Cysteine Rich Intestinal Protein 2 is a copper-responsive regulator of skeletal muscle differentiation.

Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.

Multi-omics study reveals different pathogenesis of the generation of skin lesions in SLE and IDLE patients.

Lupus erythematosus (LE) is a heterogeneous, antibody-mediated autoimmune disease. Isolate discoid LE (IDLE) and systematic LE (SLE) are traditionally regarded as the two ends of the spectrum, ranging from skin-limited damage to life-threatening multi-organ involvement. Both belong to LE, but IDLE and SLE differ in appearance of skin lesions, autoantibody panels, pathological changes, treatments, and immunopathogenesis. Is discoid lupus truly a form of LE or is it a completely separate entity? This question has not been fully elucidated. We compared the clinical data of IDLE and SLE from our center, applied multi-omics technology, such as immune repertoire sequencing, high-resolution HLA alleles sequencing and multi-spectrum pathological system to explore cellular and molecular phenotypes in skin and peripheral blood from LE patients. Based on the data from 136 LE patients from 8 hospitals in China, we observed higher damage scores and fewer LE specific autoantibodies in IDLE than SLE patients, more uCDR3 sharing between PBMCs and skin lesion from SLE than IDLE patients, elevated diversity of V-J recombination in IDLE skin lesion and SLE PBMCs, increased SHM frequency and class switch ratio in IDLE skin lesion, decreased SHM frequency but increased class switch ratio in SLE PBMCs, HLA-DRB1*03:01:01:01, HLA-B*58:01:01:01, HLA-C*03:02:02:01, and HLA-DQB1*02:01:01:01 positively associated with SLE patients, and expanded Tfh-like cells with ectopic germinal center structures in IDLE skin lesions. These findings suggest a significant difference in the immunopathogenesis of skin lesions between SLE and IDLE patients. SLE is a B cell-predominate systemic immune disorder, while IDLE appears limited to the skin. Our findings provide novel insights into the pathogenesis of IDLE and other types of LE, which may direct more accurate diagnosis and novel therapeutic strategies.

Small-Molecule Fluorescent Probes for Binding- and Activity-Based Sensing of Redox-Active Biological Metals.

Although transition metals constitute less than 0.1% of the total mass within a human body, they have a substantial impact on fundamental biological processes across all kingdoms of life. Indeed, these nutrients play crucial roles in the physiological functions of enzymes, with the redox properties of many of these metals being essential to their activity. At the same time, imbalances in transition metal pools can be detrimental to health. Modern analytical techniques are helping to illuminate the workings of metal homeostasis at a molecular and atomic level, their spatial localization in real time, and the implications of metal dysregulation in disease pathogenesis. Fluorescence microscopy has proven to be one of the most promising non-invasive methods for studying metal pools in biological samples. The accuracy and sensitivity of bioimaging experiments are predominantly determined by the fluorescent metal-responsive sensor, highlighting the importance of rational probe design for such measurements. This review covers activity- and binding-based fluorescent metal sensors that have been applied to cellular studies. We focus on the essential redox-active metals: iron, copper, manganese, cobalt, chromium, and nickel. We aim to encourage further targeted efforts in developing innovative approaches to understanding the biological chemistry of redox-active metals.

Efficient storage and regression computation for population-scale genome sequencing studies.

In the era of big data in human genetics, large-scale biobanks aggregating genetic data from diverse populations have emerged as important for advancing our understanding of human health and disease. However, the computational and storage demands of whole genome sequencing (WGS) studies pose significant challenges, especially for researchers from underfunded institutions or developing countries, creating a disparity in research capabilities. We introduce new approaches that significantly enhance computational efficiency and reduce data storage requirements for WGS studies. By developing algorithms for compressed storage of genetic data, focusing particularly on optimizing the representation of rare variants, and designing regression methods tailored for the scale and complexity of WGS data, we significantly lower computational and storage costs. We integrate our approach into PLINK 2.0. The implementation demonstrates considerable reductions in storage space and computational time without compromising analytical accuracy, as evidenced by the application to the AllofUs project data. We improve runtime of an exome-wide association analysis of 19.4 million variants and a single phenotype from 695.35 minutes (approximately 11.5 hours) on a single machine to 1.57 minutes using 30Gb of memory and 50 threads (8.67 minutes using 4 threads). Similarly, we generalize to multi-phenotype analysis. We anticipate that our approach will enable researchers across the globe to unlock the potential of population biobanks, accelerating the pace of discoveries that can improve our understanding of human health and disease.

One Carbon to Rule Them All: Formaldehyde is a One-Carbon Signal Connecting One-Carbon Metabolism and Epigenetic Methylation.

Formaldehyde is commonly thought of as an environmental toxin or laboratory fixation reagent, but there is a growing appreciation for its broader physiological contributions as a naturally generated one-carbon metabolite across all kingdoms of life. In this In Focus article, we summarize emerging advances in the field that show how formaldehyde plays diverse roles as a one-carbon signal in DNA damage, one-carbon metabolism, and epigenetic regulation.

Oxidative cyclization reagents reveal tryptophan cation-π interactions.

Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.

A Transfer Hydrogenation Approach to Activity-Based Sensing of Formate in Living Cells.

Formate is a major reactive carbon species in one-carbon metabolism, where it serves as an endogenous precursor for amino acid and nucleic acid biosynthesis and a cellular source of NAD(P)H. On the other hand, aberrant elevations in cellular formate are connected to progression of serious diseases, including cancer and Alzheimer's disease. Traditional methods for formate detection in biological environments often rely on sample destruction or extensive processing, resulting in a loss of spatiotemporal information. To help address these limitations, here we present the design, synthesis, and biological evaluation of a first-generation activity-based sensing system for live-cell formate imaging that relies on iridium-mediated transfer hydrogenation chemistry. Formate facilitates an aldehyde-to-alcohol conversion on various fluorophore scaffolds to enable fluorescence detection of this one-carbon unit, including through a two-color ratiometric response with internal calibration. The resulting two-component probe system can detect changes in formate levels in living cells with a high selectivity over potentially competing biological analytes. Moreover, this activity-based sensing system can visualize changes in endogenous formate fluxes through alterations of one-carbon pathways in cell-based models of human colon cancer, presaging the potential utility of this chemical approach to probe the continuum between one-carbon metabolism and signaling in cancer and other diseases.

Trends in Firearm Injury Prevention Research Funding, Clinical Trials, and Publications in the US, 1985-2022.

This cross-sectional study examines federal funding, registered clinical trials, and publications to quantify trends in firearm injury prevention research in the US from 1985 to 2022.

ATPase copper transporting beta (ATP7B) is a novel target for improving the therapeutic efficacy of docetaxel by disulfiram/copper in human prostate cancer.

Docetaxel has been the standard first-line chemotherapy for lethal metastatic prostate cancer (mPCa) since 2004, but resistance to docetaxel treatment is common. The molecular mechanisms of docetaxel resistance remain largely unknown and could be amenable to interventions that mitigate resistance. We have recently discovered that several docetaxel-resistant mPCa cell lines exhibit lower uptake of cellular copper and uniquely express higher levels of a copper exporter protein ATP7B. Knock-down of ATP7B by silencing RNAs (siRNAs) sensitized docetaxel resistant-mPCa cells to the growth inhibitory and apoptotic effects of docetaxel. Importantly, deletions of ATP7B in human mPCa tissues predict significantly better survival of patients after their first chemotherapy than those with wild-type ATP7B (P = 0.0006). In addition, disulfiram (DSF), an FDA-approved drug for the treatment of alcohol dependence, in combination with copper, significantly enhanced the in vivo antitumor effects of docetaxel in a docetaxel-resistant xenograft tumor model. Our analyses also revealed that DSF and copper engaged with ATP7B to decrease protein levels of COMM domain-containing protein 1 (COMMD1), S-phase kinase-associated protein 2 (Skp2), and clusterin and markedly increase protein expression of cyclin-dependent kinase inhibitor 1 (p21/WAF1). Taken together, our results indicate a copper-dependent nutrient vulnerability through ATP7B exporter in docetaxel-resistant PCa for improving the therapeutic efficacy of docetaxel.

Dysfunction in atox-1 and ceruloplasmin alters labile Cu levels and consequently Cu homeostasis in C. elegans.

Copper (Cu) is an essential trace element, however an excess is toxic due to its redox properties. Cu homeostasis therefore needs to be tightly regulated via cellular transporters, storage proteins and exporters. An imbalance in Cu homeostasis has been associated with neurodegenerative disorders such as Wilson's disease, but also Alzheimer's or Parkinson's disease. In our current study, we explored the utility of using Caenorhabditis elegans (C. elegans) as a model of Cu dyshomeostasis. The application of excess Cu dosing and the use of mutants lacking the intracellular Cu chaperone atox-1 and major Cu storage protein ceruloplasmin facilitated the assessment of Cu status, functional markers including total Cu levels, labile Cu levels, Cu distribution and the gene expression of homeostasis-related genes. Our data revealed a decrease in total Cu uptake but an increase in labile Cu levels due to genetic dysfunction, as well as altered gene expression levels of Cu homeostasis-associated genes. In addition, the data uncovered the role ceruloplasmin and atox-1 play in the worm's Cu homeostasis. This study provides insights into suitable functional Cu markers and Cu homeostasis in C. elegans, with a focus on labile Cu levels, a promising marker of Cu dysregulation during disease progression.

Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection.

Multiplexed reprogramming of T cell specificity and function can generate powerful next-generation cellular therapies. However, current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here, we develop a one-step process to enrich for unlabeled cells with knock-ins at multiple target loci using a family of repair templates named Synthetic Exon/Expression Disruptors (SEEDs). SEED engineering associates transgene integration with the disruption of a paired endogenous surface protein, allowing non-modified and partially edited cells to be immunomagnetically depleted (SEED-Selection). We design SEEDs to fully reprogram three critical loci encoding T cell specificity, co-receptor expression, and MHC expression, with up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). These methods are simple, compatible with existing clinical manufacturing workflows, and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.

Outcomes of Pleural Space Infections in Patients With Indwelling Pleural Catheters for Active Malignancies.

BACKGROUND: Pleural infections related to indwelling pleural catheters (IPCs) are an uncommon clinical problem. However, management decisions can be complex for patients with active malignancies due to their comorbidities and limited life expectancies. There are limited studies on the management of IPC-related infections, including whether to remove the IPC or use intrapleural fibrinolytics. METHODS: We conducted a retrospective cohort study of patients with active malignancies and IPC-related empyemas at our institution between January 1, 2005 and May 31, 2021. The primary outcome was to evaluate clinical outcomes in patients with malignant pleural effusions and IPC-related empyemas treated with intrapleural tissue plasminogen activator (tPA) and deoxyribonuclease (DNase) compared with those treated with tPA alone or no intrapleural fibrinolytic therapy. The secondary outcome evaluated was the incidence of bleeding complications. RESULTS: We identified 69 patients with a malignant pleural effusion and an IPC-related empyema. Twenty patients received tPA/DNase, 9 received tPA alone, and 40 were managed without fibrinolytics. Those treated with fibrinolytics were more likely to have their IPCs removed as part of the initial management strategy ( P =0.004). The rate of surgical intervention and mortality attributable to the empyema were not significantly different between treatment groups. There were no bleeding events in any group. CONCLUSION: In patients with IPC-related empyemas, we did not find significant differences in the rates of surgical intervention, empyema-related mortality, or bleeding complications in those treated with intrapleural tPA/DNase, tPA alone, or no fibrinolytics. More patients who received intrapleural fibrinolytics had their IPCs removed, which may have been due to selection bias.

Management of aero-digestive fistulas in adults: the bronchoscopist's perspective.

Aero-digestive fistulas (ADFs) are pathologic connections between the airways and gastrointestinal system. These most commonly occur between the central airways and esophagus. Fistulas may develop congenitally or be acquired from a benign or malignant process. Most fistulas presenting in adulthood are acquired, with similar rates of benign and malignant etiologies. Symptoms may severely impact a patient's quality of life and result in dyspnea, cough, and oral intolerance. ADFs have been associated with increased mortality, often related to pneumonias and malnutrition. Management is multifaceted and includes a multidisciplinary approach between the pulmonologist, gastroenterologist, and thoracic surgeon. While definitive management can be achieved with surgery, this is typically reserved for benign causes as surgical repair is often impractical in patients with advanced malignancies. With malignant causes, less invasive endoscopic and/or bronchoscopic interventions may be indicated. Stenting is the most common non-surgical invasive intervention performed. Stents can be placed in the esophagus, airway, or both. There is limited data that suggests outcomes may be better when esophageal stenting is performed with or without airway stenting. Airway stents are indicated when there is airway compromise, inadequate sealing of the fistula with an esophageal stent alone, or when an esophageal stent cannot be placed. This review will provide an overview of approaching ADFs from the bronchoscopist's perspective.

Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism.

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

Telephone-based social health screening by pharmacists in the nonadherent Medicare population.

BACKGROUND: Unmet social health needs are associated with medication nonadherence. Although pharmacists are well positioned to address medication nonadherence, there is limited experience with screening for and addressing social health needs. OBJECTIVES: To compare the prevalence of social health needs among Medicare patients with higher vs lower social health risk using a predictive model. To also evaluate pre-post changes in medication adherence and health care use following a pharmacist-initiated social health screening. METHODS: A social health screening workflow was implemented into a routine pharmacist adherence program at an integrated health care delivery system. The social health screening was conducted during medication adherence outreach phone calls with Medicare members who were overdue for statin, blood pressure, or diabetes medications. We developed a social health need predictive algorithm to flag higher-risk patients and tested this algorithm against a random subset of lower-risk patients. Screening conversations were guided by a focus group that developed open-ended questions to identify social health needs. Comparisons in social health needs were made between higher- and lower-risk patients. Use and adherence outcomes were compared pre and post for patients who accepted a referral to social health resources and patients who declined a referral. RESULTS: 1,217 patients were contacted and screened for social health needs by pharmacists. Patients flagged by the social risk algorithm were more likely to report social health needs (28.7% vs 12.7% in the unflagged group; P < 0.01). Commonly reported needs included transportation (43%), finances (34%), caregiving (22%), mental health (11%), and food access (10%). 221 patients accepted a referral to a central resource website and call center that connected patients to local services. One year after screening dates, patients who did not accept a referral spent more time in the hospital (mean change +0.7 days, SD = 7.3, P < 0.01), had fewer primary care visits (mean change -0.5 visits, SD = 6.5, P < 0.01), and had a shorter length of membership (mean change -0.4 months, SD = 1.9, P < 0.01). Patients who accepted a referral had increased statin adherence (62.3% adherent pre vs 74.7% post, P = 0.02). CONCLUSIONS: We implemented a workflow for pharmacists to screen for social health needs. The social health need prediction model doubled the identification rate of patients who have needs. Intervening on social health needs during these calls may improve statin adherence and may have no adverse effect on health care utilization or health plan membership. DISCLOSURES: Social health risk predictive model development and validation was funded by the Agency for Healthcare Research and Quality (AHRQ R18HS027343).

Ciliary localization of a light-activated neuronal GPCR shapes behavior.

Many neurons in the central nervous system produce a single primary cilium that serves as a specialized signaling organelle. Several neuromodulatory G-protein-coupled receptors (GPCRs) localize to primary cilia in neurons, although it is not understood how GPCR signaling from the cilium impacts circuit function and behavior. We find that the vertebrate ancient long opsin A (VALopA), a Gi-coupled GPCR extraretinal opsin, targets to cilia of zebrafish spinal neurons. In the developing 1-d-old zebrafish, brief light activation of VALopA in neurons of the central pattern generator circuit for locomotion leads to sustained inhibition of coiling, the earliest form of locomotion. We find that a related extraretinal opsin, VALopB, is also Gi-coupled, but is not targeted to cilia. Light-induced activation of VALopB also suppresses coiling, but with faster kinetics. We identify the ciliary targeting domains of VALopA. Retargeting of both opsins shows that the locomotory response is prolonged and amplified when signaling occurs in the cilium. We propose that ciliary localization provides a mechanism for enhancing GPCR signaling in central neurons.

In vivo bioluminescence imaging of labile iron in xenograft models and liver using FeAL-1, an iron-activatable form of D-luciferin.

Dysregulated iron homeostasis underlies diverse pathologies, from ischemia-reperfusion injury to epithelial-mesenchymal transition and drug-tolerant "persister" cancer cell states. Here, we introduce ferrous iron-activatable luciferin-1 (FeAL-1), a small-molecule probe for bioluminescent imaging of the labile iron pool (LIP) in luciferase-expressing cells and animals. We find that FeAL-1 detects LIP fluctuations in cells after iron supplementation, depletion, or treatment with hepcidin, the master regulator of systemic iron in mammalian physiology. Utilizing FeAL-1 and a dual-luciferase reporter system, we quantify LIP in mouse liver and three different orthotopic pancreatic ductal adenocarcinoma tumors. We observed up to a 10-fold increase in FeAL-1 bioluminescent signal in xenograft tumors as compared to healthy liver, the major organ of iron storage in mammals. Treating mice with hepcidin further elevated hepatic LIP, as predicted. These studies reveal a therapeutic index between tumoral and hepatic LIP and suggest an approach to sensitize tumors toward LIP-activated therapeutics.