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Introduction Among women at risk for alcohol-exposed pregnancies (AEP), smoking tobacco may be associated with increased severity of alcohol use, and risk for tobacco-exposed and other substance-exposed pregnancies (TEPs/SEPs). Our secondary data analysis of the ‘CHOICES Plus’ intervention trial explored AEP and SEP risk by smoking status. Material and Methods Eligible women (N=261) were recruited from 12 primary care clinics in a public healthcare system, not pregnant, aged 18–44 years, drinking >3 drinks/ day or >7 drinks/week, sexually active, and not using effective contraception. We compared women who did and did not smoke tobacco on alcohol and drug severity, and psychological distress (e.g. anxiety) at baseline. Results Participants were primarily Hispanic (47.1%) or non-Hispanic Black (41.8%) and reported incomes

Abstract Exercise training is a key countermeasure used to offset spaceflight-induced multisystem deconditioning. Here, we evaluated the effects of exercise countermeasures on multisystem function in a large cohort (N = 46) of astronauts on long-duration spaceflight missions. We found that during 178 ± 48 d of spaceflight, ~600 min/wk of aerobic and resistance exercise did not fully protect against multisystem deconditioning. However, substantial inter-individual heterogeneity in multisystem response was apparent with changes from pre to postflight ranging from −30% to +5%. We estimated that up to 17% of astronauts would experience performance-limiting deconditioning if current exercise countermeasures were used on future spaceflight missions. These findings support the need for refinement of current countermeasures, adjunct interventions, or enhanced requirements for preflight physiologic and functional capacity for the protection of astronaut health and performance during exploration missions to the moon and beyond.

Background: Buprenorphine treatment has been associated with reduced non-prescribed opioid use and opioid related overdose (OD). We evaluated initial outcomes of rapid induction onto extended-release injectable buprenorphine (BUP-XR) within 7 days of emergency department presentation for unintentional OD. Methods: Between February 2019-February 2021, N = 19 patients with opioid use disorder received buprenorphine/naloxone (4/1 mg), followed by BUP-XR (300 mg) at induction and continued BUP-XR outpatient for 6 months. Primary outcomes included adverse events, repeat OD, and death. Results: For patients who received at least one dose of BUP-XR, there were no treatment related serious adverse events or symptoms of precipitated withdrawal. In addition, there were no repeat visits for ODs or deaths within 6 months of the initial OD. Discussion: These preliminary findings support the need for larger controlled clinical trials to examine the safety and efficacy of rapid induction of BUP-XR in patients with opioid use disorder at high risk of opioid OD. Rapid induction onto long-lasting injectable buprenorphine may be a promising and protective treatment approach in the future.

AbstractCyanide poisoning most commonly occurs from smoke inhalation, less commonly by oral ingestion for suicide or homicide. There are rare cases of intravenous or subcutaneous parenteral cyanide. We report a fatal case of intramuscular cyanide used as a homicidal agent. A 35-year-old female was assaulted and injected with an unknown substance in her left buttock using a syringe. She was unresponsive at ED arrival and underwent immediate endotracheal intubation. After near normal vital signs at arrival (BP 130/83 mmHg, HR 102 bpm), she rapidly became hypotensive and bradycardic with worsening acidosis (pH 6.95, lactate 7.7 mmol/L). Despite vasopressors, hydroxocobalamin, and sodium thiosulfate, she succumbed. Plasma cyanide concentrations from blood drawn 1 and 4 h post exposure were both in the lethal range (3.4 and 4.1 mg/L, respectively). Our case demonstrates that intramuscular injection can result in fatal cyanide poisoning, resulting in rapid absorption, severe toxicity, and death.

IntroductionThe purpose of this study is to determine the rate of implant removal after partial carpal arthrodesis and to investigate factors associated with implant removal.MethodsCase records of 22 dogs that underwent partial carpal arthrodesis at two private veterinary referral hospitals were reviewed. Details retrieved were body weight at time of surgery, sex, neuter status, breed, age, cause of carpal hyperextension injury, joint(s) involved in carpal hyperextension injury, laterality, type of implant, administration of post-operative antibiotics, post-operative outcome and indication for implant removal. Association between these factors and implant removal was evaluated.ResultsOf 22 partial carpal arthrodesis, 12 (55%) had implant removal due to persistent lameness and 9/12 (75%) returned to full and acceptable function after implant removal. Indications for implant removal were implant interference (8), infection (4), and migration (1). When comparing type of implant, there was a significant difference when observing implant removal rates (p = 0.04). All 5 dogs with pins and wires (100%) required implant removal. Of 17 dogs with a plate, 7 (41.2%) required implant removal. Implant removal was performed on average 114 days post-operative.DiscussionImplant removal after partial carpal arthrodesis was frequent and was commonly indicated due to pin and wire fixation or plate implant interference. This study may impact how we prepare clients for potential post-operative complications and implant removal when recommending partial carpal arthrodesis.

AbstractThe evaluation of a patient after Crotalinae envenomation requires assessment of local and systemic signs and symptoms in conjunction with laboratory data. Point-of-care ultrasound (POCUS) is an emerging modality used to evaluate soft tissue changes associated with snake envenomations around the world. However, more data are needed to characterize sonographic findings of snake envenomation. Our study used POCUS to evaluate the depth of soft tissue injury, involvement of the muscles/tendons, and the proximal edge of envenomation. This was a prospective observational study evaluating the sonographic characteristics of Crotalinae envenomation. The eight patients enrolled in the study included one envenomated by a Western diamondback (Crotalus atrox), and the others were envenomated by copperheads (Agkistrodon contortrix). All the patients demonstrated initial subcutaneous cobblestoning on POCUS at the bite site. All digit envenomations revealed edema in the tendon or tendon sheath. None of these cases required acute surgical intervention. There were no sonographic signs of envenomation found below fascia or muscle. Four of the eight patients had POCUS findings of cobblestoning proximal to the edge of envenomation palpated on the extremity. The clinical significance of this is unknown.

Abstract Background Climate change models predict changes in the amount, frequency and seasonality of precipitation events, all of which have the potential to affect the structure and function of grassland ecosystems. While previous studies have examined plant or herbivore responses to these perturbations, few have examined their interactions; even fewer have included belowground herbivores. Given the ecological, economic and biodiversity value of grasslands, and their importance globally for carbon storage and agriculture, this is an important knowledge gap. To address this, we conducted a precipitation manipulation experiment in a former mesic pasture grassland comprising a mixture of C4 grasses and C3 grasses and forbs, in southeast Australia. Rainfall treatments included a control [ambient], reduced amount [50% ambient] and reduced frequency [ambient rainfall withheld for three weeks, then applied as a single deluge event] manipulations, to simulate predicted changes in both the size and frequency of future rainfall events. In addition, half of all experimental plots were inoculated with adult root herbivores (Scarabaeidae beetles). Results We found strong seasonal dependence in plant community responses to both rainfall and root herbivore treatments. The largest effects were seen in the cool season with lower productivity, cover and diversity in rainfall-manipulated plots, while root herbivore inoculation increased the relative abundance of C3, compared to C4, plants. Conclusions This study highlights the importance of considering not only the seasonality of plant responses to altered rainfall, but also the important role of interactions between abiotic and biotic drivers of vegetation change when evaluating ecosystem-level responses to future shifts in climatic conditions.

Abstract Face masks and personal respirators are used to curb the transmission of SARS-CoV-2 in respiratory droplets; filters embedded in some personal protective equipment could be used as a non-invasive sample source for applications, including at-home testing, but information is needed about whether filters are suited to capture viral particles for SARS-CoV-2 detection. In this study, we generated inactivated virus-laden aerosols of 0.3–2 microns in diameter (0.9 µm mean diameter by mass) and dispersed the aerosolized viral particles onto electrostatic face mask filters. The limit of detection for inactivated coronaviruses SARS-CoV-2 and HCoV-NL63 extracted from filters was between 10 to 100 copies/filter for both viruses. Testing for SARS-CoV-2, using face mask filters and nasopharyngeal swabs collected from hospitalized COVID-19-patients, showed that filter samples offered reduced sensitivity (8.5% compared to nasopharyngeal swabs). The low concordance of SARS-CoV-2 detection between filters and nasopharyngeal swabs indicated that number of viral particles collected on the face mask filter was below the limit of detection for all patients but those with the highest viral loads. This indicated face masks are unsuitable to replace diagnostic nasopharyngeal swabs in COVID-19 diagnosis. The ability to detect nucleic acids on face mask filters may, however, find other uses worth future investigation.

Targeting proteins to a specific membrane is crucial for proper epithelial cell function. KCa3.1, a calcium-activated, intermediate-conductance potassium channel, is targeted to the basolateral membrane (BLM) in epithelial cells. Surprisingly, the mechanism of KCa3.1 membrane targeting is poorly understood. We previously reported that targeting of KCa3.1 to the BLM of epithelial cells is Myosin-Vc-, Rab1-and Rab8-dependent. Here, we examine the role of the SNARE proteins VAMP3, SNAP-23 and syntaxin 4 (STX-4) in the targeting of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We carried out immunoblot, siRNA and Ussing chamber experiments on FRT cells, stably expressing KCa3.1-BLAP/Bir-A-KDEL, grown as high-resistance monolayers. siRNA-mediated knockdown of VAMP3 reduced BLM expression of KCa3.1 by 57 ± 5% (p ≤ 0.05, n = 5). Measurements of BLM-localized KCa3.1 currents, in Ussing chambers, demonstrated knockdown of VAMP3 reduced KCa3.1 current by 70 ± 4% (p ≤ 0.05, n = 5). Similarly, siRNA knockdown of SNAP-23 reduced the expression of KCa3.1 at the BLM by 56 ± 7% (p ≤ 0.01, n = 6) and reduced KCa3.1 current by 80 ± 11% (p ≤ 0.05, n = 6). Also, knockdown of STX-4 lowered the BLM expression of KCa3.1 by 54 ± 6% (p ≤ 0.05, n = 5) and reduced KCa3.1 current by 78 ± 11% (p ≤ 0.05, n = 5). Finally, co-immunoprecipitation experiments demonstrated associations between KCa3.1, VAMP3, SNAP-23 and STX-4. These data indicate that VAMP3, SNAP-23 and STX-4 are critical for the targeting KCa3.1 to BLM of polarized epithelial cells.

Proton CT (pCT) is a promising new imaging technique that can reconstruct relative stopping power (RSP) more accurately than x-ray CT in each cubic millimeter voxel of the patient. This, in turn, will result in better proton range accuracy and, therefore, smaller planned tumor volumes (PTV). The hardware description and some reconstructed images have previously been reported. In a series of two contributions, we focus on presenting the software algorithms that convert pCT detector data to the final reconstructed pCT images for application in proton treatment planning. There were several options on how to accomplish this, and we will describe our solutions at each stage of the data processing chain. In the first paper of this series, we present the data acquisition with the pCT tracking and energy-range detectors and how the data are preprocessed, including the conversion to the well-formatted track information from tracking data and water-equivalent path length from the data of a calibrated multi-stage energy-range detector. These preprocessed data are then used for the initial image formation with an FDK cone-beam CT algorithm. The output of data acquisition, preprocessing, and FDK reconstruction is presented along with illustrative imaging results for two phantoms, including a pediatric head phantom. The second paper in this series will demonstrate the use of iterative solvers in conjunction with the superiorization methodology to further improve the images resulting from the upfront FDK image reconstruction and the implementation of these algorithms on a hybrid CPU/GPU computer cluster.

Some youth organizations entrust adult volunteers with delivering programs and forging relationships with youth clientele. As a result, volunteers should be competent in certain knowledge, skills, and abilities that catalyze positive youth development processes to occur. This research expands upon the results of an initial study designed to address shortcomings of a volunteer competency framework. Our objective for this study was to assess and compare the discrepancies between importance and ability-to-perform ratings of adult volunteers across 6 competency areas from the Volunteer Research Knowledge Competency Taxonomy. Over 10,000 youth professionals, adult volunteers, and families of youth members responded to an online survey. Respondents rated the importance of, and volunteers’ performance in the 6 competency areas; they also provided input about the modalities they preferred for delivering training and resources. Performance means varied across the 3 groups: Volunteers’ overall performance means were the highest, followed by families, and then professionals. Mean weighted discrepancy scores were calculated to compare the importance and performance rankings across respondent groups. Based on the scores, future volunteer trainings and resources should be prioritized around the competency areas of organization, positive youth development, program management, and communication. Volunteers also preferred more self-directed approaches for future trainings. Results from this study suggest that the volunteer competency taxonomy is still a valid framework and affirms other youth worker competency frameworks. The results also help establish a baseline of data that can be used to see if future training interventions and resources are perceived as effective.

Histone ubiquitination plays a critical role in the DNA damage response pathway. Here the authors reveal how RNF168 ubiquitinates the H2A family including noncanonical variants, H2AZ and macroH2A1/2, at the divergent N-terminal tail lysine residue.

Individuals carrying a gain-of-function mutation in PKG1 develop thoracic aortic aneurysms and dissections. Here Schwaerzer et al. show that mice carrying the same mutation recapitulate the human disease, and find that treatment with anti-oxidants including cobinamide, a vitamin B12 analog, prevents disease progression.

Urocanic aciduria is caused by a deficiency in the enzyme urocanase (E.C. 4.2.1.49) encoded by the gene UROC1. In the past, deficiency of urocanase has been associated with intellectual disability in a few case studies with some suggestion that the enzyme deficiency was the causative etiology. Here, we describe two phenotypically normal siblings with compound heterozygous pathogenic variants in UROC1 and characteristic biochemical evidence of urocanase deficiency collected utilizing untargeted metabolomic analysis. These findings suggest that urocanic aciduria may represent an otherwise benign biochemical phenotype and that those individuals with concurrent developmental delay should continue to be evaluated for other underlying causes for their symptoms. Keywords: UROC1, Urocanic aciduria, Untargeted metabolomics, Cis-urocanate, Trans-urocanate, Imidazole propionate

Arsenic exposure is a global health problem. Millions of people encounter arsenic through contaminated drinking water, consumption, and inhalation. The arsenic response locus in budding yeast is responsible for the detoxification of arsenic and its removal from the cell. This locus constitutes a conserved pathway ranging from prokaryotes to higher eukaryotes. The goal of this study was to identify how transcription from the arsenic response locus is regulated in an arsenic dependent manner. An affinity enrichment strategy called CRISPR-Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) was used, which provides for the proteomic characterization of a targeted locus. CRISPR-ChAP-MS was applied to the promoter regions of the activated arsenic response locus and uncovered 40 nuclear-annotated proteins showing enrichment. Functional assays identified the histone acetyltransferase SAGA and the chromatin remodelling complex SWI/SNF to be required for activation of the locus. Furthermore, SAGA and SWI/SNF were both found to specifically organize the chromatin structure at the arsenic response locus for activation of gene transcription. This study provides the first proteomic characterization of an arsenic response locus and key insight into the mechanisms of transcriptional activation that are necessary for detoxification of arsenic from the cell.

S-Allylmercaptocysteine (CySSA) from garlic and its onion analog S-1-propenylmercaptocysteine (CySSPe) show potential anti-cancer abilities. In this study, apoptosis induction by these two S-alk(en)lymercaptocysteine (CySSR) analogs in combination with Na2SeO3 (Se) was investigated in MDA-MD-231 breast cancer cells. CySSA and CySSPe alone did not affect cell viability, whereas CySSPe + Se and CySSA + Se showed dose-dependent reduction in cell viability. Synergistic effect was confirmed by isobologram analysis. Annexin-V staining and flow cytometric analysis revealed induction of apoptosis. Mechanistically, CySSR + Se upregulated GSK-3α/β and activated p53 to trigger apoptosis. Both CySSR + Se activated JNK pathway at an early stage, but only CySSPe + Se exhibited sustained JNK activation. Inhibition of the cystine/glutamate (xC-) antiporter by sulfasalazine attenuated the antiproliferative effects of CySSR + Se suggesting the involvement of xC- in CySSR + Se− induced apoptosis. This study indicates that the onion analogue CySSPe was similarly as effective as the major studied garlic analogue CySSA in apoptosis induction in ER− breast cancer cells.

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-β1 (60-fold), IL-6 (33-fold), and TNFα (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-β1 (-88%) and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target.

Citation data have remained hidden behind proprietary, restrictive licensing agreements, which raises barriers to entry for analysts wishing to use the data, increases the expense of performing large-scale analyses, and reduces the robustness and reproducibility of the conclusions. For the past several years, the National Institutes of Health (NIH) Office of Portfolio Analysis (OPA) has been aggregating and enhancing citation data that can be shared publicly. Here, we describe the NIH Open Citation Collection (NIH-OCC), a public access database for biomedical research that is made freely available to the community. This dataset, which has been carefully generated from unrestricted data sources such as MedLine, PubMed Central (PMC), and CrossRef, now underlies the citation statistics delivered in the NIH iCite analytic platform. We have also included data from a machine learning pipeline that identifies, extracts, resolves, and disambiguates references from full-text articles available on the internet. Open citation links are available to the public in a major update of iCite (https://icite.od.nih.gov).

Broad-scale untargeted biochemical phenotyping is a technology that supplements widely accepted assays, such as organic acid, amino acid, and acylcarnitine analyses typically utilized for the diagnosis of inborn errors of metabolism. In this study, we investigate the analyte changes associated with 4-aminobutyrate aminotransferase (ABAT, GABA transaminase) deficiency and treatments that affect GABA metabolism. GABA-transaminase deficiency is a rare neurodevelopmental and neurometabolic disorder caused by mutations in ABAT and resulting in accumulation of GABA in the cerebrospinal fluid (CSF). For that reason, measurement of GABA in CSF is currently the primary approach to diagnosis. GABA-transaminase deficiency results in severe developmental delay with intellectual disability, seizures, and movement disorder, and is often associated with death in childhood. Using an untargeted metabolomics platform, we analyzed EDTA plasma, urine, and CSF specimens from four individuals with GABA-transaminase deficiency to identify biomarkers by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. Metabolomic analyses of over 1,000 clinical plasma samples revealed a rich source of biochemical information. Three out of four patients showed significantly elevated levels of the molecule 2-pyrrolidinone (Z-score ≥ 2) in plasma, and whole exome sequencing revealed variants of uncertain significance in ABAT. Additionally, these same patients also had elevated levels of succinimide or its ring-opened form, succinamic acid, in plasma, urine, and CSF and/or homocarnosine in urine and CSF. In the analysis of clinical EDTA plasma samples, the levels of succinamic acid and 2-pyrrolidinone showed a high level of correlation (R = 0.72), indicating impairment in GABA metabolism and further supporting the association with GABA-transaminase deficiency and the pathogenicity of the ABAT variants. Further analysis of metabolomic data across our patient population revealed the association of elevated levels of 2-pyrrolidinone with administration of vigabatrin, a commonly used anti-seizure medication and a known inhibitor of GABA-transaminase. These data indicate that anti-seizure medications may alter the biochemical and metabolomic data, potentially impacting the interpretation and diagnosis for the patient. Further, these data demonstrate the power of combining broad scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and support the use of metabolic phenotyping of plasma to screen for GABA-transaminase deficiency.

Wallenda (WND) is the Drosophila member of a conserved family of dual leucine-zipper kinases (DLK) active in both neuronal regeneration and degeneration. We examined the role of WND over-expression on sensory neuron morphology by driving WND in multiple subtypes of Drosophila photoreceptors. WND overexpression under control of the pan-retinal GAL4 driver GMR causes multiple photoreceptor defects including cell death, rhabdomere degeneration, and axonal sprouting. Individual photoreceptor subtypes were assayed using GAL4 drivers specific for each photoreceptor class. Many R7 and R8 cells exhibit axonal sprouting while some show cell degeneration. Delaying the onset of WND overexpression until 20 days of age showed that older adult R7 cells retain the ability to initiate new axon growth. R1–6 photoreceptor cells degenerate in response to WND expression and exhibit rhodopsin loss and rhabdomere degeneration. RNAi knockdown of the MAPK signaling components Kayak (KAY) and Hemipterous (HEP) attenuates the WND-induced loss of Rh1 rhodopsin. UAS-induced HEP expression is similar to WND expression, causing degeneration in R1–6 photoreceptors and axonal sprouting in R7 photoreceptors. These results demonstrate that WND in adult Drosophila photoreceptor cells acts through MAPK signaling activity with both regenerative and degenerative responses. These photoreceptors provide a tractable experimental model to reveal cellular mechanisms driving contradictory WND signaling responses.

Ca2+ signaling is important for many cellular and physiological processes, including cardiac function. Although sarcoplasmic reticulum (SR) proteins involved in Ca2+ signaling have been shown to be phosphorylated, the biochemical and physiological roles of protein phosphorylation within the lumen of the SR remain essentially uncharacterized. Our laboratory recently identified an atypical protein kinase, Fam20C, which is uniquely localized to the secretory pathway lumen. Here, we show that Fam20C phosphorylates several SR proteins involved in Ca2+ signaling, including calsequestrin2 and Stim1, whose biochemical activities are dramatically regulated by Fam20C mediated phosphorylation. Notably, phosphorylation of Stim1 by Fam20C enhances Stim1 activation and store-operated Ca2+ entry. Physiologically, mice with Fam20c ablated in cardiomyocytes develop heart failure following either aging or induced pressure overload. We extended these observations to show that non-muscle cells lacking Fam20C display altered ER Ca2+ signaling. Overall, we show that Fam20C plays an overarching role in ER/SR Ca2+ homeostasis and cardiac pathophysiology.

Feline obesity elicits a plethora of metabolic responses leading to comorbidities, with potential reversal during weight loss. The specific metabolic alterations and biomarkers of organ dysfunction are not entirely understood. Untargeted, high-throughput metabolomic technologies may allow the identification of biological components that change with weight status in cats, increasing our understanding of feline metabolism. The objective of this study was to utilize untargeted metabolomic techniques to identify biomarkers and gain mechanistic insight into the serum metabolite changes associated with reduced food intake and weight loss in overweight cats. During a four-wk baseline period, cats were fed to maintain body weight. For 18 wk following baseline, cats were fed to lose weight at a rate of ~1.5% body weight/wk. Blood serum metabolites were measured at wk 0, 1, 2, 4, 8, 12, and 16. A total of 535 named metabolites were identified, with up to 269 of them being altered (p- and q-values < 0.05) at any time point. A principal component analysis showed a continual shift in metabolite profile as weight loss progressed, with early changes being distinct from those over the long term. The majority of lipid metabolites decreased with weight loss; however, ketone bodies and small lipid particles increased with weight loss. The majority of carbohydrate metabolites decreased with weight loss. Protein metabolites had a variable result, with some increasing, but others decreasing with weight loss. Metabolic mediators of inflammation, oxidative stress, xenobiotics, and insulin resistance decreased with weight loss. In conclusion, global metabolomics identified biomarkers of reduced food intake and weight loss in cats, including decreased markers of inflammation and/or altered macronutrient metabolism.

Altered function of the muscle LIM protein (MLP) causes dilated cardiomyopathy in mice and humans. Lange et al. explain the molecular role of MLP in the heart by showing that it affects the signalling complex at the intercalated discs of failing hearts that consists of PKCα, PLCβ1 and CARP by inhibiting PKCα auto-phosphorylation and function.

TRPV4 channels are calcium-permeable cation channels that are activated by several physicochemical stimuli. Accordingly, TRPV4 channels have been implicated in the regulation of osmosensing, mechanotransduction, thermosensation, and epithelial/endothelial barrier functions. Whether TRPV4 is also mechanistically implicated in melanoma cell proliferation is not clear. Here, we hypothesized that TRPV4 is expressed in human melanoma and that pharmacological activation interferes with cell proliferation.TRPV4 functions were studied in melanoma cell lines (A375, SK-MEL-28, MKTBR), immortalized non-cancer keratinocytes (HaCaT), and murine 3T3 fibroblasts by patch-clamp, qRT-PCR, intracellular calcium measurements, cell proliferation, and flow cytometric assays of apoptosis and cell cycle. The selective TRPV4-activator, GSK1016790A, elicited non-selective cation currents with TRPV4-typical current-voltage-relationship in all cell lines. GSK1016790A-induced currents were blocked by the TRPV4-blocker, HC067047. TRPV4 mRNA expression was demonstrated by qRT-PCR. In A375 cells, TRPV4 activation was frequently paralleled by co-activation of calcium/calmodulin-regulated KCa3.1 channels. Light microscopy showed that TRPV4-activation produced rapid cellular disarrangement, nuclear densification, and detachment of a large fraction of all melanoma cell lines and HaCaT cells. TRPV4-activation induced apoptosis and drastically inhibited A375 and HaCaT proliferation that could be partially prevented by HC067047.Our study showed that TRPV4 channels were functionally expressed in human melanoma cell lines and in human keratinocytes. Pharmacological TRPV4 activation in human melanoma cells and keratinocytes caused severe cellular disarrangement, necrosis and apoptosis. Pharmacological targeting of TRPV4 could be an alternative or adjuvant therapeutic strategy to treat melanoma progression and other proliferative skin disorders.

We previously demonstrated endocytosis of KCa2.3 is caveolin-1-, dynamin II- and Rab5-dependent. KCa2.3 then enters Rab35/EPI64C- and RME-1-containing recycling endosomes and is returned to the plasma membrane (PM). Herein, we report on the mechanism by which KCa2.3 is inserted into the PM during recycling and following exit from the Golgi. We demonstrate KCa2.3 colocalizes with SNAP-23 and Syntaxin-4 in the PM of HEK and endothelial cells by confocal immunofluorescence microscopy. We further show KCa2.3 can be co-immunoprecipitated with SNAP-23 and Syntaxin-4. Overexpression of either Syntaxin-4 or SNAP-23 increased PM expression of KCa2.3, whereas shRNA-mediated knockdown of these SNARE proteins significantly decreased PM KCa2.3 expression, as assessed by cell surface biotinylation. Whole-cell patch clamp studies confirmed knockdown of SNAP-23 significantly decreased the apamin sensitive, KCa2.3 current. Using standard biotinylation/stripping methods, we demonstrate shRNA mediated knockdown of SNAP-23 inhibits recycling of KCa2.3 following endocytosis, whereas scrambled shRNA had no effect. Finally, using biotin ligase acceptor peptide (BLAP)-tagged KCa2.3, coupled with ER-resident biotin ligase (BirA), channels could be biotinylated in the ER after which we evaluated their rate of insertion into the PM following Golgi exit. We demonstrate knockdown of SNAP-23 significantly slows the rate of Golgi to PM delivery of KCa2.3. The inhibition of both recycling and PM delivery of newly synthesized KCa2.3 channels likely accounts for the decreased PM expression observed following knockdown of these SNARE proteins. In total, our results suggest insertion of KCa2.3 into the PM depends upon the SNARE proteins, Syntaxin-4 and SNAP-23.

In epithelia, the intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane (BLM) where this channel plays numerous roles in absorption and secretion. A growing body of research suggests that the membrane resident population of KCa3.1 may be critical in clinical manifestation of diseases. In this study, we investigated the key molecular components that regulate the degradation of KCa3.1 using a Fisher rat thyroid cell line stably expressing KCa3.1. Using immunoblot, Ussing chamber, and pharmacological approaches, we demonstrated that KCa3.1 is targeted exclusively to the BLM, provided a complete time course of degradation of KCa3.1 and degradation time courses of the channel in the presence of pharmacological inhibitors of ubiquitylation and deubiquitylation to advance our understanding of the retrograde trafficking of KCa3.1. We provide a complete degradation profile of KCa3.1 and that the degradation is via an ubiquitin-dependent pathway. Inhibition of E1 ubiquitin activating enzyme by UBEI-41 crippled the ability of the cells to internalize the channel, shown by the increased BLM surface expression resulting in an increased function of the channel as measured by a DCEBIO sensitive K+ current. Additionally, the involvement of deubiquitylases and degradation by the lysosome were also confirmed by treating the cells with PR-619 or leupeptin/pepstatin, respectively; which significantly decreased the degradation rate of membrane KCa3.1. Additionally, we provided the first evidence that KCa3.1 channels were not deubiquitylated at the BLM. These data further define the retrograde trafficking of KCa3.1, and may provide an avenue for therapeutic approach for treatment of disease.

Long-duration spaceflight results in muscle atrophy and a loss of bone mineral density. In skeletal muscle tissue, acute exercise and protein (e.g., essential amino acids) stimulate anabolic pathways (e.g., muscle protein synthesis) both independently and synergistically to maintain neutral or positive net muscle protein balance. Protein intake in space is recommended to be 12%–15% of total energy intake (≤1.4 g∙kg−1∙day−1) and spaceflight is associated with reduced energy intake (~20%), which enhances muscle catabolism. Increasing protein intake to 1.5–2.0 g∙kg−1∙day−1 may be beneficial for skeletal muscle tissue and could be accomplished with essential amino acid supplementation. However, increased consumption of sulfur-containing amino acids is associated with increased bone resorption, which creates a dilemma for musculoskeletal countermeasures, whereby optimizing skeletal muscle parameters via essential amino acid supplementation may worsen bone outcomes. To protect both muscle and bone health, future unloading studies should evaluate increased protein intake via non-sulfur containing essential amino acids or leucine in combination with exercise countermeasures and the concomitant influence of reduced energy intake.

Abstract: The epithelial intermediate-conductance calcium/calmodulin-regulated KCa3.1 channel is considered to be a regulator of intestine function by controlling chloride secretion and water/salt balance. Yet, little is known about the functional importance of KCa3.1 in the intestinal epithelium in vivo. Our objective was to determine the impact of epithelial-specific inducible overexpression of a KCa3.1 transgene (KCa3.1+) and of inducible suppression (KCa3.1−) on intestinal homeostasis and function in mice. KCa3.1 overexpression in the duodenal epithelium of doxycycline (DOX)-treated KCa3.1+ mice was 40-fold above the control levels. Overexpression caused an inflated duodenum and doubling of the chyme content. Histology showed conserved architecture of crypts, villi, and smooth muscle. Unaltered proliferating cell nuclear antigen (PCNA) immune reactivity and reduced amounts of terminal deoxynucleotide transferase mediated X-dUTP nick end labeling (TUNEL)-positive apoptotic cells in villi indicated lower epithelial turnover. Myography showed a reduction in the frequency of spontaneous propulsive muscle contractions with no change in amplitude. The amount of stool in the colon was increased and the frequency of colonic contractions was reduced in KCa3.1+ animals. Senicapoc treatment prevented the phenotype. Suppression of KCa3.1 in DOX-treated KCa3.1− mice caused no overt intestinal phenotype. In conclusion, inducible KCa3.1 overexpression alters intestinal functions by increasing the chyme content and reducing spontaneous contractions and epithelial apoptosis. Induction of epithelial KCa3.1 can play a mechanistic role in the process of adaptation of the intestine.

The goal of this study was to provide insight into the mechanism by which bariatric surgical procedures led to weight loss and improvement or resolution of diabetes. Global biochemical profiling was used to evaluate changes occurring in nondiabetic and type 2 diabetic (T2D) patients experiencing either less extreme sleeve gastrectomy or a full gastric bypass. We were able to identify changes in metabolism that were affected by standard preoperation liquid weight loss diet as well as by bariatric surgery itself. Preoperation weight-loss diet was associated with a strong lipid metabolism signature largely related to the consumption of adipose reserves for energy production. Glucose usage shift away from glycolytic pyruvate production toward pentose phosphate pathway, via glucose-6-phosphate, appeared to be shared across all patients regardless of T2D status or bariatric surgery procedure. Our results suggested that bariatric surgery might promote antioxidant defense and insulin sensitivity through both increased heme synthesis and HO activity or expression. Changes in histidine and its metabolites following surgery might be an indication of altered gut microbiome ecology or liver function. This initial study provided broad understanding of how metabolism changed globally in morbidly obese nondiabetic and T2D patients following weight-loss surgery.

To address the question as to whether echocardiographic and/or microcomputed tomography (microCT) analysis can be utilized to assess the extent of Coxsackie B virus (CVB) induced myocarditis in the absence of left ventricular dysfunction in the mouse.Viral myocarditis is a significant clinical problem with associated inflammation of the myocardium and myocardial injury. Murine models of myocarditis are commonly used to study the pathophysiology of the disease, but methods for imaging the mouse myocardium have been limited to echocardiographic assessment of ventricular dysfunction and, to a lesser extent, MRI imaging.Using a murine model of myocarditis, we used both echocardiography and microCT to assess the extent of myocardial involvement in murine myocarditis using both wild-type mice and CVB cleavage-resistant dystrophin knock-in mice.Areas of increased echogenicity were only observed in the myocardium of Coxsackie B virus infected mice. These echocardiographic abnormalities correlated with the extent of von Kossa staining (a marker of membrane permeability), inflammation, and fibrosis. Given that calcium phosphate uptake as imaged by von Kossa staining might also be visualized using microCT, we utilized microCT imaging which allowed for high-resolution, 3-dimensional images of radiodensities that likely represent calcium phosphate uptake. As with echocardiography, only mice infected with Coxsackie B virus displayed abnormal accumulation of calcium within individual myocytes indicating increased membrane permeability only upon exposure to virus.These studies demonstrate new, quantitative, and semi-quantitative imaging approaches for the assessment of myocardial involvement in the setting of viral myocarditis in the commonly utilized mouse model of viral myocarditis.

Our understanding of the modulation of proteins has shifted in direction with the discovery of microRNAs (miRs) over twenty years ago. MiRs are now in the ‘limelight’ as these non-coding pieces of RNA (generally ~22 nucleotides long) result in altered translation and function of proteins. Indeed, miRs are now reported to be potential biomarkers of disease. Epithelial K channels play many roles in electrolyte and fluid homeostasis of the human body and have been suggested to be therapeutic targets of disease. Interestingly, the role of miRs in modulating K channels of epithelial tissues is only emerging now. This minireview focuses on recent novel findings into the role of miRs in the regulation of K channels of epithelia.

Obesity promotes insulin resistance and chronic inflammation. Disrupting any of several distinct steps in lipid synthesis decreases adiposity, but it is unclear if this approach coordinately corrects the environment that propagates metabolic disease. We tested the hypothesis that inactivation of FAS in the hypothalamus prevents diet-induced obesity and systemic inflammation. Ten weeks of high-fat feeding to mice with inactivation of FAS (FASKO) limited to the hypothalamus and pancreatic β cells protected them from diet-induced obesity. Though high-fat fed FASKO mice had no β-cell phenotype, they were hypophagic and hypermetabolic, and they had increased insulin sensitivity at the liver but not the periphery as demonstrated by hyperinsulinemic-euglycemic clamps, and biochemically by increased phosphorylated Akt, glycogen synthase kinase-3beta, and FOXO1 compared with wild-type mice. High-fat fed FASKO mice had decreased excretion of urinary isoprostanes, suggesting less oxidative stress and blunted tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) responses to endotoxin, suggesting less systemic inflammation. Pair-feeding studies demonstrated that these beneficial effects were dependent on central FAS disruption and not merely a consequence of decreased adiposity. Thus, inducing central FAS deficiency may be a valuable integrative strategy for treating several components of the metabolic syndrome, in part by correcting hepatic insulin resistance and suppressing inflammation.

In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP). Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A). In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior.

Cardiac arrest induces whole body ischemia, which causes damage to multiple organs particularly the heart and the brain. There is clinical and preclinical evidence that neurological injury is responsible for high mortality and morbidity of patients even after successful cardiopulmonary resuscitation. A better understanding of the metabolic alterations in the brain during ischemia will enable the development of better targeted resuscitation protocols that repair the ischemic damage and minimize the additional damage caused by reperfusion.A validated whole body model of rodent arrest followed by resuscitation was utilized; animals were randomized into three groups: control, 30 minute asphyxial arrest, or 30 minutes asphyxial arrest followed by 60 min cardiopulmonary bypass (CPB) resuscitation. Blood gases and hemodynamics were monitored during the procedures. An untargeted metabolic survey of heart and brain tissues following cardiac arrest and after CPB resuscitation was conducted to better define the alterations associated with each condition.After 30 min cardiac arrest and 60 min CPB, the rats exhibited no observable brain function and weakened heart function in a physiological assessment. Heart and brain tissues harvested following 30 min ischemia had significant changes in the concentration of metabolites in lipid and carbohydrate metabolism. In addition, the brain had increased lysophospholipid content. CPB resuscitation significantly normalized metabolite concentrations in the heart tissue, but not in the brain tissue.The observation that metabolic alterations are seen primarily during cardiac arrest suggests that the events of ischemia are the major cause of neurological damage in our rat model of asphyxia-CPB resuscitation. Impaired glycolysis and increased lysophospholipids observed only in the brain suggest that altered energy metabolism and phospholipid degradation may be a central mechanism in unresuscitatable brain damage.

Pistachio nut ingestion (3 oz./d, two weeks) was tested for effects on exercise performance and 21-h post-exercise recovery from inflammation, oxidative stress, immune dysfunction, and metabolite shifts.Using a randomized, crossover approach, cyclists (N = 19) engaged in two 75-km time trials after 2-weeks pistachio or no pistachio supplementation, with a 2-week washout period. Subjects came to the lab in an overnight fasted state, and ingested water only or 3 oz. pistachios with water before and during exercise. Blood samples were collected 45 min pre-exercise, and immediately post-, 1.5-h post-, and 21-h post-exercise, and analyzed for plasma cytokines, C-reactive protein (CRP), F2-isoprostanes (F2-IsoP), granulocyte phagocytosis (GPHAG) and oxidative burst activity (GOBA), and shifts in metabolites.Performance time for the 75-km time trial was 4.8% slower under pistachio conditions (2.84 ± 0.11 and 2.71 ± 0.07 h, respectively, P = 0.034). Significant time effects were shown for plasma cytokines, CRP, F2-IsoP, GPHAG, and GOBA, with few group differences. Metabolomics analysis revealed 423 detectable compounds of known identity, with significant interaction effects for 19 metabolites, especially raffinose, (12Z)-9,10-Dihydroxyoctadec-12-enoate (9,10-DiHOME), and sucrose. Dietary intake of raffinose was 2.19 ± 0.15 and 0.35 ± 0.08 mg/d during the pistachio and no pistachio periods, and metabolomics revealed that colon raffinose and sucrose translocated to the circulation during exercise due to increased gut permeability. The post-exercise increase in plasma raffinose correlated significantly with 9,10-DiHOME and other oxidative stress metabolites.In summary, 2-weeks pistachio nut ingestion was associated with reduced 75-km cycling time trial performance and increased post-exercise plasma levels of raffinose, sucrose, and metabolites related to leukotoxic effects and oxidative stress.ClinicalTrials.gov NCT01821820.

The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells.

Ankrd1/CARP, Ankrd2/Arpp, and Ankrd23/DARP belong to a family of stress inducible ankyrin repeat proteins expressed in striated muscle (MARPs). The MARPs are homologous in structure and localized in the nucleus where they negatively regulate gene expression as well as in the sarcomeric I-band, where they are thought to be involved in mechanosensing. Together with their strong induction during cardiac disease and the identification of causative Ankrd1 gene mutations in cardiomyopathy patients, this suggests their important roles in cardiac development, function, and disease. To determine the functional role of MARPs in vivo, we studied knockout (KO) mice of each of the three family members. Single KO mice were viable and had no apparent cardiac phenotype. We therefore hypothesized that the three highly homologous MARP proteins may have redundant functions in the heart and studied double and triple MARP KO mice. Unexpectedly, MARP triple KO mice were viable and had normal cardiac function both at basal levels and in response to mechanical pressure overload induced by transverse aortic constriction as assessed by echocardiography and hemodynamic studies. Thus, CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to mechanical pressure overload.

Dr. Robert K. Crane made major contributions to our understanding of carbohydrate metabolism and transport of the intestine over a very long and productive career. This Perspective examines, briefly, his early life and academic positions, but more importantly, this Perspective highlights his contributions to the understanding of coupled Na+-glucose absorption by the small intestine. I discuss how his early hypothesis of a ‘cotransport’ of sodium and glucose ushered in and provided the physiological explanation for the clinical treatment of acute diarrhea and cholera when using oral rehydration therapy (ORT). ORT saves millions of lives each year. Certainly, humankind is better off because of Crane’s hypothesis of the Na+-glucose cotransporter which he put forth over 50 years ago?

Polyphenol supplementation was tested as a countermeasure to inflammation and oxidative stress induced by 3-d intensified training.Water soluble polyphenols from blueberry and green tea extracts were captured onto a polyphenol soy protein complex (PSPC). Subjects were recruited, and included 38 long-distance runners ages 19-45 years who regularly competed in road races. Runners successfully completing orientation and baseline testing (N = 35) were randomized to 40 g/d PSPC (N = 17) (2,136 mg/d gallic acid equivalents) or placebo (N = 18) for 17 d using double-blinded methods and a parallel group design, with a 3-d running period inserted at day 14 (2.5 h/d, 70% VO2max). Blood samples were collected pre- and post-14 d supplementation, and immediately and 14 h after the third day of running in subjects completing all aspects of the study (N = 16 PSPC, N = 15 placebo), and analyzed using a metabolomics platform with GC-MS and LC-MS.Metabolites characteristic of gut bacteria metabolism of polyphenols were increased with PSPC and 3 d running (e.g., hippurate, 4-hydroxyhippurate, 4-methylcatechol sulfate, 1.8-, 1.9-, 2.5-fold, respectively, P

The field of ubiquitylation and dubiquitylation of proteins in molecular physiology is growing at a rapid rate. Our understanding of molecular physiology of these processes may become limited by the advancement of technologies that scientists can employ. Therefore, it is important to approach physiological questions of ubiquitylation and dubiquitylation of proteins from a multiple methodological direction. Indeed, the role of ubiquitylation and dubiquitylation of proteins in cellular function has been implicated in the pathophysiology of human diseases including cancer, viral diseases and neurodegenerative disorders. There are many modulators (activators and inhibitors) of ubiquitylation and dubiquitylation. Therefore, the link is being able to rapidly assess potential modulators of ubiquitylation and dubiquitylation and determine which specific modulators play a role(s) within a particular physiological setting. After the specific modulators have been identified, further experimentation is required to assess the downstream use as potential clinical target for a particular disease. The first step is to identify the specific modulators. This perspective highlights a multi-prong technologies approach that uses three novel techn

A significant current challenge in human genetics is the identification of interacting genetic loci mediating complex polygenic disorders. One of the best characterized polygenic diseases is Down syndrome (DS), which results from an extra copy of part or all of chromosome 21. A short interval near the distal tip of chromosome 21 contributes to congenital heart defects (CHD), and a variety of indirect genetic evidence suggests that multiple candidate genes in this region may contribute to this phenotype. We devised a tiered genetic approach to identify interacting CHD candidate genes. We first used the well vetted Drosophila heart as an assay to identify interacting CHD candidate genes by expressing them alone and in all possible pairwise combinations and testing for effects on rhythmicity or heart failure following stress. This comprehensive analysis identified DSCAM and COL6A2 as the most strongly interacting pair of genes. We then over-expressed these two genes alone or in combination in the mouse heart. While over-expression of either gene alone did not affect viability and had little or no effect on heart physiology or morphology, co-expression of the two genes resulted in ≈50% mortality and severe physiological and morphological defects, including atrial septal defects and cardiac hypertrophy. Cooperative interactions between DSCAM and COL6A2 were also observed in the H9C2 cardiac cell line and transcriptional analysis of this interaction points to genes involved in adhesion and cardiac hypertrophy. Our success in defining a cooperative interaction between DSCAM and COL6A2 suggests that the multi-tiered genetic approach we have taken involving human mapping data, comprehensive combinatorial screening in Drosophila, and validation in vivo in mice and in mammalian cells lines should be applicable to identifying specific loci mediating a broad variety of other polygenic disorders.

Several injectable materials have been shown to preserve or improve cardiac function as well as prevent or slow left ventricular (LV) remodeling post-myocardial infarction (MI). However, it is unclear as to whether it is the structural support or the bioactivity of these polymers that lead to beneficial effects. Herein, we examine how passive structural enhancement of the LV wall by an increase in wall thickness affects cardiac function post-MI using a bio-inert, non-degradable synthetic polymer in an effort to better understand the mechanisms by which injectable materials affect LV remodeling.Poly(ethylene glycol) (PEG) gels of storage modulus G' = 0.5±0.1 kPa were injected and polymerized in situ one week after total occlusion of the left coronary artery in female Sprague Dawley rats. The animals were imaged using magnetic resonance imaging (MRI) at 7±1 day(s) post-MI as a baseline and again post-injection 49±4 days after MI. Infarct wall thickness was statistically increased in PEG gel injected vs. control animals (p