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Julita Chlebowicz,1 Radha Akella,1 John M Humphreys,1 Haixia He,1 Ashari R Kannangara,1 Shuguang Wei,2 Bruce Posner,2 Elizabeth J Goldsmith1 1Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX, USA; 2Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX, USACorrespondence: Elizabeth J Goldsmith, Department of Biophysics, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390-8816, USA, Tel +1 214 645 6376, Email Elizabeth.goldsmith@utsouthwestern.eduIntroduction: WNK [with no lysine (K)] kinases are serine/threonine kinases associated with familial hyperkalemic hypertension (FHHt). WNKs are therapeutic targets for blood pressure regulation, stroke and several cancers including triple negative breast cancer and glioblastoma. Here, we searched for and characterized novel WNK kinase inhibitors.Methods: We used a ~210,000-compound library in a high-throughput screen, re-acquisition and assay, commercial specificity screens and crystallography to identify WNK-isoform-selective inhibitors.Results: We identified five classes of compounds that inhibit the kinase activity of WNK1: quinoline compounds, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-derived compounds. The compounds are strongly pan-WNK selective, inhibiting all four WNK isoforms. A class of quinoline compounds was identified that further shows selectivity among the WNK isoforms, being more potent toward WNK3 than WNK1. The crystal structure of the quinoline-derived SW120619 bound to the kinase domain of WNK3 reveals active site binding, and comparison to the WNK1 structure reveals the potential origin of isoform specificity.Discussion: The newly discovered classes of compounds may be starting points for generating pharmacological tools and potential drugs treating hypertension and cancer.Graphical Abstract: Keywords: WNK, inhibition screen, kinase inhibitor, WNK3-specific, structure–activity relationship, crystallography

An aggregate planning issue that directly influences the cost to a manufacturing facility of supplier quality and reliability was explored. The level of optimal production and the volume of raw material order, which can reduce the total expected cost, were calculated at the same time. It was shown that this strategy can lead to considerable savings. The cost of reliable raw material supplies were assessed and characterized, and a method of assigning capacity to various products was discussed.

Abstract Fabry disease (FD) is a treatable X linked lysosomal storage disorder with a wide phenotypic spectrum. There is a scarcity of published data on the burden of FD in India. This study evaluates the clinical and molecular spectrum of Indian patients with FD. In this multicentric study involving 10 tertiary referral centers in India, we analyzed the clinical course and genotype of 54 patients from 37 families. Family screening identified 19 new patients (35%) from 12 index cases. Then, 33 GLA gene variants were identified in 49/54 (90.7%) which included 11 novel and 22 known pathogenic variants. Of the 54 patients in our cohort, 40 patients had “classical” and 10 patients had a “nonclassical” presentation. The symptoms and signs included kidney dysfunction in 38/54 (70.3%), neuropathic pain in 34/54 (62.9%), left ventricular hypertrophy in 22/49 (44.8%) and stroke in 5/54 (9.2%). Female heterozygotes were 10/54 (18.5%) of whom 2 were index cases. There was a significant delay in reaching the diagnosis of 11.7 years. Enzyme replacement therapy was initiated in 28/54 (51.8%) patients with significant improvement of neuropathic pain and gastrointestinal symptoms. This study highlights the clinical presentation and mutational spectrum of FD in India and suggests that family screening and screening of high‐risk groups (hypertrophic cardiomyopathy, idiopathic chronic renal failure and cryptogenic stroke) could be the most cost‐effective strategies for early identification of FD.

Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1 and CWN2). Here, we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride-binding site are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here, we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand., Competing Interests: LT, RA, JH, HH, EG No competing interests declared, (© 2023, Teixeira et al.)

Previous study has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell-volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N 2 gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N 2 gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed.

Background-Previous studies on coronavirus disease 2019 (COVID-19) were limited to specific geographical locations and small sample sizes. Therefore, we used the National Inpatient Sample (NIS) 2020 database to determine the risk factors for severe outcomes and mortality in COVID-19. Methods-We included adult patients with COVID-19. Univariate and multivariate logistic regression was performed to determine the predictors of severe outcomes and mortality in COVID-19. Results-1,608,980 (95% CI 1,570,803-1,647,156) hospitalizations with COVID-19 were included. Severe complications occurred in 78.3% of COVID-19 acute respiratory distress syndrome (ARDS) and 25% of COVID-19 pneumonia patients. The mortality rate for COVID-19 ARDS was 54% and for COVID-19 pneumonia was 16.6%. On multivariate analysis, age > 65 years, male sex, government insurance or no insurance, residence in low-income areas, non-white races, stroke, chronic kidney disease, heart failure, malnutrition, primary immunodeficiency, long-term steroid/immunomodulatory use, complicated diabetes mellitus, and liver disease were associated with COVID-19 related complications and mortality. Cardiac arrest, septic shock, and intubation had the highest odds of mortality. Conclusions-Socioeconomic disparities and medical comorbidities were significant determinants of mortality in the US in the pre-vaccine era. Therefore, aggressive vaccination of high-risk patients and healthcare policies to address socioeconomic disparities are necessary to reduce death rates in future pandemics.

With No lysine (K) [WNK] kinases are structurally unique serine/threonine protein kinases that have therapeutic potential for blood pressure regulation and cancer. A novel class of trihalo-sulfone compounds was identified by high-throughput screening. Trihalo-sulfone 1 emerged as an effective inhibitor of WNK1 with an IC 50 value of 1.6 μM. Herein, we define chemical features necessary for inhibition of WNK1 using chemical synthesis and X-ray crystallography. Analogues that probed the role of specific functional groups to the inhibitory activity were synthesized. X-ray structures of trihalo-sulfone 1 and a second trihalo-sulfone 23 bound to WNK1 revealed active site binding to two of the three previously defined canonical inhibitor binding pockets as well as a novel binding site for the trihalo-sulfone moiety. The elucidation of these novel interaction sites may allow for the strategic design of even more selective and potent WNK inhibitors., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Objective In this study, we aimed to explore the association of demographic characteristics and comorbidities with the risk of venous thromboembolism (VTE) in cancer inpatients, as well as to delineate the mortality risk in cancer inpatients with VTE. Methods We conducted a retrospective cohort analysis based on the National Inpatient Sample (NIS) 2012-2014, involving 339,395 inpatients with a primary diagnosis of cancer subdivided into cohorts without VTE (n=331,695) and with VTE (n=7,700). We used a binomial logistic regression model to evaluate the odds ratio (OR) of demographics, comorbidities, and in-hospital mortality rate with respect to cancer inpatients with VTE. Results A higher proportion of cancer inpatients with VTE were 36-50 years in age (83.1%), male (50%), and of black (19.3%) and Hispanic ethnicity (17.2%) compared to the non-VTE cohort. The prevalence of comorbidities was higher in the VTE cohort, including HIV/AIDS, congestive heart failure (CHF), chronic pulmonary disease, diabetes, hypertension, and obesity. CHF demonstrated the highest risk of association with VTE (OR: 2.68, 95% CI: 2.30-3.12), followed by hypertension (OR: 1.23, 95% CI: 1.16-1.29), diabetes (OR: 1.16, 95% CI: 1.07-1.26), and chronic pulmonary disease (OR: 1.13, 95% CI: 1.05-1.22). Conversely, valvular diseases, obesity, and drug abuse were not significantly associated with VTE in cancer inpatients. The in-hospital mortality rate was higher in cancer inpatients with VTE (12% vs. 2.1%), thereby increasing the in-hospital mortality risk (OR: 3.87, 95% CI: 3.58-4.18). Conclusion VTE risk was significantly higher in cancer patients with comorbid CHF, hypertension, diabetes, and chronic pulmonary disease. The risk of all-cause in-hospital mortality was increased by four times in cancer inpatients with VTE., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Rijal et al.)

Nicolaides-Baraitser's syndrome is a rare, dominantly inherited well-delineated syndrome caused by mutations in the SMARCA2 gene which is located on the small arm of chromosome 9. In this study, a de novo missense variant, which was identified in a 3-year-old boy by whole exome sequencing is reported. The de novo heterozygous V1198M missense variant in SMARCA2 gene in exon 25 is novel. Identifying the condition is crucial for the long-term management and family counseling. Follow-up over 4 years revealed improvements in overall performance., Competing Interests: Conflict of Interest None declared., (Thieme. All rights reserved.)

With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.

With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low- or high-potassium diet. We, therefore, investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of Drosophila WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either ∼13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na + /K + -ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule Drosophila WNK activity. In vitro, potassium increased the melting temperature of Drosophila WNK, WNK1, and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of Drosophila WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, Ste20-related proline/alanine-rich kinase (SPAK). The greatest sensitivity of WNK4 to potassium occurred in the range of 80-180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of Drosophila and mammalian WNK kinases through direct effects of potassium ion on the kinase.

Background With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule. Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux. Results In vitro , autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, Drosophila WNK activity acutely increased. Drosophila WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK , with or without Drosophila Mo25 , did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux. Conclusions Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium., (Copyright © 2018 by the American Society of Nephrology.)

In this paper, we propose a framework to dynamically estimate the probability that a patient is readmitted after he is discharged from the ICU and transferred to a lower level care. We model this probability as a latent state which evolves over time using Dynamical Linear Models (DLM). We use as an input a combination of numerical and text features obtained from the patient Electronic Medical Records (EMRs). We process the text from the EMRs to capture different diseases, symptoms and treatments by means of noun phrases and ontologies. We also capture the global context of each text entry using Statistical Topic Models. We fill out the missing values using a Expectation Maximization based method (EM). Experimental results show that our method outperforms other methods in the literature terms of AUC, sensitivity and specificity. In addition, we show that the combination of different features (numerical and text) increases the prediction performance of the proposed approach.

Crucial information on a patient's physical or mental conditions is provided by mentions of disorders, such as disease, syndrome, injury, and abnormality. Identifying disorder mentions is one of the most significant steps in clinical text analysis. However, there are many surface forms of the same concept documented in clinical notes. Some are even recorded disjointedly, briefly, or intuitively. Such difficulties have challenged the information extraction systems that focus on identifying explicit mentions. In this study, we proposed a hybrid approach to disorder extraction, which leverages supervised machine learning, rule-based annotation, and an unsupervised NLP system. To identify different surface forms, we exploited rich features, especially the semantic, syntactic, and sequential features, for better capturing implicit relationships among words. We evaluated our method on the CLEF 2013 eHealth dataset. The experiments showed that our hybrid approach achieves a 0.776 F-score under strict evaluation standards, outperforming any participating systems in the Challenge.

Patients often search for information on the web about treatments and diseases after they are discharged from the hospital. However, searching for medical information on the web poses challenges due to related terms and synonymy for the same disease and treatment. In this paper, we present a method that combines Statistical Topics Models, Language Models and Natural Language Processing to retrieve healthcare related documents. In addition, we test if the incorporation of terms extracted from the patient's discharge summary improves the retrieval performance. We show that the proposed framework outperformed the winner of the retrieval CLEF eHealth 2013 challenge by 68% in the MAP measure (0:5226 vs 0:3108), and by 13% in NDCG (0:5202 vs 0:3637). Compared with standard language models, we obtain an improvement of 92% in MAP (0:2666) and 45% in NDCG. (0:3637).

The MAP kinase cascades, composed of a MAP3K, a MAP2K, and a MAPK, control switch responses to extracellular stimuli and stress in eukaryotes. The most important feature of these modules is thought to be the two double phosphorylation reactions catalyzed by MAP3Ks and MAP2Ks. We addressed whether the reactions are sequential or random in the p38 MAP kinase module. Mass spectrometry was used to track the phosphorylation of the MAP2K MEK6 by two MAP3Ks, TAO2 and ASK1, and the subsequent phosphorylation of p38α by MEK6/S*T* (where S (Ser) and T (Thr) are the two phosphorylation sites and * denotes phosphorylation). Both double phosphorylation reactions are precisely ordered. MEK6 is phosphorylated first on Thr-211 and then on Ser-207 by both MAP3Ks. This is the first demonstration of a precise reaction order for a MAP2K. p38α is phosphorylated first on Tyr-182 and then on Thr-180, the same reaction order observed previously in ERK2. Thus, intermediates were MEK6/ST* and p38α/TY*. Similarly, the phosphorylation of the p38α transcription factor substrate ATF2 occurs in a precise sequence. Progress curves for the appearance of intermediates were fit to kinetic models. The models confirmed the reaction order, revealed processivity in the phosphorylation of MEK6 by ASK1, and suggested that the order of phosphorylation is dictated by both binding and catalysis rates.

MAPKs engage substrates, MAP2Ks, and phosphatases via a docking groove in the C-terminal domain of the kinase. Prior crystallographic studies on the unphosphorylated MAPKs p38α and ERK2 defined the docking groove and revealed long-range conformational changes affecting the activation loop and active site of the kinase induced by peptide. Solution NMR data presented here for unphosphorylated p38α with a MEK3b-derived peptide (p38α/pepMEK3b) validate these findings. Crystallograhic data from doubly phosphorylated active p38α (p38α/T∗GY∗/pepMEK3b) reveal a structure similar to unphosphorylated p38α/MEK3b, and distinct from phosphorylated p38γ (p38γ/T∗GY∗) and ERK2 (ERK2/T∗EY∗). The structure supports the idea that MAP kinases adopt three distinct conformations: unphosphorylated, phosphorylated, and a docking peptide-induced form., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A synthetic approach to palau'amine is described that exploits veiled symmetry in the structure. Bis-alkylidenes i have been prepared and found susceptible to halogenative desymmetrization using t-BuOCl. This oxidation forms the imbedded spirocyclopentane motif observed in the natural product. A host of atypical reactions and processes developed during these studies are discussed, as are plans for completing total syntheses of this compound class.

Motivation: With the increasing volume of scientific papers and heterogeneous nomenclature in the biomedical literature, it is apparent that an improvement over standard pattern matching available in existing search engines is required. Cognition Search Information Retrieval (CSIR) is a natural language processing (NLP) technology that possesses a large dictionary (lexicon) and large semantic databases, such that search can be based on meaning. Encoded synonymy, ontological relationships, phrases, and seeds for word sense disambiguation offer significant improvement over pattern matching. Thus, the CSIR has the right architecture to form the basis for a scientific search engine., Result: Here we have augmented CSIR to improve access to the MEDLINE database of scientific abstracts. New biochemical, molecular biological and medical language and acronyms were introduced from curated web-based sources. The resulting system was used to interpret MEDLINE abstracts. Meaning-based search of MEDLINE abstracts yields high precision (estimated at >90%), and high recall (estimated at >90%), where synonym, ontology, phrases and sense seeds have been encoded. The present implementation can be found at http://MEDLINE.cognition.com., Contact: Elizabeth.goldsmith@UTsouthwestern.edu Kathleen.dahlgren@cognition.com.

MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/DeltaN/DD) has been solved at 2.3 angstroms resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The enzyme adopts an inactive conformation, based upon structural queues, despite the phosphomimetic mutations. Gel filtration and small-angle X-ray scattering analysis confirm that the crystallographically observed ellipsoidal dimer is a feature of MEK6/DeltaN/DD and full-length unphosphorylated wild-type MEK6 in solution. The interface includes the phosphate binding ribbon of each subunit, part of the activation loop, and a rare "arginine stack" between symmetry-related arginine residues in the N-terminal lobe. The autoinhibited structure likely confers specificity on active MAP2Ks. The dimer may also serve the function in unphosphorylated MEK6 of preventing activation loop phosphorylation by inappropriate kinases.

Previous studies have revealed that transforming growth factor-beta-activated protein kinase 1 (TAB1) interacts with p38alpha and induces p38alpha autophosphorylation. Here, we examine the sequence requirements in TAB1 and p38alpha that drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38alpha. Furthermore, a cryptic D-domain-like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in the phi(B)+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38alpha is involved in this interaction, whereas the CD domain and ED domain are not. Furthermore, chimeric analysis with p38beta (which does not bind to TAB1) revealed a previously unidentified locus of p38alpha comprising Thr218 and Ile275 that is essential for specific binding of p38alpha to TAB1. Converting either of these residues to the corresponding amino acid of p38beta abolishes p38alpha interaction with TAB1. These p38alpha mutants still can be fully activated by p38alpha upstream activating kinase mitogen-activated protein kinase kinase 6, but their basal activity and activation in response to some extracellular stimuli are reduced. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38alpha substrates and activators. This suggests that TAB1-induced autophosphorylation of p38alpha results from conformational changes that are similar but unique to those seen in p38alpha interactions with its substrates and activating kinases.

The structures of the MAP kinase p38 in complex with docking site peptides containing a phi(A)-X-phi(B) motif, derived from substrate MEF2A and activating enzyme MKK3b, have been solved. The peptides bind to the same site in the C-terminal domain of the kinase, which is both outside the active site and distinct from the "CD" domain previously implicated in docking site interactions. Mutational analysis on the interaction of p38 with the docking sites supports the crystallographic models and has uncovered two novel residues on the docking groove that are critical for binding. The two peptides induce similar large conformational changes local to the peptide binding groove. The peptides also induce unexpected and different conformational changes in the active site, as well as structural disorder in the phosphorylation lip.