bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulome‐Seq: Searching for Single Nucleotide Variants (SNVs) Associated with Disease Beyond Protein‐Coding Regions
Authors: Pinsach‐Abuin Mel·lina , Mates Jesus , del Olmo Bernat , Allegue Catarina , Brugada Ramon , Garcia‐Bassets Ivan , Pagans Sara 
Published: November 2023

Heart disease is a leading cause of morbidity and mortality in children and adults and it largely depends on genetic factors. Genome‐wide association studies suggest that single nucleotide variants (SNVs) in cis‐regulatory regions increase the susceptibility to cardiac disease. Here we describe the development of an efficient strategy for selective capturing and sequencing of cis‐regulatory regions from human patients (Regulome‐seq) and the application of this strategy for the comprehensive evaluation of cis‐regulatory regions in patients clinically diagnosed with Brugada Syndrome (BrS). BrS is an electrical disease in the heart with high susceptibility to ventricular arrhythmia and sudden cardiac death (SCD). The main genes involved in BrS pathogenesis are SCN5A (which encodes for the cardiac sodium channel and represents 20% of the cases), SCN2B, SCN3B, CACNA1C, CACNA2D1 and CACNB2. SNVs in exonic regions of BrS‐associated genes account for 25–30% of BrS cases. We hypothesize that SNVs at cis‐regulatory regions of BrS‐associated genes could be the cause of a number of BrS cases that currently cannot be explained by exonic SNVs (orphan BrS cases). We identified approximately 1,500 cis‐regulatory regions potentially relevant to BrS by combining information of topological organization in the human genome, chromatin accessibility, histone marks, and binding of transcriptional regulators over six BrS‐associated genes in cardiac cells. Then, we prepared DNA libraries, enriched with the 1,500 cis‐regulatory regions, from genomic DNA of 72 BrS orphan cases using Nextera Rapid Capture (NRC). DNA libraries were sequenced on a HiSeq 2500. After computational integration of the sequencing data, we identified a total of 114,937 variants (>2,500 variants per patient). From those variants identified, 79% represent single nucleotide polymorphisms (SNPs), while 20% represent insertions and deletions (INDELs). The highest percentage (46.15%) of these variants is located within the topological domains of beta subunit genes (SCN2B and SCN3B). Beta subunits are known to modulate the cardiac sodium channel function. In summary, our Regulome‐seq approach gives the possibility to analyze a massive number of human samples at the same time because it simplifies and reduces the cost of the analysis of genetic variants and it could be used for the future diagnosis of patients. Regulome‐seq could potentially provide novel therapeutic targets for pharmacological intervention in the treatment of genetic diseases.Support or Funding InformationPredoctoral fellowship, Generalitat de Catalunya, Spain (MP).Marie‐Curie International Reintegration Grant, IRG07‐GA‐2010‐268395 (SP).
Oxime Reactivation Mechanisms for Organophosphate Conjugated Human Acetylcholinesterase
Authors: Hou William C. , Radic Zoran , Sharpless K. Barry , Taylor Palmer 
Published: November 2023

Organophosphates (OP) interacting with acetylcholinesterase (AChE) carry narrowly prescribed therapeutic benefits and are effective, biodegradable pesticides when judiciously used. However, certain volatile OP's have been employed insidiously in terrorism. To develop efficacious OP antidotes and scavenging agents, we have adopted a new chemistry with zwitterionic oximes in order for the antidote to cross the blood‐brain barrier and enable reactivation of AChE in the central nervous system. Such compounds also exhibit appropriate oral bioavailability, eliminating parenteral administration in field situations. Studies of oxime catalysis of low molecular weight OP esters show that the oximate is the general base, nucleophile in solution. To investigate whether a similar mechanism occurs in the active center of AChE, we examined the pH dependency of oxime reactivation on the OP conjugated human AChE. Quaternary pyridinium aldoximes, such as pralidoxime (2‐PAM), and a series of zwitterionic oxime species, such as RS 41A and RS194B, were compared at various oxime concentrations to deconstruct the overall kinetic constant for reactivation, kov, into an apparent dissociation constant, Kox and a maximal rate, k2, of reactivation. General base catalysis predicts that, since the anionic oximate is the attacking species, rate constants would be expected to increase with pH, but oximolysis of the conjugated OP in the human AChE shows a broad pH dependence with a peak reactivation rate constant at pH values around 7.5 at both partial and full saturation of the active center. These findings suggest the possibility of transient electric fields and hydrogen bonding allow for proton tunneling of the oxime hydrogen for oximate formation in the active center gorge of AChE.Support or Funding InformationSupported by USPHS grants GM 18360 and NS 058046.
Directly binding to HNE and up‐regulating α1‐AT mediate the anti‐migration effects of EGCG on neutrophil elastase‐induced migration in A549 cells
Authors: LI Xuejun , Xiaokaiti Yilixiati 
Published: November 2023

EGCG has been extensively investigated as a multi‐targeting anti‐tumor and anti‐inflammatory compound. Lung cancer is a complex process that occurs in unregulated inflammatory environment. In this study, we demonstrated a novel mechanism by which EGCG inhibits the neutrophil elastase‐induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1‐antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1‐antitrypsin was up‐regulated after EGCG treatment in neutrophil elastase treated A549 cells. We preliminarily discovered that the EGCG‐mediated induction of α1‐antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase‐induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1‐antitrypsin by regulating the PI3K/AKT pathway.Support or Funding InformationThis work was supported by grants from the National Natural Science Foundation of China (No. 91129727, 81473235 and 81020108031) and the Research Fund from the Ministry of Education of China (111 Project, No. B07001).
Salubrinal inhibits glioma growth and sensitizes malignant gliomas to ER stress‐ and Temozolomide‐induced death
Authors: Li Zhaozhong , Bezalel Spencer , Chen Jin , Belal Cherine , Al'Khafaji Aziz , Ameli Neema J , Baharani Akanksha , Chigurupati Srinivasulu , Mughal Mohamed , Wersto Robert R , Cheng Zixi Jack , Chan Sic L 
Published: November 2023

Glioblastoma multiforme represents a particularly difficult‐to‐treat type of cancer with a dismal prognosis. Recent studies showed that human malignant gliomas adapt to their local cellular environments by triggering the endoplasmic reticulum (ER) stress response. This response not only activates critical adaptive pathways for cell survival but also switches to apoptosis when ER stress signaling is prolonged or excessive. Therefore, approaches that either down‐regulate the protective ER stress response or enhance ER stress signaling in malignant gliomas may significantly improve treatment outcome. Here we report that Salubrinal (Sal), an inhibitor of phospho‐eukaryotic translation initiation factor (p‐eIF2α) dephosphorylation, impedes glioma proliferation and invasiveness. The anti‐tumorigenic properties of Sal are associated with impaired receptor‐mediated calcium signaling resulting from the down‐regulation of the ER‐resident calcium release channel protein, inositol triphosphate receptor (IP3R). We also found that Sal elevates ER stress signaling and sensitizes gliomas to apoptosis in the presence of ER stress‐inducing agents. Furthermore, Sal sensitizes gliomas to apoptosis induced by Temozolomide (TMZ), a methylating drug widely used to treat malignant gliomas. In contrast, Sal protected primary astrocytes and neurons against ER stressors suggesting that this differential cell type‐specific response to Sal could be harnessed to selectively sensitize malignant gliomas. Collectively, our findings indicate that Sal should be evaluated alone or in combination, as a safe and effective anti‐cancer therapy.Support or Funding InformationFlorida Center for Brain Tumor Research to Sic L Chan and Zixi (Jack) Cheng
AXL Knockdown For Therapy of Uterine Cancer
Authors: Palisoul Marguerite , Nguyen Mai , Pan Hua , Lohrey Anne , Desai Riva , Powell Matthew , Mutch David , Wickline Samuel , Fuh Katherine 
Published: November 2023

ObjectiveTo validate that siAXL‐p5RHH can be a potential therapeutic inhibitor in uterine cancer.MethodsA novel serum‐stable, cell‐penetrating, endosomolytic amphipathic peptide delivery system (p5RHH) was used for genetic knock‐down of a receptor tyrosine kinase, AXL. p5RHH peptide was dissolved in 10mM in DNase‐, RNAse‐, and protease‐free sterile purified water. The p5RHH siRNA nanoparticles were prepared by mixing equal volumes of the p5RHH peptide and siRNA at a peptide/siRNA ratio of 1:20 in HBSS with Ca2+ and Mg2+. This preparation results in a nanoparticle size of 55nm after applying a stabilizing albumin coating. Total RNA and total cell lysates were recovered from p5RHH‐siAXL nanoparticle treated uterine cancer tumor cells to evaluate for RNA and protein expression knockdown. AXL knockdown by siRNA was compared with either a traditional transfection reagent (DharmaFECT) or with the novel serum‐stable, cell‐penetrating, endosomolytic amphipathic peptide delivery system (p5RHH). Invasion assays were performed using Boyden chambers coated with Matrigel, and migration assays were performed using Boyden chambers.ResultsWestern blotting confirmed over‐expression of AXL in uterine cancer cell lines. The novel delivery system, p5RHH, was loaded with siAXL, and shown to inactivate AXL in ARK1 uterine cancer cells at the RNA and protein level to a similar extent as traditional transfection technique (8.4‐fold vs 8.7‐fold decrease for RNA, respectively; 67% vs 83% knockdown at 48h and 84% vs 97% knockdown at 96 hours for protein, respectively). Uterine cancer cells with genetic inactivation of AXL (through shRNA to AXL) showed less invasive cells than those with AXL (shRNA to scrambled Control) of 32 vs 117 cells/high power field (hpf), p=0.0001 as well as less migratory cells (10 vs 35 cells/hpf, p<0.001).ConclusionsAXL expression is associated with metastatic uterine cancer cell lines. AXL plays a role in uterine cancer invasion and migration. p5RHH‐siAXL is able to inactivate AXL and can be a potential therapeutic inhibitor in uterine cancer.
Organophosphorus Pesticides Promote Cytokine Production in Human Macrophages
Authors: Grodzki Cristina , Fryer Allison , Lein Pamela 
Published: November 2023

Macrophages are sentinel immune cells that reside in most if not all tissues and play a critical role in innate immunity. We previously established that exposure to organophosphorus pesticides (OPs) increases airway hyperreactivity in animal models. OPs are neurotoxicants that cause acute cholinergic toxicity via inhibition of the enzyme acetylcholinesterase (AChE); however, our studies demonstrated that the OP parathion causes airway hyperreactivity independent of inhibition of AChE activity. Rather, parathion‐induced airway hyperreactivity is mediated by dysfunction of autoinhibitory muscarinic M2 receptors expressed on the parasympathetic nerves that innervate airway smooth muscle. Recently, we reported that the airway hyperreactivity triggered by parathion requires macrophages and immune cytokines. Here, we test the hypothesis that OPs interact directly with macrophages to stimulate them to express cytokines that modulate neuronal M2 receptors. Using the human THP‐1 monocyte line, we evaluated cytokine expression, NFkB activation through a secreted embryonic alkaline phosphatase (SEAP) reporter expression and nuclear localization of the NFkB p65 subunit and intracellular calcium mobilization in PMA‐differentiated THP‐1 cells exposed to different OPs and their metabolites. Preliminary experiments indicate that in differentiated THP‐1 macrophages, OPs significantly increase the mRNA expression of the pro‐inflammatory cytokines TNFα and IL‐1β. OPs also significantly increased NFkB activation but did not increase intracellular calcium levels. Therefore, our in vitro data strongly indicate that OPs interact directly with macrophages to provoke release of pro‐inflammatory mediators via activation of the NFkB signaling pathway. Ongoing studies are exploring the action of carbamates and pyrethrins on macrophages and further evaluating the mechanism by which these pesticides stimulate macrophages.Support or Funding InformationThis work was supported by funding from the National Institutes of Health grant R01 ES017592.
Refining the Half‐Site Reaction Mechanism of SULT1B1 Using Engineered Heterodimers
Authors: Tibbs Zachary Evan , Falany Charles N 
Published: November 2023

The cytosolic sulfotransferases (SULTs) biotransform drugs and hormones into sulfate esters, aiding in the deactivation and/or excretion of the compounds from the cell and body. Each human SULT isoform is a homodimer interfacing along a conserved motif with the consensus sequence KxxxTVxxxE. Despite conservation of the dimerization domain across all fourteen human SULT isoforms, its contribution to SULT activity is not fully understood. Based on results from recent molecular dynamic simulations (MDS) of dimeric SULT1B1 in the presence and absence of the cofactor, 3′‐phosphoadenosine 5′phosphosulfate (PAPS), our laboratory has developed a mechanistic model for SULT dimer half‐site reactivity. In this proposed reaction mechanism, the binding of PAP(S) to the enzyme surface elicits structural changes which are communicated to the partnering subunit through the dimerization domain. The result of this communication is a dimer with a single active subunit at any given time. The dimer subunits oscillate between active and inactive states, “taking turns” as the active subunit. The advantage of this oscillating mechanism is thought to be an overall increased reaction rate, primarily propelled by a more favorable koff for the reaction byproduct, 3′ 5′‐diphosphoadenosine (PAP). As a means for testing the oscillating activity hypothesis in vitro, we sought to disrupt the activity of one subunit of the dimer while leaving the activity of the neighboring subunit fully in tact. Comparison of the heterodimer's kinetic parameters to those of the wildtype isoform would discern the involvement of the partnering subunit in the overall reaction mechanism. Two key amino acid residues were mutated to disrupt the activity of SULT1B1. Mutation of Arg258 to a Lys rendered the SULT deficient in its ability to bind PAPS (Kd ≈ 200 uM), whereas mutation of the catalytic base, His109, to a Tyr rendered the SULT incapable of catalyzing sulfonate transfer while leaving substrate binding unaffected. Unfortunately, the favorable kon for SULT dimerization complicated isolation of heterodimeric SULT species (only one subunit with the desired mutation). To overcome this limitation, we engineered SULT1B1 isoforms capable of selective dimerization, propelled by amino acid charge repulsion/attraction properties. This heterodimer‐generating system allowed us to manipulate each individual subunit independently prior to dimerization. Therefore, we combined the heterodimer‐generating system with the activity‐altering mutations (R258K and H109Y) to isolate SULT dimers with one fully active subunit. The resulting heterodimeric SULT isoforms are currently the subjects of in vitro sulfation activity analyses.Support or Funding InformationNIH Grants: GM113980 and ES022606
Effects of Cimaglermin alfa Administration on LV Function in Rats with Chronic Heart Failure Induced by LAD Occlusion
Authors: Ganguly Anindita , Zolty Ronald , Troy Erika , Iaci Jennifer , Hackett Craig , Caggiano Anthony , Parry Tom J 
Published: November 2023

IntroductionNeuregulin growth factors are essential for the normal development and function of the heart. Neuregulins have been found to protect and restore left ventricular (LV) function in a variety of species with heart failure (HF) induced by myocardial infarction (MI). No report so far has shown the effect of neuregulin administration on LV function when treatment is delayed until 20‐weeks post‐MI in rats.AimThe goal of this study was to examine the effects of delayed treatment with the neuregulin, cimaglermin, on LV function (echocardiographic) and characterize the effects of withdrawal and re‐initiation of treatment in rats with chronic HF induced by LAD ligation.MethodsLAD ligation was performed in (n=10–12/group) 8–10 week old male Sprague Dawley rats. At 20 weeks post‐ligation, rats were randomly assigned to vehicle or cimaglermin (0.5 and 2.6 mg/kg IV or 2.6 and 10 mg/kg SC) treatment twice weekly for 1 month. This was followed by a 1 month treatment‐free period and a re‐treatment period similar to the first. Echocardiographic parameters were assessed at least once weekly throughout the study.ResultsEjection fraction (%EF) and end systolic volume (ESV) improved with intravenous and subcutaneous cimaglermin treatment. Both %EF and ESV reverted to vehicle‐treated levels during a 4 week washout of cimaglermin with both routes of administration. At an intravenous dose of 0.5 mg/kg, the changes in %EF and ESV were significantly different from vehicle control during the re‐treatment period. At 2.6 mg/kg IV, the %EF (see graph) and ESV improved significantly during both treatment periods (p<0.05). For subcutaneous dosing, %EF improved significantly vs vehicle treatment during the initial and re‐treatment periods (p<0.05) at both SC doses while ESV trended toward improvement compared to vehicle controls at the high SC dose.ConclusionsCimaglermin treatment initiated at 20 weeks post‐MI produced significant improvement in LV function in rats. Cimaglermin re‐treatment following a 4 week withdrawal period produced LV functional improvements similar to those seen with initial treatment.
Pharmacological Perturbations of Eicosanoids in vitro and ex vivo in a Novel, Broad‐spectrum, Plasma Lipidomics Assay
Authors: Mazaleuskaya Liudmila L , Ricciotti Emanuela , Lawson John A , Li Xuanwen , Grant Gregory , Grosser Tilo , FitzGerald Garret A 
Published: November 2023

Eicosanoids are a family of bioactive lipids formed from arachidonic acid and include prostaglandins, leukotrienes and related compounds. Nonsteroidal anti‐inflammatory drugs (NSAIDs) suppress prostaglandins and are widely used for anti‐pyretic, analgesic and anti‐inflammatory effects. NSAIDs selective for inhibition of cyclooxygenase (COX)‐2 have fewer gastro‐intestinal side effects, but predispose patients to increased cardiovascular risk. The cardiovascular complications prompted interest in microsomal prostaglandin synthase 1 (mPGES‐1), downstream of COX‐2, as a novel drug target. Unlike COX‐2, global deletion of mPGES‐1 in mice suppresses prostaglandin E2 (PGE2) and augments prostacyclin (PGI2) through substrate rediversion, and does not predispose to thrombogenesis and hypertension. However, cell‐specific deletions of mPGES‐1 reveal that the predominant prostaglandin product of substrate rediversion varies by cell type. We hypothesized that inhibition of mPGES‐1 leads to changes in bioactive lipids beyond the prostaglandin pathway and aimed 1) to develop a mass spectrometry‐based, broad‐spectrum lipidomics approach to measure arachidonate‐derived products additional to prostanoids and 2) to develop a human whole blood assay (hWBA) to study substrate rediversion upon mPGES‐1 vs COX‐2 inhibition. We have developed a novel, reproducible, analytical method to scan the impact of drugs on the bioactive lipidome in human whole blood in vitro and ex vivo. We observed substrate rediversion in both COX and LOX (lipoxygenase) pathways after mPGES‐1 inhibition, which was distinct from inhibiting COX‐1/2. We validated our in vitro assay by its application ex vivo after administration of a COX‐2 inhibitor to human subjects. Overall, we have shown that blockade, alone and in combination, of diverse targets in the eicosanoid synthesis/response cascade results in both expected and unanticipated signals in the circulating human lipidome. Such novel aspects of drug action prompt reconsideration of how drugs in these pathways might be utilized in the alleviation of disease.Support or Funding InformationThis study was supported by The Personalized NSAID Therapeutics Consortium (PENTACON: HL117798) and by T32 H107971.
Angiotensin II Induces Prostaglandin E2 Production and Oxidative Stress in the Renal Cortex
Authors: Alanazi Wael Abdullah , Fakhruddin Selim , Jackson Keith 
Published: November 2023

Angiotensin II (AngII) is a systemic vasoconstrictor and body fluid regulator. Virtually all released AngII binds to AT1 receptors, which stimulates cell signaling pathways in various cell types. In kidneys, AngII enhances arachidonic acid release by increasing the activity of phospholipases enzymes and cyclooxygenase‐2 (COX‐2), leading to an elevation of PGs predominantly prostaglandin E2 (PGE2). Thus, the current study hypothesizes that acute AngII infusion induces the local production of PGE2 and oxidative stress in the renal cortex, which results in renal damage. Male Sprague Dawley rats (200 – 300g) were anesthetized and microdialysis probes were placed in the renal cortex. After a stabilization period, a 4 hour control period was followed by vehicle or AngII (7ng/kg/min, IV) treatment for 4 hours. Blood pressure was elevated significantly during AngII infusion from (109.8 ± 0.085mmHg) to (129.36 ± 0.1mmHg). Urine flow, urinary Na+/K+ excretion, glomerular filtration rate, and renal blood flow were decreased in comparison with control. Through renal microdialysis, we found that PGE2 levels were significantly induced by AngII treatment when compared to control. In addition, acute AngII infusion augmented the present oxidative stress in the renal tissues. Superoxide and peroxynitrite levels were evaluated by EPR using CPH and CMH as spin traps. In the current study, we found that local effects of acute AngII infusion stimulate PGE2 and free radical release producing a disruption in normal renal hemodynamic function. In summary, the current study demonstrates a potential mechanism by which acute elevations in AngII promotes hypertension induced end organ kidney damage.Support or Funding InformationSaudi Arabian Cultural Mission (SACM), INBRE