA demethylation in vivo and may therefore be regulated by vitamin C. We found that vitamin C induces a massive transcriptomic shift (n = 3,848 genes) in primary cultured Schwann cells while simultaneously producing a global increase in genomic 5-hydroxymethylcytosine (5hmC), a DNA demethylation intermediate which regulates transcription. Vitamin C up-regulates 10 pro-myelinating genes which exhibit elevated 5hmC content in both the promoter and gene body regions of these loci following treatment. Using a mouse model of human vitamin C metabolism, we found that maternal dietary vitamin C deficiency causes peripheral nerve hypomyelination throughout early development in resulting offspring. Additionally, dietary vitamin C intake regulates the expression of myelin-related proteins such as periaxin (PRX) and myelin basic protein (MBP) during development and remyelination after injury in mice. Taken together, these results suggest that vitamin C cooperatively promotes myelination through 1) increased DNA demethylation and transcription of pro-myelinating genes, and 2) its known role in stabilizing collagen helices to form the basal lamina that is necessary for myelination.

In vitro derived female hPGCLCs are unable to complete meiosis in embryoid bodies.

The fundamental question about the functionality of in vitro derived human primordial germ cell-like cells remains unanswered, despite ongoing research in this area. Attempts have been made to imitate the differentiation of human primordial germ cells (hPGCs) and meiocytes in vitro from human pluripotent stem cells (hPSCs). A defined system for developing human haploid cells in vitro is the challenge that scientists face to advance the knowledge of human germ cell development. To develop human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) that are capable of giving rise to haploid cells, we applied a sequential induction protocol via the early mesodermal push of female human embryonic and induced pluripotent stem cells. BMP4-induced early mesoderm-like cells showed significant alterations in their expression profiles toward early (PRDM1 and NANOS3) and late (VASA and DAZL) germ cell markers. Furthermore, using retinoic acid (RA), we induced hPGCLCs in embryoid bodies and identified positive staining for the meiotic initiation marker STRA8. Efforts to find the cells exhibiting progression to meiosis were unsuccessful. The validation by the expression of SCP3 did not correspond to the natural pattern. Regarding the 20-day meiotic induction, the derived hPGCLCs containing two X-chromosomes were unable to complete the meiotic division. We observed the expression of the oocyte marker PIWIL1 and PIWIL4. RNAseq analysis and cluster dendrogram showed a similar clustering of hPGCLC groups and meiotic like cell groups as compared to previously published data. This reproducible in vitro model for deriving hPGCLCs provides opportunities for studying the molecular mechanisms involved in the specification of hPGCs. Moreover, our results will support a further elucidation of gametogenesis and meiosis of female hPGCs.

Mitochondrial Transfer of the Mutant Human ND6T14484C Gene Causes Visual Loss and Optic Neuropathy.

Purpose: To evaluate the long-term effects of mitochondrial gene transfer of mutant human NADH ubiquinone oxidoreductase subunit VI (hND6T14484C) in the mouse eye. Methods: Adult mice were injected intravitreally with mitochondrial-targeted adeno-associated virus carrying either hND6T14484C or mitochondrial encoded mCherry. The delivery and expression of the interest gene were detected by polymerase chain reaction (PCR), quantitative PCR (qPCR), and immunostaining. The pathologic effects of the mutant gene in live mice were assessed with RNA-seq, serial spectral domain optical coherence tomography (SD-OCT), and pattern electroretinogram (PERG). Results: Delivered hND6 was found 30-fold greater than endogenous mouse ND6 in microdissected retinal ganglion cells of hND6-injected mice. Compared to controls injected with mCherry, PERG amplitude of hND6 mice dropped significantly at 3 (P = 0.0023), 6 (P = 0.0058), and 15 (P = 0.031) months after injection. SD-OCT revealed swelling of the optic nerve head followed by the progressive retinal and optic nerve atrophy in hND6 mice. Furthermore, RNA-seq data showed a change in 381 transcripts' expression in these mice compared to mCherry mice. Postmortem analysis showed hND6 mice had marked atrophy of the entire optic nerve, from the globe to the optic chiasm, and a significant loss of retinal ganglion cells compared to age-matched control mice (P = 1.7E-9). Conclusions: Delivered hND6T14484C induces visual loss and optic neuropathy in mice, the hallmarks of human Leber's hereditary optic neuropathy (LHON). Translational Relevance: Results from this study will help establish a novel strategy not only to generate an LHON animal model but also to provide a potential to treat this or any other mitochondrial diseases.

Magnetic separation of peripheral nerve-resident cells underscores key molecular features of human Schwann cells and fibroblasts: an immunochemical and transcriptomics approach.

Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translational research but their use can be limited due to the risk of fibroblast overgrowth. Fibroblasts are an ill-defined population consisting of highly proliferative cells that, contrary to human SCs, do not undergo senescence in culture. We initiated this study by performing an exhaustive immunological and functional characterization of adult nerve-derived human SCs and fibroblasts to reveal their properties and optimize a protocol of magnetic-activated cell sorting (MACS) to separate them effectively both as viable and biologically competent cells. We next used immunofluorescence microscopy imaging, flow cytometry analysis and next generation RNA sequencing (RNA-seq) to unambiguously characterize the post-MACS cell products. High resolution transcriptome profiling revealed the identity of key lineage-specific transcripts and the clearly distinct neural crest and mesenchymal origin of human SCs and fibroblasts, respectively. Our analysis underscored a progenitor- or stem cell-like molecular phenotype in SCs and fibroblasts and the heterogeneity of the fibroblast populations. In addition, pathway analysis of RNA-seq data highlighted putative bidirectional networks of fibroblast-to-SC signaling that predict a complementary, yet seemingly independent contribution of SCs and fibroblasts to nerve regeneration. In sum, combining MACS with immunochemical and transcriptomics approaches provides an ideal workflow to exhaustively assess the identity, the stage of differentiation and functional features of highly purified cells from human peripheral nerve tissues.

Single-cell resolution analysis of the human pancreatic ductal progenitor cell niche.

We have described multipotent progenitor-like cells within the major pancreatic ducts (MPDs) of the human pancreas. They express PDX1, its surrogate surface marker P2RY1, and the bone morphogenetic protein (BMP) receptor 1A (BMPR1A)/activin-like kinase 3 (ALK3), but not carbonic anhydrase II (CAII). Here we report the single-cell RNA sequencing (scRNA-seq) of ALK3bright+-sorted ductal cells, a fraction that harbors BMP-responsive progenitor-like cells. Our analysis unveiled the existence of multiple subpopulations along two major axes, one that encompasses a gradient of ductal cell differentiation stages, and another featuring cells with transitional phenotypes toward acinar tissue. A third potential ducto-endocrine axis is revealed upon integration of the ALK3bright+ dataset with a single-cell whole-pancreas transcriptome. When transplanted into immunodeficient mice, P2RY1+/ALK3bright+ populations (enriched in PDX1+/ALK3+/CAII- cells) differentiate into all pancreatic lineages, including functional ß-cells. This process is accelerated when hosts are treated systemically with an ALK3 agonist. We found PDX1+/ALK3+/CAII- progenitor-like cells in the MPDs of types 1 and 2 diabetes donors, regardless of the duration of the disease. Our findings open the door to the pharmacological activation of progenitor cells in situ.

Genetic compensation in a stable slc25a46 mutant zebrafish: A case for using F0 CRISPR mutagenesis to study phenotypes caused by inherited disease.

A phenomenon of genetic compensation is commonly observed when an organism with a disease-bearing mutation shows incomplete penetrance of the disease phenotype. Such incomplete phenotypic penetrance, or genetic compensation, is more commonly found in stable knockout models, rather than transient knockdown models. As such, these incidents present a challenge for the disease modeling field, although a deeper understanding of genetic compensation may also hold the key for novel therapeutic interventions. In our study we created a knockout model of slc25a46 gene, which is a recently discovered important player in mitochondrial dynamics, and deleterious mutations in which are known to cause peripheral neuropathy, optic atrophy and cerebellar ataxia. We report a case of genetic compensation in a stable slc25a46 homozygous zebrafish mutant (hereafter referred as "mutant"), in contrast to a penetrant disease phenotype in the first generation (F0) slc25a46 mosaic mutant (hereafter referred as "crispant"), generated with CRISPR/Cas-9 technology. We show that the crispant phenotype is specific and rescuable. By performing mRNA sequencing, we define significant changes in slc25a46 mutant's gene expression profile, which are largely absent in crispants. We find that among the most significantly altered mRNAs, anxa6 gene stands out as a functionally relevant player in mitochondrial dynamics. We also find that our genetic compensation case does not arise from mechanisms driven by mutant mRNA decay. Our study contributes to the growing evidence of the genetic compensation phenomenon and presents novel insights about Slc25a46 function. Furthermore, our study provides the evidence for the efficiency of F0 CRISPR screens for disease candidate genes, which may be used to advance the field of functional genetics.

Oscillatory cAMP signaling rapidly alters H3K4 methylation.

Epigenetic variation reflects the impact of a dynamic environment on chromatin. However, it remains elusive how environmental factors influence epigenetic events. Here, we show that G protein-coupled receptors (GPCRs) alter H3K4 methylation via oscillatory intracellular cAMP. Activation of Gs-coupled receptors caused a rapid decrease of H3K4me3 by elevating cAMP, whereas stimulation of Gi-coupled receptors increased H3K4me3 by diminishing cAMP. H3K4me3 gradually recovered towards baseline levels after the removal of GPCR ligands, indicating that H3K4me3 oscillates in tandem with GPCR activation. cAMP increased intracellular labile Fe(II), the cofactor for histone demethylases, through a non-canonical cAMP target-Rap guanine nucleotide exchange factor-2 (RapGEF2), which subsequently enhanced endosome acidification and Fe(II) release from the endosome via vacuolar H+-ATPase assembly. Removing Fe(III) from the media blocked intracellular Fe(II) elevation after stimulation of Gs-coupled receptors. Iron chelators and inhibition of KDM5 demethylases abolished cAMP-mediated H3K4me3 demethylation. Taken together, these results suggest a novel function of cAMP signaling in modulating histone demethylation through labile Fe(II).

Vitamin C supplementation expands the therapeutic window of BETi for triple negative breast cancer.

BACKGROUND: Bromodomain and extra-terminal inhibitors (BETi) have shown efficacy for the treatment of aggressive triple negative breast cancer (TNBC). However, BETi are plagued by a narrow therapeutic window as manifested by severe toxicities at effective doses. Therefore, it is a limitation to their clinical implementation in patient care. METHODS: The impact of vitamin C on the efficacy of small compounds including BETi was assessed by high-throughput screening. Co-treatment of TNBC by BETi especially JQ1 and vitamin C was evaluated in vitro and in vivo. FINDINGS: High-throughput screening revealed that vitamin C improves the efficacy of a number of structurally-unrelated BETi including JQ1, I-BET762, I-BET151, and CPI-203 in treating TNBC cells. The synergy between BETi and vitamin C is due to suppressed histone acetylation (H3ac and H4ac), which is in turn caused by upregulated histone deacetylase 1 (HDAC1) expression upon vitamin C addition. Treatment with JQ1 at lower doses together with vitamin C induces apoptosis and inhibits the clonogenic ability of cultured TNBC cells. Oral vitamin C supplementation renders a sub-therapeutic dose of JQ1 able to inhibit human TNBC xenograft growth and metastasis in mice. INTERPRETATION: Vitamin C expands the therapeutic window of BETi by sensitizing TNBC to BETi. Using vitamin C as a co-treatment, lower doses of BETi could be used to achieve an increased therapeutic index in patients, which will translate to a reduced side effect profile. FUND: University of Miami Sylvester Comprehensive Cancer Center, Bankhead Coley Cancer Research program (7BC10), Flight Attendant Medical Research Institute, and NIH R21CA191668 (to GW) and 1R56AG061911 (to CW and CHV).