Molecular details of ruthenium red pore block in TRPV channels.

Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.

Structural mechanism of TRPV5 inhibition by econazole.

The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular details of TRPV5 modulation by exogenous and endogenous molecules. However, the details of TRPV5 inhibition by the antifungal agent econazole (ECN) remain elusive due to the low resolution of the currently available structure. In this study, we employ cryo-EM to comprehensively examine how the ECN inhibits TRPV5. By combining our structural findings with site-directed mutagenesis, calcium measurements, electrophysiology, and molecular dynamics simulations, we determined that residues F472 and L475 on the S4 helix, along with residue W495 on the S5 helix, collectively constitute the ECN-binding site. Additionally, the structure of TRPV5 in the presence of ECN and PI(4,5)P2, which does not show the bound activator, reveals a potential inhibition mechanism in which ECN competes with PI(4,5)P2, preventing the latter from binding, and ultimately pore closure.

Structural basis of the activation of TRPV5 channels by long-chain acyl-Coenzyme-A.

Long-chain acyl-coenzyme A (LC-CoA) is a crucial metabolic intermediate that plays important cellular regulatory roles, including activation and inhibition of ion channels. The structural basis of ion channel regulation by LC-CoA is not known. Transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6) are epithelial calcium-selective ion channels. Here, we demonstrate that LC-CoA activates TRPV5 and TRPV6 in inside-out patches, and both exogenously supplied and endogenously produced LC-CoA can substitute for the natural ligand phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in maintaining channel activity in intact cells. Utilizing cryo-electron microscopy, we determined the structure of LC-CoA-bound TRPV5, revealing an open configuration with LC-CoA occupying the same binding site as PI(4,5)P2 in previous studies. This is consistent with our finding that PI(4,5)P2 could not further activate the channels in the presence of LC-CoA. Our data provide molecular insights into ion channel regulation by a metabolic signaling molecule.

Plasma signatures of Congenital Generalized Lipodystrophy patients identified by untargeted lipidomic profiling are not changed after a fat-containing breakfast meal.

BACKGROUND: The incapacity to store lipids in adipose tissue in Congenital Generalized Lipodystrophy (CGL) causes hypoleptinemia, increased appetite, ectopic fat deposition and lipotoxicity. CGL patients experience shortened life expectancy. The plasma lipidomic profile has not been characterized fully in CGL, nor has the extent of dietary intake in its modulation. The present work investigated the plasma lipidomic profile of CGL patients in comparison to eutrophic individuals at the fasted state and after a breakfast meal. METHOD: Blood samples from 11 CGL patients and 10 eutrophic controls were collected after 12 h fasting (T0) and 90 min after an ad libitum fat-containing breakfast (T90). The lipidomic profile of extracted plasma lipids was characterized by non-target liquid chromatography mass spectrometry. RESULTS: Important differences between groups were observed at T0 and at T90. Several molecular species of fatty acyls, glycerolipids, sphingolipids and glycerophospholipids were altered in CGL. All the detected fatty acyl molecular species, several diacylglycerols and one triacylglycerol species were upregulated in CGL. Among sphingolipids, one sphingomyelin and one glycosphingolipid species showed downregulation in CGL. Alterations in the glycerophospholipids glycerophosphoethanolamines, glycerophosphoserines and cardiolipins were more complex. Interestingly, when comparing T90 versus T0, the lipidomic profile in CGL did not change as intensely as it did for control participants. CONCLUSIONS: The present study found profound alterations in the plasma lipidomic profile of complex lipids in CGL patients as compared to control subjects. A fat-containing breakfast meal did not appear to significantly influence the CGL profile observed in the fasted state. Our study may have implications for clinical practice, also aiding to a deeper comprehension of the role of complex lipids in CGL in view of novel therapeutic strategies.

Frizzled2 receives WntA signaling during butterfly wing pattern formation.

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. Although the secreted ligand WntA has been shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologs of the Frizzled-family of Wnt receptors. Here, we show that CRISPR mosaic knockouts of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss of function in multiple nymphalids. Whereas WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning and shed light on the functional diversity of insect Frizzled receptors.

Definition and Clinical Significance of the Monoclonal Gammopathy of Undetermined Significance-Like Phenotype in Patients With Monoclonal Gammopathies.

PURPOSE: The existence of patients with multiple myeloma (MM) and light-chain (AL) amyloidosis who present with a monoclonal gammopathy of undetermined significance (MGUS)-like phenotype has been hypothesized, but methods to identify this subgroup are not standardized and its clinical significance is not properly validated. PATIENTS AND METHODS: An algorithm to identify patients having MGUS-like phenotype was developed on the basis of the percentages of total bone marrow (BM) plasma cells (PC) and of clonal PC within the BM PC compartment, determined at diagnosis using flow cytometry in 548 patients with MGUS and 2,011 patients with active MM. The clinical significance of the algorithm was tested and validated in 488 patients with smoldering MM, 3,870 patients with active MM and 211 patients with AL amyloidosis. RESULTS: Patients with smoldering MM with MGUS-like phenotype showed significantly lower rates of disease progression (4.5% and 0% at 2 years in two independent series). There were no statistically significant differences in time to progression between treatment versus observation in these patients. In active newly diagnosed MM, MGUS-like phenotype retained independent prognostic value in multivariate analyses of progression-free survival (PFS; hazard ratio [HR], 0.49; P = .001) and overall survival (OS; HR, 0.56; P = .039), together with International Staging System, lactate dehydrogenase, cytogenetic risk, transplant eligibility, and complete remission status. Transplant-eligible patients with active MM with MGUS-like phenotype showed PFS and OS rates at 5 years of 79% and 96%, respectively. In this subgroup, there were no differences in PFS and OS according to complete remission and measurable residual disease status. Application of the algorithm in two independent series of patients with AL predicted for different survival. CONCLUSION: We developed an open-access algorithm for the identification of MGUS-like patients with distinct clinical outcomes. This phenotypic classification could become part of the diagnostic workup of MM and AL amyloidosis.

Systematic Review and Critical Appraisal of Five of the Most Recurrently Validated Child Maltreatment Assessment Instruments from 2010 to 2020.

Assessment of child maltreatment has been inconsistent across literature due to its complexity, multidimensionality, and the variety of conceptualizations of this construct. Five instruments have recurrently examined psychometric properties across the last years of research: Childhood Trauma Questionnaire, Maltreatment and Abuse Chronology of Exposure, Child Abuse Potential Inventory, Identification of Parents at Risk for child Abuse and Neglect, and Psychosocial Screening Tool. This article aims to examine and wrap up the knowledge regarding the psychometric properties of these instruments. A systematic review was performed through three of the most relevant databases in order to identify the most validated instruments to assess child maltreatment from 2010 to 2020, and 19 research articles were identified. Results indicate that there is a lack of information regarding some psychometric properties and therefore, in the light of this information, it is not possible to clearly determine if there are instruments with stronger scientific evidence for their psychometric properties, although the Maltreatment and Abuse Chronology of Exposure Scale (MACE) obtained the strongest psychometric evidence. This systematic review provided a comprehensive review on the main psychometric properties of five child maltreatment instruments in order to facilitate researchers and child welfare professionals the selection of the most suitable instrument for their specific purpose. We recommend addressing these gaps of information by further examining the psychometric properties of these instruments, and developing valid and reliable instruments for early detection in child maltreatment.

What structures did, and did not, reveal about the function of the epithelial Ca2+ channels TRPV5 and TRPV6.

Transient Receptor Potential Vanilloid 5 and 6 (TRPV5 and TRPV6) are Ca2+ selective epithelial ion channels. They are the products of a relatively recent gene duplication in mammals, and have high sequence homology to each other. Their functional properties are also much more similar to each other than to other members of the TRPV subfamily. They are both constitutively active, and this activity depends on the endogenous cofactor phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Both channels undergo Ca2+-induced inactivation, which is mediated by direct binding of the ubiquitous Ca2+ binding protein calmodulin (CaM) to the channels, and by a decrease in PI(4,5)P2 levels by Ca2+ -induced activation of phospholipase C (PLC). Recent cryo electron microscopy (cryo-EM) and X-ray crystallography structures provided detailed structural information for both TRPV5 and TRPV6. This review will discuss this structural information in the context of the function of these channels focusing on the mechanism of CaM inhibition, activation by PI(4,5)P2 and binding of pharmacological modulators.

Gating and anion selectivity are reciprocally regulated in TMEM16A (ANO1).

Numerous essential physiological processes depend on the TMEM16A-mediated Ca2+-activated chloride fluxes. Extensive structure-function studies have helped to elucidate the Ca2+ gating mechanism of TMEM16A, revealing a Ca2+-sensing element close to the anion pore that alters conduction. However, substrate selection and the substrate-gating relationship in TMEM16A remain less explored. Here, we study the gating-permeant anion relationship on mouse TMEM16A expressed in HEK 293 cells using electrophysiological recordings coupled with site-directed mutagenesis. We show that the apparent Ca2+ sensitivity of TMEM16A increased with highly permeant anions and SCN- mole fractions, likely by stabilizing bound Ca2+. Conversely, mutations at crucial gating elements, including the Ca2+-binding site 1, the transmembrane helix 6 (TM6), and the hydrophobic gate, impaired the anion permeability and selectivity of TMEM16A. Finally, we found that, unlike anion-selective wild-type channels, the voltage dependence of unselective TMEM16A mutant channels was less sensitive to SCN-. Therefore, our work identifies structural determinants of selectivity at the Ca2+ site, TM6, and hydrophobic gate and reveals a reciprocal regulation of gating and selectivity. We suggest that this regulation is essential to set ionic selectivity and the Ca2+ and voltage sensitivities in TMEM16A.

Circulating Tumor Cells for the Staging of Patients With Newly Diagnosed Transplant-Eligible Multiple Myeloma.

PURPOSE: Patients with multiple myeloma (MM) may show patchy bone marrow (BM) infiltration and extramedullary disease. Notwithstanding, quantification of plasma cells (PCs) continues to be performed in BM since the clinical translation of circulating tumor cells (CTCs) remains undefined. PATIENTS AND METHODS: CTCs were measured in peripheral blood (PB) of 374 patients with newly diagnosed MM enrolled in the GEM2012MENOS65 and GEM2014MAIN trials. Treatment included bortezomib, lenalidomide, and dexamethasone induction followed by autologous transplant, consolidation, and maintenance. Next-generation flow cytometry was used to evaluate CTCs in PB at diagnosis and measurable residual disease (MRD) in BM throughout treatment. RESULTS: CTCs were detected in 92% (344 of 374) of patients with newly diagnosed MM. The correlation between the percentages of CTCs and BM PCs was modest. Increasing logarithmic percentages of CTCs were associated with inferior progression-free survival (PFS). A cutoff of 0.01% CTCs showed an independent prognostic value (hazard ratio: 2.02; 95% CI, 1.3 to 3.1; P = .001) in multivariable PFS analysis including the International Staging System, lactate dehydrogenase levels, and cytogenetics. The combination of the four prognostic factors significantly improved risk stratification. Outcomes according to the percentage of CTCs and depth of response to treatment showed that patients with undetectable CTCs had exceptional PFS regardless of complete remission and MRD status. In all other cases with detectable CTCs, only achieving MRD negativity (and not complete remission) demonstrated a statistically significant increase in PFS. CONCLUSION: Evaluation of CTCs in PB outperformed quantification of BM PCs. The detection of ≥ 0.01% CTCs could be a new risk factor in novel staging systems for patients with transplant-eligible MM.

A gas chromatography-mass spectrometry method for quantifying squalane and squalene in aerosol emissions of electronic cigarette, or vaping, products.

Numerous chemicals of unknown inhalational toxicity have been measured in electronic cigarette, or vaping, products (EVPs). In addition, little is known about the liquid-to-aerosol transmission and deliveries of these chemicals, including oil-like terpenes such as squalene (SQE) and squalane (SQA). To provide information on the aerosol deliveries of these compounds from EVPs, we developed and validated a quantitative method to measure squalene and squalane in EVP aerosol emissions. Validation parameters include measurement repeatability (SQA and SQE %RSD <6%), intermediate precision (SQA: %RSD 11%, SQE: %RSD 17%), accuracy (SQA: 86-107%, SQE: 104-113%), matrix effects, method robustness, and analyte stability. Limits of detection were 6.06 ng/mL puffed air volume for both squalene and squalane. The method was used to measure squalene and squalane in aerosol emissions of 153 EVPs associated with case patients from a recent outbreak of e-cigarette, or vaping, product use associated lung injury (EVALI). The EVPs analyzed were organized into nicotine, cannabidiol, and tetrahydrocannabinol products by the percentage of nicotine, cannabidiol, and tetrahydrocannabinol in total particulate matter after vaping. In case-associated tetrahydrocannabinol products the detection rates and mean concentrations were 82.4% and 33.0 ng/mL puffed air for squalene and 4.41% and 7.80 ng/mL puffed air for squalane.

Isotope-Dilution Gas Chromatography-Mass Spectrometry Method for the Selective Detection of Nicotine and Menthol in E-Cigarette, or Vaping, Product Liquids and Aerosols.

We developed a quantitative method for analyzing nicotine and menthol in e-cigarette, or vaping, products (EVPs). These products may adversely impact health through inhalational exposure to addictive and harmful chemicals. The presence of unknown substances in do-it-yourself e-liquids, counterfeits, or unregulated products may increase exposure to harmful chemicals, as underscored by the 2019 EVP use-associated lung injury (EVALI) outbreak. To minimize these risks, it is important to accurately quantify nicotine and menthol in e-liquids and aerosol emissions to evaluate EVP authenticity, verify product label accuracy, and identify potentially hazardous products. We developed a simple, versatile, high-throughput method using isotope-dilution gas chromatography-mass spectrometry for quantifying nicotine and menthol concentrations in both e-liquid contents and machine-generated aerosol emissions of EVPs. Rigorous validation has demonstrated that the method is specific, precise (CV<2.71%), accurate (percent error ≤7.0%), and robust. Linear calibration ranges from 0.01 to 1.00 mg/ml for both analytes was achieved, corresponding to expected analyte levels in e-liquids and machine-generated EVP aerosols. Limits of detection (LODs) in the final 10-ml sample extract were 0.4 µg/ml for nicotine and 0.2 µg/ml for menthol. The method was used to analyze aerosol emissions of 141 EVPs associated with the 2019 EVALI outbreak; detectable levels of nicotine (2.19-59.5 mg/g of aerosol) and menthol (1.09-10.69 mg/g of aerosol) were observed in 28 and 11%, respectively, of the samples analyzed. Nicotine was not detected in any of the tetrahydrocannabinol (THC), cannabidiol (CBD), or oil-based products, while menthol (2.95 mg/g of aerosol) was only detected in one of these products (THC-labeled). The analytical method can be used to quantify nicotine and menthol concentrations in the e-liquids and aerosols from a range of EVPs, and these findings highlight a difference between e-cigarette and other vaping products.

Gas Chromatography-Tandem Mass Spectrometry Method for the Selective Detection of Glycols and Glycerol in the Liquids and Aerosols of E-Cigarette, or Vaping, Products.

The long-term health effects of using e-cigarette, or vaping, products (EVPs; also known as e-cigarettes, electronic nicotine delivery systems, and vape pens) remain largely unknown. The inhalation of excipients, such as propylene glycol (PG) and glycerin (GLY), may have long-term health effects. In addition to the direct health effects of PG and GLY, glycerin-containing products can be contaminated with toxic ethylene glycol (EG) and diethylene glycol (DEG). To assess this issue, we developed a simple, versatile, high-throughput isotope dilution gas chromatography-tandem mass spectrometry method for quantifying these common excipients and contaminants. The method is applicable to both the liquid contents and machine-generated aerosols of EVPs. Our rigorous method validation demonstrates that the new method is specific, precise, accurate, and rugged/robust. The calibration range is linear from 0.1-7 mg for the excipients and 2.5-1,000 µg for the contaminants. These ranges encompass expected excipients levels in EVP e-liquids and their machine-generated aerosols and the relevant maximum residue safety limit of 1 mg/g, or 0.1% (w/w), for the contaminants. The calculated limits of detection for PG, GLY, EG, and DEG were determined as 0.0109 mg, 0.0132 mg, 0.250 µg, and 0.100 µg, respectively. The method was applied to the aerosol emissions analysis of 141 EVPs associated with the 2019 lung injury outbreak, and found typical levels of PG (120.28-689.35 mg/g of aerosol) and GLY (116.83-845.96 mg/g of aerosol) in all nicotine-containing products; PG (81.58-491.92 mg/g of aerosol) and GLY (303.86-823.47 mg/g of aerosol) in 13% of cannabidiol (CBD) products; PG (74.02-220.18 mg/g of aerosol) and GLY (596.43-859.81 mg/g of aerosol) in products with neither nicotine nor CBD; and none detected in tetrahydrocannabinol (THC) products. No products contained glycol contaminants above the recommended maximum residue safety limit.

Electro-steric opening of the CLC-2 chloride channel gate.

The widely expressed two-pore homodimeric inward rectifier CLC-2 chloride channel regulates transepithelial chloride transport, extracellular chloride homeostasis, and neuronal excitability. Each pore is independently gated at hyperpolarized voltages by a conserved pore glutamate. Presumably, exiting chloride ions push glutamate outwardly while external protonation stabilizes it. To understand the mechanism of mouse CLC-2 opening we used homology modelling-guided structure-function analysis. Structural modelling suggests that glutamate E213 interacts with tyrosine Y561 to close a pore. Accordingly, Y561A and E213D mutants are activated at less hyperpolarized voltages, re-opened at depolarized voltages, and fast and common gating components are reduced. The double mutant cycle analysis showed that E213 and Y561 are energetically coupled to alter CLC-2 gating. In agreement, the anomalous mole fraction behaviour of the voltage dependence, measured by the voltage to induce half-open probability, was strongly altered in these mutants. Finally, cytosolic acidification or high extracellular chloride concentration, conditions that have little or no effect on WT CLC-2, induced reopening of Y561 mutants at positive voltages presumably by the inward opening of E213. We concluded that the CLC-2 gate is formed by Y561-E213 and that outward permeant anions open the gate by electrostatic and steric interactions.

Chemical Composition of JUUL Pods Collected From Students in California High Schools.

PURPOSE: To examine the chemical composition of JUUL pods collected from a convenience sample of 16 high schools in California to identify possible consumer modification or counterfeit use. METHODS: Using Gas Chromatography-Mass Spectrometry, we quantitatively analyzed the nicotine, propylene glycol (PG), and vegetable glycerin (VG) in JUUL pods (n = 26) collected from California high schools and compared results to commercial 3% (n = 15) and 5% (n = 24) JUUL pods purchased online. RESULTS: Most of the collected JUUL pods (24/26 pods) had a nicotine concentration (43.3 mg/ml, 95% PI: 21.5-65.1) outside the prediction intervals (PI) of the 3% (33.5 mg/ml, 95% PI: 31.8-35.2) and 5% (55.0 mg/ml, 95% PI: 51.5-58.3) commercial JUUL pods. Most (73%) collected JUUL pods had VG concentrations (583.5 mg/ml, PI: 428.9-738.1) lower than the 3% (722.2 mg/ml, PI: 643.0-801.4) and 5% (710.5 mg/ml, PI: 653.1-767.8) commercial JUUL pods. CONCLUSIONS: Used JUUL products collected from high school students or found on school grounds were not chemically consistent with the manufacturer's stated formulations.

Effectiveness of a mobile app intervention to prevent dating violence in residential child care / La eficacia de una intervención con aplicación móvil sobre actitudes asociadas a la violencia de pareja en los adolescentes de centros de acogida

This study focuses on the effectiveness of an app-based, monitored intervention using the Liad@s app in a residential youth-care SETTING: The aim of this intervention is to reduce maladaptive beliefs and attitudes linked to dating violence: distortions or myths about romantic love and hostile and benevolent dimensions of sexism. A quasi-experimental pre-post study with a control group was carried out. Participants were 71 adolescents from 9 group homes in Valencia (Spain). The outcomes measures were hostile and benevolent sexism (Ambivalent Sexism Inventory - ASI), ambivalence and prejudice towards men (Ambivalence toward Men Inventory - AMI), and myths about romantic love. The results of the intervention were assessed using multivariate analysis of variance (MANOVA). A post-intervention, statistically significant reduction with a medium-large effect size was observed in all dimensions for the treatment group, with eta-square of .25 for the sexism dimensions, .38 for myths, and .21 for ambivalence and prejudices. The experimental group demonstrated significantly more change than the control group on all measures. The benefits of the intervention did not vary by participants' sex. These results may be helpful to professionals involved in child and youth care, who can profit from adolescents' proclivity toward online communication

Este estudio se centra en la de eficacia de una intervención monitorizada, basada en el uso de la app Liad@s en centros de acogida para jóvenes. El objetivo de esta intervención es reducir creencias desadaptativas y actitudes asociadas a violencia en las relaciones de pareja: mitos del amor romántico y sexismo tanto en su dimensión hostil como benévola. Se realizó un estudio pre-post cuasi experimental con grupo control. Participaron 71 adolescentes de 9 centros de acogida de Valencia (España). Se tomaron medidas del cambio en sexismo hostil y benévolo (Inventario de Sexismo Ambivalente - ASI), de ambivalencia y prejuicio hacia los hombres (Inventario Ambivalencia hacia Hombres - AMI) y medida de mitos del amor romántico. Los resultados de la intervención fueron evaluados usando MANOVA (análisis multivariado de varianza). Se obtuvo una reducción significativa con tamaño del efecto entre medio y grande en todas las dimensiones esperadas en el grupo que usó la app. Las etas cuadrado fueron de .25 para las dimensiones de sexismo, .38 para mitos y .21 para ambivalencia y prejuicios. El grupo experimental mostró significativamente más cambio que el control en todas las medidas. Las ventajas de la intervención no varían en función del sexo de los participantes. Estos resultados pueden ser útiles para profesionales implicados en atención y cuidado a jóvenes, quienes pueden beneficiarse a su vez de su facilidad para comunicarse online

Biological and clinical significance of dysplastic hematopoiesis in patients with newly diagnosed multiple myeloma.

Risk of developing myelodysplastic syndrome (MDS) is significantly increased in both multiple myeloma (MM) and monoclonal gammopathy of undetermined significance, suggesting that it is therapy independent. However, the incidence and sequelae of dysplastic hematopoiesis at diagnosis are unknown. Here, we used multidimensional flow cytometry (MFC) to prospectively screen for the presence of MDS-associated phenotypic alterations (MDS-PA) in the bone marrow of 285 patients with MM enrolled in the PETHEMA/GEM2012MENOS65 trial (#NCT01916252). We investigated the clinical significance of monocytic MDS-PA in a larger series of 1252 patients enrolled in 4 PETHEMA/GEM protocols. At diagnosis, 33 (11.6%) of 285 cases displayed MDS-PA. Bulk and single-cell-targeted sequencing of MDS recurrently mutated genes in CD34+ progenitors (and dysplastic lineages) from 67 patients revealed clonal hematopoiesis in 13 (50%) of 26 cases with MDS-PA vs 9 (22%) of 41 without MDS-PA; TET2 and NRAS were the most frequently mutated genes. Dynamics of MDS-PA at diagnosis and after autologous transplant were evaluated in 86 of 285 patients and showed that in most cases (69 of 86 [80%]), MDS-PA either persisted or remained absent in patients with or without MDS-PA at diagnosis, respectively. Noteworthy, MDS-associated mutations infrequently emerged after high-dose therapy. Based on MFC profiling, patients with MDS-PA have altered hematopoiesis and T regulatory cell distribution in the tumor microenvironment. Importantly, the presence of monocytic MDS-PA at diagnosis anticipated greater risk of hematologic toxicity and was independently associated with inferior progression-free survival (hazard ratio, 1.5; P = .02) and overall survival (hazard ratio, 1.7; P = .01). This study reveals the biological and clinical significance of dysplastic hematopoiesis in newly diagnosed MM, which can be screened with moderate sensitivity using cost-effective MFC.

Voltage-Dependent Protonation of the Calcium Pocket Enable Activation of the Calcium-Activated Chloride Channel Anoctamin-1 (TMEM16A).

Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca2+-activated Cl- channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca2+-binding pocket; the voltage-dependent binding of two intracellular Ca2+ ions to the pocket gates the pore. However, in the absence of intracellular Ca2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca2+.