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We analyzed the P2X4 receptor structure-activity relationship of a known antagonist

Background: Regulation of vascular permeability is critical to maintaining tissue metabolic homeostasis. VEGF (vascular endothelial growth factor) is a key stimulus of vascular permeability in acute and chronic diseases including ischemia reperfusion injury, sepsis, and cancer. Identification of novel regulators of vascular permeability would allow for the development of effective targeted therapeutics for patients with unmet medical need. Methods: In vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and phosphatidylinositol 4,5-bisphosphate levels were evaluated with and without modulation of PLC (phospholipase C) β2. Results: Global knock-out of PLCβ2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and transendothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knockdown of PLCβ2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCβ2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of phosphatidylinositol 4,5-bisphosphate compared to control cells. Finally, loss of PLCβ2 in both a hyperoxia-induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared with wild-type controls. Conclusions: The results implicate PLCβ2 as a key positive regulator of VEGF-induced vascular permeability through regulation of both calcium flux and phosphatidylinositol 4,5-bisphosphate levels at the cellular level. Targeting of PLCβ2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.

Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-seq technology to identify the gene expression profiles, pathway analysis, and qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 KO and littermate WT mice to ischemic stroke. After 3 days, mice were sacrificed, total RNA was isolated using Trizol, and subjected to RNA-seq and Nanostring-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify mRNA transcript expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs WT tissue after stroke. Out of these DEGs, 1920 gene were downregulated, and 325 genes were upregulated in KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 (IL-6), an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha (Ctla2a), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.

Background and Purpose: Identification of a new potential drug target protein and their plausible mechanism for stroke treatment is critically needed. We earlier showed that genetic deletion as well as short term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides acute neuroprotection and thus can be potential drug targets to treat ischemic stroke. However, potential mechanisms remain unknown. Therefore, in this study, we employed RNA-seq technology to identify the gene expression profiles, pathways analysis and qPCR validation of differentially expressed genes (DEGs). This analysis will identify role of those DEGs in certain biological processes responsible for P2X4R dependent neuroprotection after stroke. Methods: We subjected young (8-12 weeks-old n= 4/group), and aged (12-18-month-old; n=8/group) male and female Global P2X4 KO and littermate WT mice to right middle cerebral artery occlusion MCAo for 60 min followed by 3 day of reperfusion. After 3 days, mice were sacrificed and prefrontal cortex tissue was isolated to extract total RNA using Trizol and used for RNA-seq sample preparations as well as for validation by Nanostring mediated qPCR technique. DESeq2 and Gene Ontology (GO) and /or Ingenuity Pathway Analysis (IPA) were used to identify mRNA transcript expression profiles and biological pathway. qPCR was analyzed with nSolver Data Analysis Support system. Results: We found 2246 DEGs in P2X4R KO vs WT tissue after stroke. Out of these DEGs 1920 gene were downregulated and 325 genes were upregulated in KO. GO/IPA analysis of top 300 DEGs suggests an enrichment of ion channel transport system, inflammation and extracellular matrix component genes. QPCR validation of top 30 DEGs revealed down regulation of two common age independent genes: Lnterleukin-6 ( IL-6) , an inflammatory cytokine and Cytotoxic T Lymphocyte-Associated Protein 2 alpha ( Ctla2a ), an immunosuppressive factor KO group. Conclusion: This data suggests P2X4R mediated neuroprotection after stroke is brought by attenuation of immune modulatory pathways in both young and aged mice of both sexes. Future studies will delineate the detailed role of IL-6 and Ctla2a in P2X4R mediated neuroprotection mechanisms after stroke.

Background and Purpose: Identification of a novel treatment for ischemic stroke is urgently needed. We previously showed that genetic deletion as well as short term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides acute neuroprotection and thus can be potential drug targets to treat ischemic stroke. Here we synthesized and pharmacologically validated those experimental drugs for the treatment of ischemic stroke. Methods: We first performed the P2X4 receptor structure activity (SAR) relationship based on known heterocyclic antagonist P2X4 antagonists including NP-1815-PX Following extensive modification of the reported synthetic route, we synthesized several analogues that maintained or enhanced antagonist affinity. We used [Ca 2+ ] i influx assay in P2X4R overexpressing human HEK cells to identify potent and specific P2X4R antagonist. Two most potent leads (MRS4719 and 4596 given subcutaneously for 3 days post stroke) were then tested for their neuroprotective activity using middle cerebral artery occlusion (MCAo) model of ischemic stroke (60 min MCAo followed by 3 days of reperfusion) using both sexes of young and aged mice. MRS4719 was also tested in human primary monocyte derived macrophage obtained from the blood of de-identified aged healthy subjects (55±10 years). Results: Analogues MRS 4719 and MRS4596 were most potent at human (h) P2X4R (IC 50 0.503 and 1.38 μM, respectively), and were highly selective versus hP2X1R, hP2X2/3R, hP2X3R). Both MRS 4719 and MRS 4596 showed neuroprotective effects after 3 days of ischemic stroke in a dose dependent manner in young mice (8-12 weeks old). Optimal dose of MRS4719 (1.5 mg/kg/day) also significantly reduced infarct volume (59.6±5.3 vs. 27.47±10.23 mm 3 ; p2+ ] i influx in primary human monocyte-derived macrophages, consistent with its blocking effect at endogenously expressed P2X4R. Conclusions: This study indicates MRS4719 is a potent P2X4R antagonist and has translational potential for the treating of ischemic stroke across the age.

Cardiovascular disease (CVD) is a leading cause of premature mortality in the US and the world. CVD comprises of several complex and mostly heritable conditions, which range from myocardial infarction to congenital heart disease. Here, we report our findings from an integrative analysis of gene expression, disease-causing gene variants, and associated phenotypes among CVD populations, with a focus on high-risk Heart Failure (HF) patients. We built a cohort using electronic health records (EHR) of consented patients with available samples, and then performed high-throughput whole-genome and RNA sequencing (RNA-seq) of key genes responsible for HF and other CVD pathologies. We also incorporated a translational aspect to our study by integrating genomics findings with patient medical records. This involved linking ICD-10 codes with our gene expression and variant data to identify associations with HF and other CVDs. Our in-depth gene expression analysis revealed differentially expressed genes associated with HF (41 genes) and other CVDs (23 genes). Furthermore, a variant analysis of whole-genome sequence data of CVD patients identified genes with altered gene expression (FLNA, CST3, LGALS3, and HBA1) with functional and nonfunctional mutations in these genes. Our study highlights the importance of an integrative approach that leverages gene expression, genetic mutations, and clinical data that will allow the prioritization of key driver genes for complex diseases to improve personalized healthcare.

Background: Fatigue is one of the most common acute complications of radiation therapy (RT). It can have a serious impact on a patient’s quality of life and ability to engage in treatment, and has been associated with lower recurrence-free and overall survival in patients with breast cancer. The pathogenesis of radiation-induced fatigue remains elusive, and factors implicated include biological (low hemoglobin and elevated pro-inflammatory cytokines and chemokines) and psychological (pain, depression and anxiety). Although it is a common clinical issue, optimal treatment strategies are lacking, and a better understanding of the mechanisms involved is needed to devise effective interventions. The purpose of this exploratory, non-randomized, prospective study was to examine a number of possible biological and psychological factors influencing fatigue in patients undergoing breast irradiation for early stage breast cancer. Methods: All subjects were assessed at five time points: immediately before RT, mid-point of RT, end of RT, and 6 months and 1 year after completion of RT. Clinical evaluation of skin toxicity and cosmetic outcome and laboratory measures evaluating anemia and hepatic toxicity were performed. Laboratory markers of systemic inflammatory/stress response included salivary cortisol, CRP and cytokines. Caspase-1 and caspase-3, novel markers of apoptosis, were also collected. Fatigue, distress, depression, anxiety, sleep, energy level and pain were assessed at each time point using validated measures. A two-sample student t-test or a non-parametric Wilcoxon rank sum test was used to identify significant associations between fatigued and non-fatigued subjects at each time point. Results: Fifty-three subjects completed the study. Subjects were predominantly white and non-Hispanic, middle to upper-middle class, with a mean age of 59. Fatigued subjects were more likely than non-fatigued subjects to have a history of anxiety and/or depression. Across all subjects, fatigue increased during treatment and returned to or near baseline by 6 months after treatment. Fatigue was significantly associated with overall distress, energy level, and some measures of physical and functional well-being during treatment; these associations were not present at baseline and resolved by 6 months. Fatigued subjects were more likely to have increases in depression scores by the end of treatment, although mean depression scores did not reach clinical significance. Fatigue was significantly associated with breast-specific pain by the end of treatment, and this resolved by 6 months. Fatigue was significantly associated with some measures of sleep at baseline, and this persisted during treatment and resolved by 6 months to 1 year. Fatigue was not associated with measures of skin toxicity and cosmetic outcome and laboratory measures of anemia, hepatic toxicity, salivary cortisol, caspase-1, caspase-3, and IL-10; most other cytokines were undetectable. Conclusions: This was a comprehensive, longitudinal study evaluating the association of biological and psychological factors with the development of fatigue in patients undergoing breast irradiation. As expected, in patients experiencing heightened fatigue during breast irradiation, fatigue was greatest at the end of treatment and returned to or near baseline by 6 months after treatment. Important associations with the development of fatigue included physical and functional parameters, and of particular significance were patients with a prior history of mental health diagnoses. In contrast to previous studies, we did not find an increase in pro-inflammatory or in novel biomarkers including caspase-1 and caspase-3. Identification of and interventions directed at those patients at risk could impact positively on the experience associated with fatigue and radiation. Citation Format: Edward Perry, Sudhanshu Mulay, Jayesh Kamath, Robert Dowsett, Rong Wu, Xiaoyan Wang, Matthew Greenwood, Jacob Neuwirth, James Grady, Bruce Liang, Susan Tannenbaum. Factors influencing fatigue in breast cancer patients undergoing breast irradiation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS9-29.

BackgroundRegulation of vascular permeability (VP) is critical to maintaining tissue metabolic homeostasis. Vascular endothelial growth factor (VEGF) is a key stimulus of VP in acute and chronic diseases including ischemia reperfusion injury, sepsis and cancer. Identification of novel regulators of VP would allow for the development of effective targeted therapeutics for patients with unmet medical need.MethodsIn vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and PIP2 levels were evaluated with and without modulation of PLCβ2.ResultsGlobal knock-out of PLCβ2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and trans-endothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knock-down of PLCβ2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCβ2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of PIP2 compared to control cells. Finally, loss of PLCβ2 in both a hyperoxia induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared to WT controls.ConclusionsThe results implicate PLCβ2 as a key positive regulator of VEGF-induced VP through regulation of both calcium flux and PIP2 levels at the cellular level. Targeting of PLCβ2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.Graphic AbstractHighlightsPLCβ2 promotes VEGFA induced vascular permeability.Loss of PLCβ2 prevents VEGFA vascular permeability via repression of cellular calcium flux and membrane PIP2 levels.Loss of PLCβ2 reduces vascular permeability and improves outcomes in a hyperoxic lung damage model and a cardiac ischemia:reperfusion model in vivo.Targeting PLCβ2 inhibition may lead to a novel therapeutic for diseases such as stroke and myocardial infarction.

Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting β

BackgroundCharacterization of neutralization antibodies to SARS-CoV-2 infection or vaccination in children and young adults with inflammatory bowel disease (IBD) receiving biologic therapies is crucial.MethodsWe performed a prospective longitudinal cohort study evaluating SARS-CoV-2 Spike protein receptor binding domain (S-RBD) IgG positivity along with consistent clinical symptoms in patients with IBD receiving infliximab or vedolizumab. Serum was also obtained following immunization with approved vaccines. IgG antibody to the spike protein binding domain of SARS-CoV-2 was assayed with a fluorescent bead-based immunoassay that takes advantage of the high dynamic range of fluorescent molecules using flow cytometry. A sensitive and high-throughput neutralization assay that incorporates SARS-CoV-2 Spike protein onto a lentivirus and measures pseudoviral entry into ACE2 expressing HEK-293 cells was used.Results436 patients were enrolled (mean age 17 years, range 2-26 years, 58% male, 71% Crohn’s disease, 29% ulcerative colitis, IBD-unspecified). 44 (10%) of enrolled subjects had SARS-CoV-2 S-RBD IgG antibodies. Compared to non-IBD adults (ambulatory) and hospitalized pediatric patients with PCR documented SARS-CoV-2 infection, S-RBD IgG antibody levels were significantly lower in the IBD cohort and by 6 months post infection most patients lacked neutralizing antibody. Following vaccination (n=33) patients had a 15-fold higher S-RBD antibody response in comparison to natural infection, and all developed neutralizing antibodies to both wild type and variant SARS-CoV-2.Conclusions and RelevanceThe lower and less durable SARS-CoV-2 S-RBD IgG response to natural infection in IBD patients receiving biologics puts them at risk of reinfection. The robust response to immunization is likely protective.SummaryOur study showed a low and poorly durable SARS-CoV-2 S-RBD neutralizing IgG response to natural infection in IBD patients receiving biologics potentially putting them at risk of reinfection. However, they also had a robust response to immunization that is likely protective

Background The last decade has seen a dramatic increase in the availability of scientific data, where human-related biological databases have grown not only in count but also in volume, posing unprecedented challenges in data storage, processing, analysis, exchange, and curation. Next generation sequencing (NGS) advancements have facilitated and accelerated the process of identifying genetic variations. Adopting NGS with Whole-Genome and RNA sequencing in a diagnostic context has the potential to improve disease-risk detection in support of precision medicine and drug discovery. Several bioinformatics pipelines have been developed to strengthen variant interpretation by efficiently processing and analyzing sequence data, whereas many published results show how genomics data can be proactively incorporated into medical practices and improve utilization of clinical information. To utilize the wealth of genomics and health, there is a crucial need to generate appropriate gene-disease annotation repositories accessed through modern technology. Results Our focus here is to create a comprehensive database with mobile access to actionable genes and classified diseases, considered the foundation for clinical genomics and precision medicine. We present a publicly available iOS app, PAS-Gen, which invites global users to freely download it on iPhone and iPad devices, quickly adopt its easy to use interface, and search for genes and related diseases. PAS-Gen was developed using Swift, XCODE, and PHP scripting that uses Web and MySQL database servers, which includes over 59,000 protein-coding and non-coding genes, and over 90,000 classified gene-disease associations. PAS-Gen is founded on the clinical and scientific premise that easier healthcare and genomics data sharing will accelerate future medical discoveries. Conclusions We present a cutting-edge gene-disease database with a smart phone application, integrating information on classified diseases and related genes. The PAS-Gen app will assist researchers, medical practitioners, and pharmacists by providing a broad and view of genes that may be implicated in the likelihood of developing certain diseases. This tool with accelerate users’ abilities to understand the genetic basis of human complex diseases and by assimilating genomic and phenotypic data will support future work to identify gene-specific designer drugs, target precise molecular fingerprints for tumors, suggest appropriate drug therapies, predict individual susceptibility to disease, and diagnose and treat rare illnesses.

It’s been over 100 years since the word `gene’ is around and progressively evolving in several scientific directions. Time-to-time technological advancements have heavily revolutionized the field of genomics, especially when it’s about, e.g. triple code development, gene number proposition, genetic mapping, data banks, gene–disease maps, catalogs of human genes and genetic disorders, CRISPR/Cas9, big data and next generation sequencing, etc. In this manuscript, we present the progress of genomics from pea plant genetics to the human genome project and highlight the molecular, technical and computational developments. Studying genome and epigenome led to the fundamentals of development and progression of human diseases, which includes chromosomal, monogenic, multifactorial and mitochondrial diseases. World Health Organization has classified, standardized and maintained all human diseases, when many academic and commercial online systems are sharing information about genes and linking to associated diseases. To efficiently fathom the wealth of this biological data, there is a crucial need to generate appropriate gene annotation repositories and resources. Our focus has been how many gene–disease databases are available worldwide and which sources are authentic, timely updated and recommended for research and clinical purposes. In this manuscript, we have discussed and compared 43 such databases and bioinformatics applications, which enable users to connect, explore and, if possible, download gene–disease data.

Backgrounds: An acute ischemic stroke (AIS) triggers rapid infiltration of circulating immune cells in the brain. P2X4R, a receptor for adenosine triphosphate ATP, regulate activation of circulating monocytes after stroke injury. Over-stimulation of P2X4R contributes to ischemic injury. CD14 ++ CD16 – classical, CD14 ++ CD16 + intermediate, and CD14 + CD16 ++ non-classical monocytes are three primary subsets of monocytes. Alterations in activity of circulating monocyte subsets may independently predict pathogenesis of AIS, however, the role of P2X4R in the activation of these monocyte subsets is not known. Methods: Consecutive AIS patients (60-90 years) undergoing endovascular clot retrieval and healthy control subjects both young (18-45 years) and aged (60-90 years) of both sexes were recruited and informed consent obtained. Flow cytometric analysis of whole blood derived monocytes at 0-2 days (acute, n=10), 3-7 days (subacute, n=9), and 65±20 days (chronic, n=7) after stroke onset were compared with healthy subjects (n=9-10/ age group). Results: Both number of total monocyte counts and P2X4R intensity significantly increase with age when compared between healthy young and aged control (P++ CD16 + intermediate monocytes were significantly reduced after stroke ( p ++ CD16 + intermediate, and CD14 + CD16 ++ non-classical monocytes showed a significant increased median fluorescent intensity (P Conclusions: P2X4R expression increases with age and after stroke. Disappearance of the CD14 + CD16 ++ non-classical monocyte subpopulation from circulation during stroke recovery suggests potential migration of these cells to the site of injury, consistent with their potential role in inflammation/phagocytosis. Increased P2X4R expression in intermediate and non-classical monocytes subpopulation suggest its specific role in selective activation of these monocytes subtype. Detailed molecular characterization of P2X4R response in intermediate and non-classical monocyte subpopulations may provide novel insights into P2X4R’s therapeutic potential during AIS.

Development of antibody protection during SARS-CoV-2 (CoV-2) infection is a pressing question for public health and for vaccine development. We developed highly sensitive CoV-2-specific antibody and neutralization assays. CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n=87) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. CoV-2 neutralization was determined in COVID-19 and convalescent plasma up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which was also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Further, subjects who donated plasma further out from the diagnosis of COVID-19 appeared to have lower titers. Interestingly, some COVID-19 patients also contained NAbs against SARS Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

Stroke remains a leading cause of disability in the United States. Despite recent advances, interventions to reduce damage and enhance recovery after stroke are lacking. P2X4R, a receptor for adenosine triphosphate (ATP), regulates activation of myeloid immune cells (infiltrating monocytes/macrophages and brain-resident microglia) after stroke injury. However, over-stimulation of P2X4Rs due to excessive ATP release from dying or damaged neuronal cells can contribute to ischemic injury. Therefore, we pharmacologically inhibited P2X4R to limit the over-stimulated myeloid cell immune response and improve both acute and chronic stroke recovery. We subjected 8–12-week-old male and female wild type mice to a 60 minute right middle cerebral artery occlusion (MCAo) followed by 3 or 30 days of reperfusion. We performed histological, RNA sequencing, behavioral (sensorimotor, anxiety, and depressive), and biochemical (Evans blue dye extravasation, western blot, quantitative PCR, and flow cytometry) analyses to determine the acute (3 days after MCAo) and chronic (30 days after MCAo) effects of P2X4R antagonist 5-BDBD (1 mg/kg P.O. daily × 3 days post 4 h of MCAo) treatment. 5-BDBD treatment significantly (p

Background Anthracycline induced cardiomyopathy is a major cause of mortality and morbidity among pediatric cancer survivors. It has been postulated that oxidative stress induction and inflammation may play a role in the pathogenesis of this process. Accordingly, the present study performed an assessment of biomarker profiles and functional imaging parameters focused upon potential early determinants of anthracycline induced cardiomyopathy. Methods Patients (10–22 years) were prospectively enrolled between January 2013 and November 2014. Thirteen subjects completed the study and underwent serial cardiac magnetic resonance imaging and plasma biomarker profiling performed 24–48 h after the first anthracycline dose and at set dose intervals. In addition, we collected plasma samples from 62 healthy controls to examine normal plasma biomarker profiles. Results Left ventricular ejection fraction (LVEF) decreased from 64.3 ± 6.2 at the first visit to 57.5 ± 3.3 (p = 0.004) 1 year after chemotherapy. A decline in longitudinal strain magnitude occurred at lower cumulative doses. A differential inflammatory/matrix signature emerged in anthracycline induced cardiomyopathy patients compared to normal including increased interleukin-8 and MMP levels. With longer periods of anthracycline dosing, MMP-7, a marker of macrophage proteolytic activation, increased by 165 ± 54% whereas interleukin-10 an anti-inflammatory marker decreased by 75 ± 13% (both p

To efficiently fathom the wealth of genomics and clinical data, there is a crucial need to generate appropriate gene-disease annotation repositories accessed through modern resources. Our focus was to create a comprehensive database with mobile access to authentic Single Nucleotide Polymorphisms (SNPs) and classified diseases worldwide, considered as the foundation for clinical and genomics research, epidemiology and precision medicine. In this manuscript, we present non-profit, academic and publicly available iOS application, PAS-SNP, which invites global users to freely download it on iPhone & iPad devices, effortlessly adopt it's easy to use interface and search for SNPs, genes and related diseases. PAS-SNP is developed with Swift multi-paradigm programming language, XCODE integrated development environment for MacOS, and PHP scripting that uses a MySQL server database, which includes over 67,000 SNPs reported for over 19,000 genes, from patients located in over 1000 regions, published in over 3000 articles in over 415 journals available at the PubMed, and over 100,000 classified SNP-disease combinations. It includes SNPs released and organized by the Genome-wide Association Study (GWAS). PAS-SNP is founded on the clinical and scientific premise to support and promote healthcare and genomics data sharing with technological advancements.

Today, we stand on the threshold of the new medical revolution, and learning to read the code of life, and this revolution offers big hope to people suffering from all kinds of diseases. The complexity and variability in disease classification is a great challenge to overcome. Disease classification is routinely composed of healthcare units streaming data from multiple sources simultaneously e.g., pathology, genomics, imaging, and electrophysiology etc. To efficiently fathom the wealth of healthcare, there is a crucial need to generate appropriate disease-treatment annotation repositories accessed through modern technologies. One platform that has proven to be an efficient tool in several areas including healthcare, is the multi-purpose mobile computing devices e.g., smart phone and tablet computer. Our focus here was to develop a mobile application with a comprehensive database for efficient management and access to International Classification of Diseases (ICD) and National Drug Code (NDC) for epidemiology, health management, clinical support, and scientific research. Innovative and smart systems are necessary to improve the quality and transition of healthcare by understanding heterogeneous healthcare and related data. In this manuscript, we present PAS-Code, an iOS app developed with Swift and PHP scripting that uses a MySQL server database, which includes over 80,000 ICDs maintained by the World Health Organization and over 123,000 NDCs approved by the US Food and Drug Administration. It enables users worldwide to install the application on Apple mobile devices and search for medical classified codes, disease, drugs, and related information such as, signs and symptoms, abnormal findings, complaints, social circumstances, and external causes of injury. PAS-Code is an exclusively academic application founded on the clinical, scientific, and modern technology premise to support healthcare and enable scientific data retrieval using efficient mobile-based tools. It has the potential to assist clinical researchers by providing enhanced exposure to ICD and NDC with greater visibility and easy browsing. These set of codes are highly significant at every level of healthcare.

Accumulating evidence supports a therapeutic role of purinergic signaling in cardiac diseases. Previously, efficacy of systemically infused MRS2339, a charged methanocarba derivative of 2-Cl-adenosine monophosphate, was demonstrated in animal models of heart failure. We now test the hypothesis that an uncharged adenine nucleoside phosphonate, suitable as an oral agent with a hydrolysis-resistant phospho moiety, can prevent the development of cardiac dysfunction in a post-infarction ischemic or pressure overload-induced heart failure model in mice. The diester-masked uncharged phosphonate MRS2978 was efficacious in preventing cardiac dysfunction with improved left ventricular (LV) fractional shortening when administered orally at the onset of ischemic or pressure overload-induced heart failure. MRS2925, the charged, unmasked MRS2978 analog, prevented heart dysfunction when infused subcutaneously but not by oral gavage. When administered orally or systemically, MRS2978 but not MRS2925 could also rescue established cardiac dysfunction in both ischemic and pressure overload heart failure models. The diester-masked phosphate MRS4074 was highly efficacious at preventing the development of dysfunction as well as in rescuing pressure overload-induced and ischemic heart failure. MRS2978 was orally bioavailable (57–75%) giving rise to MRS2925 as a minor metabolite in vivo, tested in rats. The data are consistent with a novel therapeutic role of adenine nucleoside phosphonates in systolic heart failure. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11302-020-09688-0) contains supplementary material, which is available to authorized users.

Healthcare data includes information about patients’ life style, medical history, claims data, metabolomics and genomics profiles. Adequate and analytic access to healthcare data has potential to revolutionize the field of medicine by detecting diseases at earlier stages and modelling complex biological interactions by integrating and analyzing knowledge in a holistic manner. To improve the quality and transition of healthcare at reduced cost, innovative platforms are necessary to analyze heterogeneous clinical data of huge volume, velocity, variety and veracity. Healthcare data analytic process implementation is not straightforward and to effectively implement it, various big data challenges have to be overcome, which requires significant efforts from the experts in various disciplines. In response to these challenges and promoting significant medical transformation in public health, we showcase a new HIPAA compliant and high performance computing based large scaled big data system: Management, Analysis and Visualization of Clinical Data (MAV-clic), developed at UConn Health. MAV-clic successfully implements healthcare data analytic process and major analytic functionalities dealing with healthcare data consisting of three different modules: Cohort building, Data analysis, and Customized Measurement analysis. MAV-clic contains medical history of over 800,000 patients, and information about over all providers, medications and diagnosis codes. To fulfill the growing interests in implementing the health information system, MAV-clic fulfills the requirements of data owners as well as data users in the healthcare system. To give better understanding of the MAV-clic, we highlight background, and discuss its system designs, methods, development details and analysis capabilities in this manuscript.

Introduction: Stroke remains a leading cause of disability in the United States. Despite recent advances, interventions to reduce damage and enhance recovery after stroke is lacking. Therefore, validating new drug targets is critical for stroke treatment. P2X4, a receptor for adenosine triphosphate ATP, regulate activation of myeloid immune cells (both infiltrating monocytes/macrophages and brain-resident microglia) after stroke injury. Over-stimulation of P2X4Rs, due to excessive ATP release from the dying or damaged neuronal cells, contributes to ischemic injury. In this study, we pharmacologically targeted P2X4R to control immune response of myeloid cells during acute stroke phase and studied recovery at acute or chronic time point after stroke. Methods: We subjected 8-12 weeks old wild type mice of both sexes to 60 min right middle cerebral artery occlusion (MCAo) followed by 3 (acute) or 30 (chronic) days of reperfusion. We performed histological, behavioral (sensorimotor, anxiety and depressive), and flow cytometric analyses to determine the acute and chronic outcomes after P2X4R inhibitor 5-BDBD (1mg/kg P.O daily x3 days started after 4 hrs. of MCAo) treatment. Results: Treatment with 5-BDBD for 3 days not only significantly (*P+hi pro-inflammatory monocytes was reduced in 5-BDBD vs. vehicle treated mice. Conclusions: Acute P2X4R inhibition protects against ischemic injury at both acute and chronic time point after stroke. Reduced number of infiltrated Ly6C +hi proinflammatory monocyte in 5-BDBD treated mice suggests its potential mechanism of neuroprotection after stroke.

Rapid screening of red blood cells for active infection of COVID-19 is presented using a compact and field-portable, 3D-printed shearing digital holographic microscope. Video holograms of thin blood smears are recorded, individual red blood cells are segmented for feature extraction, then a bi-directional long short-term memory network is used to classify between healthy and COVID positive red blood cells based on their spatiotemporal behavior. Individuals are then classified based on the simple majority of their cells’ classifications. The proposed system may be beneficial for under-resourced healthcare systems. To the best of our knowledge, this is the first report of digital holographic microscopy for rapid screening of COVID-19.

We previously reported the development of an amphiphilic brush-like block copolymer composed of polynorbornene-cholesterol/polyethylene glycol (P(NBCh9-b-NBPEG)) that self-assembles in aqueous media to form long circulating nanostructures capable of encapsulating doxorubicin (DOX-NPs). Biodistribution studies showed that this formulation preferentially accumulates in tumor tissue with markedly reduced accumulation in the heart and other major organs. The aim of the current study was to evaluate the in vivo efficacy and toxicity of DOX containing self-assembled polymer nanoparticles in a mouse xenograft tumor model and compare its effects with the hydrochloride non-encapsulated form (free DOX). DOX-NPs significantly reduced the growth of tumors without inducing any apparent toxicity. Conversely, mice treated with free DOX exhibited significant weight loss, early toxic cardiomyopathy, acute toxic hepatopathy, reduced hematopoiesis and fatal toxicity. The improved safety profile of the polymeric DOX-NPs can be explained by the low circulating concentration of non-nanoparticle-associated drug as well as the reduced accumulation of DOX in non-target organs. These findings support the use of P(NBCh9-b-NBPEG) nanoparticles as delivery platforms for hydrophobic anticancer drugs intended to reduce the toxicity of conventional treatments.

Introduction Acute ischemic injury leads to severe neuronal loss. One of the key mechanisms responsible for this effect is inflammation, which is characterized by the activation of myeloid cells, including resident microglia and infiltrating monocytes/macrophages. P2X4 receptors (P2X4Rs) present on these immune cells modulate the inflammatory response. For example, excessive release of adenosine triphosphate during acute ischemic stroke triggers stimulation of P2X4Rs, leading to myeloid cell activation and proliferation and further exacerbating post-ischemic inflammation. In contrast, during recovery P2X4Rs activation on microglia leads to the release of brain-derived neurotrophic factor (BDNF), which alleviate depression, maintain synaptic plasticity and hasten post-stroke behavioral recovery. Therefore, we hypothesized that deletion of the P2X4R specifically from myeloid cells would have differential effects on acute versus chronic recovery following stroke. Methods We subjected global or myeloid-specific (MS) P2X4R knock-out (KO) mice and wild-type littermates of both sexes to right middle cerebral artery occlusion (60 min). We performed histological, behavioral (sensorimotor and depressive), and biochemical (quantitative PCR and flow cytometry) analyses to determine the acute (three days after occlusion) and chronic (30 days after occlusion) effects of receptor deletion. Results Global P2X4R deletion led to reduced infarct size in both sexes. In MS P2X4R KO mice, only females showed reduced infarct size, an effect that did not change with ovariectomy. MS P2X4R KO mice of both sexes showed swift recovery from sensorimotor deficits during acute recovery but exhibited a more pronounced post-stroke depressive behavior phenotype that was independent of infarct size. Quantitative PCR analysis of whole cell lysate as well as flow-sorted myeloid cells from the perilesional cortex showed increased cellular interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) mRNA levels but reduced plasma levels of these cytokines in MS P2X4R KO mice after stroke. The expression levels of BDNF and other depression-associated genes were reduced in MS P2X4R KO mice after stroke. Conclusions P2X4R deletion protects against stroke acutely but predisposes to depression-like behavior chronically after stroke. Thus, a time-sensitive approach should be considered when targeting P2X4Rs after stroke.

Background: Sirtuin3 (Sirt3), NAD+-dependent deacetylase, regulates several key proteins in both mitochondria and nucleus. The majority of early studies have shown neuroprotective effects of Sirt3 activation in various models of CNS injury. Recently, it was found that genetic loss of Sirt3 is beneficial in ischemia/reperfusion (I/R) injury. Therefore, this study was designed to explore the effects of both genetic and pharmacological inhibition of Sirt3 in a middle cerebral artery occlusion (MCAo) model of stroke. Methods: Focal cerebral ischemia was induced by transient right MCAo for 60min followed by 0, 6, 24 and 72 hrs of reperfusion. A 72hrs post ischemic injury time point was chosen to measure infarction in various treatment groups. The Sirt3 inhibitor AGK7 (sc- 204281 Santa cruz,) that selectively inhibits Sirt3 over Sirt1 and Sirt2, was given as single intraperitoneal injection (0.15, 0.5 and 1.5mg/kg) 3hrs after stroke onset. Activity or expression of Sirt3, was examined with Sirt3 activity kit or western blot using anti-Sirt3, anti-acetylated lysine antibodies. Results: Sirt3 knockout mice showed a significant reduction in hemispheric infarct volume compared to WT littermate controls (37.72±5.21 vs 52.8±6.76, p Summary and Conclusions: Changes in subcellular translocation and activity of Sirt3 after I/R stress suggests it important role in target protein deacetylation in stroke pathophysiology. Surprisingly, genetic deletion but not pharmacological inhibition led to neuroprotection, indicating the need to carefully examine target protein by both genetic and pharmacological approaches.

Background— Heart failure (HF), despite continuing progress, remains a leading cause of mortality and morbidity. P2X4 receptors (P2X4R) have emerged as potentially important molecules in regulating cardiac function and as potential targets for HF therapy. Transgenic P2X4R overexpression can protect against HF, but this does not explain the role of native cardiac P2X4R. Our goal is to define the physiological role of endogenous cardiac myocyte P2X4R under basal conditions and during HF induced by myocardial infarction or pressure overload. Methods and Results— Mice established with conditional cardiac-specific P2X4R knockout were subjected to left anterior descending coronary artery ligation–induced postinfarct or transverse aorta constriction–induced pressure overload HF. Knockout cardiac myocytes did not show P2X4R by immunoblotting or by any response to the P2X4R-specific allosteric enhancer ivermectin. Knockout hearts showed normal basal cardiac function but depressed contractile performance in postinfarct and pressure overload models of HF by in vivo echocardiography and ex vivo isolated working heart parameters. P2X4R coimmunoprecipitated and colocalized with nitric oxide synthase 3 (eNOS) in wild-type cardiac myocytes. Mice with cardiac-specific P2X4R overexpression had increased S-nitrosylation, cyclic GMP, NO formation, and were protected from postinfarct and pressure overload HF. Inhibitor of eNOS, L-N 5 -(1-iminoethyl)ornithine hydrochloride, blocked the salutary effect of cardiac P2X4R overexpression in postinfarct and pressure overload HF as did eNOS knockout. Conclusions— This study establishes a new protective role for endogenous cardiac myocyte P2X4R in HF and is the first to demonstrate a physical interaction between the myocyte receptor and eNOS, a mediator of HF protection.

Cardiac fibrosis is the excessive deposition of extracellular matrix proteins by cardiac fibroblasts and myofibroblasts, and is a hallmark feature of most heart diseases, including arrhythmia, hypertrophy, and heart failure. This maladaptive process occurs in response to a variety of stimuli, including myocardial injury, inflammation, and mechanical overload. There are multiple signaling pathways and various cell types that influence the fibrogenesis cascade. Fibroblasts and myofibroblasts are central effectors. Although it is clear that Ca2+ signaling plays a vital role in this pathological process, what contributes to Ca2+ signaling in fibroblasts and myofibroblasts is still not wholly understood, chiefly because of the large and diverse number of receptors, transporters, and ion channels that influence intracellular Ca2+ signaling. Intracellular Ca2+ signals are generated by Ca2+ release from intracellular Ca2+ stores and by Ca2+ entry through a multitude of Ca2+-permeable ion channels in the plasma membrane. Over the past decade, the transient receptor potential (TRP) channels have emerged as one of the most important families of ion channels mediating Ca2+ signaling in cardiac fibroblasts. TRP channels are a superfamily of non-voltage-gated, Ca2+-permeable non-selective cation channels. Their ability to respond to various stimulating cues makes TRP channels effective sensors of the many different pathophysiological events that stimulate cardiac fibrogenesis. This review focuses on the mechanisms of Ca2+ signaling in fibroblast differentiation and fibrosis-associated heart diseases and will highlight recent advances in the understanding of the roles that TRP and other Ca2+-permeable channels play in cardiac fibrosis.

Aims To determine the role of CD13 as an adhesion molecule in trafficking of inflammatory cells to the site of injury in vivo and its function in wound healing following myocardial infarction induced by permanent coronary artery occlusion. Methods and results Seven days post-permanent ligation, hearts from CD13 knockout (CD13KO) mice showed significant reductions in cardiac function, suggesting impaired healing in the absence of CD13. Mechanistically, CD13KO infarcts showed an increase in small, endothelial-lined luminal structures, but no increase in perfusion, arguing against an angiogenic defect in the absence of CD13. Cardiac myocytes of CD13KO mice showed normal basal contractile function, eliminating myocyte dysfunction as a mechanism of adverse remodelling. Conversely, immunohistochemical and flow cytometric analysis of CD13KO infarcts demonstrated a dramatic 65% reduction in infiltrating haematopoietic cells, including monocytes, macrophages, dendritic, and T cells, suggesting a critical role for CD13 adhesion in inflammatory trafficking. Accordingly, CD13KO infarcts also contained fewer myofibroblasts, consistent with attenuation of fibroblast differentiation resulting from the reduced inflammation, leading to adverse remodelling. Conclusion In the ischaemic heart, while compensatory mechanisms apparently relieve potential angiogenic defects, CD13 is essential for proper trafficking of the inflammatory cells necessary to prime and sustain the reparative response, thus promoting optimal post-infarction healing.

Activation of a cardiac myocyte P2X4 receptor protects against heart failure. 5'-Phosphonate and 5'-phosphate analogues of AMP containing a (N)-methanocarba (bicyclo[3.1.0]hexane) system could protect from heart failure by potentially activating this cardioprotective channel. Phosphoesters and phosphonodiesters were synthesized and administered in vivo via a miniosmotic pump in a mouse ischemic heart failure model and most significantly increased intact heart contractile function (echocardiography) compared to vehicle infusion. Several new thio and deuterated phosphate derivatives were protective in a calsequestrin (CSQ) overexpressing heart failure model. Diethyl (7, MRS4084) and diisopropyl (8, MRS4074) phosphotriesters were highly protective in the ischemic model. Substitution of 2-Cl with iodo reduced protection in the CSQ model. Diisopropyl ester 16 (MRS2978) of (1'S,2'R,3'S,4'R,5'S)-4'-(6-amino-2-chloropurin-9-yl)-2',3'-(dihydroxy)-1'-(phosphonoethylene)bicyclo[3.1.0]hexane was highly efficacious (CSQ), while lower homologue 1'-phosphonomethylene derivative 14 was inactive. Thus, we identified uncharged carbocyclic nucleotide analogues that represent potential candidates for the treatment of heart failure, suggesting this as a viable and structurally broad approach.

Objective: Stroke is a sexually dimorphic disease. A growing body of literature from both clinical and experimental studies suggests that the outcomes after stroke differ in males versus females. Peripheral immune cells play a key role in stroke outcomes and also show sexual dimorphism. However, general immunosuppression has not shown an overall benefit in stroke patients. Therefore, identifying novel targets to improve stroke outcomes are urgently needed. P2X4 receptors (P2X4R) and P2X7 receptors (P2X7R) are the predominant subtypes expressed on immune and neural cells. The P2X7R and P2X4R interact and may physically associate with each other. Recent evidence demonstrates that P2X4R is necessary for the pro-inflammatory function of P2X7R. The goal of this study is to test the hypothesis that genetic deletion of P2X4R in myeloid immune cells impacts on stroke outcome and that such outcome differs in males vs. female mice. Methods: P2X4R myeloid knockout mice and wild type littermates of both sexes (∼20-25g; C57BL/6), were randomized and subjected to right middle cerebral artery occlusion (MCAO-90min) followed by 3 days of reperfusion. At 72h after stroke mice were perfused and infarcts were quantified from 30μicron cut cresyl violet stained sections. The effect of myeloid P2X4R deletion on chronic functional recovery was assessed with neurological scores, corner test, Open field and the novel object recognition test. Data are expressed as mean±sem. ANOVA was performed and a P < .05 was set for statistical significance. Results: A significant neuroprotective effect is seen in P2X4R KO compared to WT females (Cortex 36.1±1.5 vs 27.5±4.7; Striatum 67.4±1.5 vs 56.3±7.0; Total 34.8±1.6 vs 26.9±2.8. P Conclusions: There is a sex-specific effect of immune cell P2X4R deletion. Myeloid specific deletion of P2X4R protects females from ischemia/reperfusion injury but has no salutary effect in males. This is the first study to demonstrate a deleterious effect of myeloid P2X4R on stroke outcomes. These initial findings implicate myeloid P2X4 as a novel therapeutic approach to improve ischemic outcomes in females.

The A(3) adenosine receptor (A(3)AR) coupled to G(i) (inhibitory regulative guanine nucleotide-binding protein) mediates anti-inflammatory, anticancer and anti-ischemic protective effects. The receptor is overexpressed in inflammatory and cancer cells, while low expression is found in normal cells, rendering the A(3)AR as a potential therapeutic target. Highly selective A(3)AR agonists have been synthesized and molecular recognition in the binding site has been characterized. In this article, we summarize preclinical and clinical human studies that demonstrate that A(3)AR agonists induce specific anti-inflammatory and anticancer effects through a molecular mechanism that entails modulation of the Wnt and the NF-κB signal transduction pathways. At present, A(3)AR agonists are being developed for the treatment of inflammatory diseases, including rheumatoid arthritis (RA) and psoriasis; ophthalmic diseases such as dry eye syndrome and glaucoma; liver diseases such as hepatocellular carcinoma and hepatitis.

We have previously demonstrated that in response to traumatic injury in skeletal muscle, there is a dysregulation of the matrix metalloproteases (MMPs) and their inhibitors (TIMPs), a response hypothesized to interfere with proper skeletal muscle regeneration. Moreover, we have shown that pharmacological activation of the adenosine A3 receptor by Cl-IBMECA in skeletal muscle can protect against ischemia-reperfusion and eccentric exercise injury. However, the mechanism by which Cl-IBMECA protects muscle tissue is poorly defined. This study evaluated the effects of Cl-IBMECA on MMP/TIMP expression in skeletal muscle and tested the hypothesis that adenosine A3 receptor-stimulated protection of skeletal muscle following traumatic injury is associated with a blunting of MMPs involved in inflammatory processes and collagen degradation, and an increase in MMPs associated with extracellular matrix remodeling. Sixty C57BL/6J male mice were injected with Cl-IBMECA ( n = 30) or a vehicle ( n = 30), and Evans blue dye. Injury was induced by applying a cold steel probe (−79°C) to the tibialis anterior (TA) muscle for 10 s. TA muscles from uninjured and injured legs were collected 3, 10, and 24 h postinjury for analysis of muscle injury and MMP/TIMP mRNA and protein levels. Twenty-four hours postinjury, 56.8% of the fibers were damaged in vehicle-treated mice vs. 35.4% in Cl-IBMECA-treated mice ( P = 0.02). Cl-IBMECA treatment reduced membrane type 1 (MT1)-MMP, MMP-3, MMP-9, and TIMP-1 mRNA expression 2- to 20-fold compared with vehicle-treated mice ( P < 0.05). Cl-IBMECA decreased protein levels of latent/shed MT1-MMP 23–2,000%, respectively, 3–10 h postinjury. In Cl-IBMECA-treated mice, latent MMP-2 was decreased 20% 3 h postinjury, active MMP-3 was decreased 64% 3 h postinjury, and latent/active MMP-9 was decreased 417,631% 3 h postinjury and 20% 10 h postinjury. Protein levels of active MMP-2 and latent MMP-3 were increased 25% and 74% 3 h postinjury, respectively. The present study elucidates a new protective role of adenosine A3 receptor stimulation in posttraumatic skeletal muscle injury.