bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pathophysiological role of NNAT in ER+ breast cancer
Authors: Semenikhina Marharyta , Plasterer Cody , Tsaih Shirng‐Wern , Flister Michael , Palygin Oleg 
Published: May 2022

Neuronatin (NNAT) was recently identified as a novel mediator of estrogen receptor‐positive (ER+) breast cancer cell proliferation and migration, which correlated with decreased tumorigenic potential and prolonged patient survival. However, despite these observations, the molecular and pathophysiological mechanism(s) of NNAT in ER+ breast cancer remain unclear. Based on high protein homology with phospholamban, we hypothesized that NNAT mediates homeostasis of intracellular calcium [Ca2+]i) levels and endoplasmic reticulum (EndoR) function, which is frequently disrupted in ER+ breast cancer and other malignancies.To evaluate the role of NNAT on [Ca2+]i homeostasis, we used intracellular labeling and subcellular confocal imaging of multiple breast cancer models (ER+ breast cancer cell lines T47D and ZR75, MCF10A normal mammary epithelial cells, and MDA‐MB‐231 triple‐negative breast cancer cell lines originally obtained from ATCC (American Type Culture Collection) to better understand the role of NNAT in Ca2+‐mediated cellular functions.Our data indicate that NNAT localizes predominantly to EndoR and lysosome, and genetic manipulation of NNAT levels demonstrated that NNAT modulates [Ca2+]i influx and maintaining Ca2+homeostasis. The application of thapsigargin and consecutive inhibition of SERCA in ER+ breast cancer cells overexpressed NNAT show a significant elevation in the amount of Ca2+ in EndoR (184 ± 5 %, p<0.001 comparing to GFP). At the same time, overexpression of NNAT increased basal cytosolic Ca2+ concentration up to 50% (p<0.001 comparing to GFP). Pharmacological inhibition of calcium channels revealed that NNAT regulates [Ca2+]i levels in breast cancer cells through the interaction with ORAI1 but not the TRPC3 signaling cascade. Furthermore, NNAT transcriptionally regulated by NRF1, PPARα, and PPARγ and is strongly upregulated by oxidative stress via the ROS and PPAR signaling cascades.Collectively, these data suggest that NNAT regulates Ca2+homeostasis in response to oxidative stress, thus providing a molecular link between the longstanding observation that accumulating ROS and altered Ca2+signaling are key oncogenic drivers of cancer.
Optimization and Validation of Nanopore Based Sequencing Method for Molecular Testing of CNS Tumours
Authors: Mimosa Mashiat L. , Al‐ameri Wafa , Nakhla Michael , Simpson Jared T. , Boissinot Karel , Munoz David G. , Das Sunit , Fattouh Ramzi , Saleeb Rola 
Published: May 2022

BackgroundThe World Health Organization (WHO) introduced molecular identifiers for the diagnosis and prognosis of CNS tumors including the mutational status of isocitrate dehydrogenase or IDHgenes in glial tumors. Currently used immunohistochemistry (IHC) is not capable of detecting the non‐canonical mutations, and sequencing is often required as a follow‐up. Current next‐generation sequencing (NGS) technologies used in tumor molecular marker detection introduce key challenges including high capital cost, complex infrastructure requirements, and long turnaround times. These challenges considerably limit the ability to perform NGS testing in many pathology laboratories. In this study, we aimed to use third generation nanopore sequencing technology to resolve these limitations. The Oxford Nanopore MinION, a pocket‐sized nanopore sequencing device, has minimal capital costs and infrastructural requirements, and shorter turnaround times. However, the nanopore technology has not been validated in clinical practice and has not been optimized on formalin‐fixed paraffin‐embedded (FFPE) tissue.MethodsDNA extraction of selective tumor areas was performed from the corresponding FFPE tissue blocks from a cohort of gliomas with confirmed IDH1and IDH2gene statuses (n=65). A PCR amplicon‐based approach was used to amplify hot spots of the IDH1and IDH2genes, starting with 30ng DNA material. The amplicon libraries were sequenced for 2 hours in multiplex on the MinION device and IDHSNPs were called with the Nanopolish software. ASIP Abstract Mashiat MimosaResults26 IDH mutant samples were identified: 21 IDH1R132H, 2 IDH1R132G, 2 IDH2R172G, and 1 IDH2D177H. All cases showed concordant IDHmutational status when compared to the reference methods (IHC or NGS) and both analytical sensitivity and specificity were 100%. Precision analysis of variant allele frequency (VAF) showed the coefficient of variation was less than 5% (both inter and intra runs), and the limit of detection for VAF was 3%. The range of read depth obtained was 882X to 43,000x with an average of 20,000x. This assay revealed a $50‐$100 material cost per sample, and the time taken from extracted nucleic acid to final result generation was 1‐2 business days.ConclusionThis project is the first to optimize and validate an approach to detect SNP mutations in FFPE samples using nanopore technology. It has demonstrated the feasibility and efficacy of the nanopore amplicon sequencing method in cancer FFPE tissue with excellent test performance characteristics, significantly shorter turnaround times at considerably lower costs and without any infrastructural needs. Thus, it can be used to circumvent challenges to current NGS testing platforms and can be the milestone that would make cancer NGS testing available for every laboratory.
Cerebrovascular Carbon Dioxide Reactivity with Hyperoxia and Hypoxia in Humans with Treated Hypertension
Authors: Fan Jui‐Lin , Sayegh Ana L. C. , Dawes Mathew , Paton Julian F. R. , Fisher James P. 
Published: May 2022

Hypertension increases the risk of cerebrovascular diseases such as stroke and dementia, but the underlying mechanisms are currently unclear. While chronic hypertension can lead to anatomical and functional adaptations within the cerebral circulation, its effect on cerebral blood flow (CBF) control remains poorly understood. We tested the hypothesis that the CBF responses to CO2 with either high or low oxygen levels are impaired in treated hypertensive patients compared to normotensive aged‐matched controls.In 14 treated hypertensive patients (age: 68±5 years, mean±SD) and 8 normotensive age‐matched controls (age: 66±6 years), we assessed cardiorespiratory and cerebrovascular variables at rest, during voluntary hyperventilation (partial pressure of end‐tidal CO2 [PETCO2] ~25 mmHg), and hyperoxic and hypoxic rebreathing. Using transcranial Doppler ultrasound and finger photoplethysmography, we measured beat‐to‐beat middle cerebral artery blood velocity (MCAv: index of CBF) and BP, respectively, while PETCO2 was assessed using a gas analyzer. Linear regression was used to determine the MCAv‐CO2 slope (i.e., cerebrovascular CO2 reactivity).At rest, mean BP was higher in the hypertensive patients (108±9 mmHg) compared to normotensive controls (93±10 mmHg, p=0.001), while MCAv (46.8±11.4 vs. 53.3±10.4 cm/s) and PETCO2 (41±4 vs. 42±3 mmHg) were not different between groups (p<0.05). Hyperventilation‐induced hypocapnia elicited similar reductions in MCAv in the hypertensive and normotensive groups (Δ14.8±4.7 vs. Δ16.5±5.7 cm/s, p=0.484). Similarly, the MCAv‐CO2slope was not different between the hypertensive and normotensive groups during either hyperoxic rebreathing (1.53±1.05 vs. 1.98±0.56 cm/s/mmHg, p=0.320) or hypoxic rebreathing (1.94±1.04 vs. 2.21±1.13 cm/s/mmHg, p=0.815).These preliminary findings indicate that in treated hypertensive patients, the MCAv responses to hypocapnia, and hypercapnia administrated with either high or low oxygen levels, do not differ from that observed in age‐matched normotensive controls.
Late Gestation Exposure to PolyI:C‐Induced Maternal Immune Activation Results in Sex‐Dependent Antiviral Responses in Rat Placenta
Authors: Thomas Ruth , De Oliveira Cleusa , Rajakumar Nagalingam , Renaud Stephen 
Published: May 2022

IntroductionViral infections are a major cause of pregnancy‐related complications and can lead to neurodevelopmental deficits in offspring. Viral infections stimulate maternal immune activation (MIA), which disrupts the delicate immune balance required for pregnancy. The placenta is a temporary organ that forms the interface between mom and baby during pregnancy and provides nutrition and protection for the developing fetus. Placental responses to MIA, and how MIA alters placental function, are not well understood. Polyinosinic:polycytidylic acid (PolyI:C), a synthetic double‐stranded RNA and viral mimetic elicits MIA in rodent models. PolyI:C‐induced MIA is sufficient to cause neurobehavioural consequences in offspring, with male offspring typically exhibiting more severe outcomes than female offspring.ObjectiveThe aim of this project was to elucidate how MIA affects the immunological profiles of the nascent male and female placenta and brain from a late prenatal PolyI:C stimulation.HypothesisWe hypothesize that injection of PolyI:C into pregnant rats at late gestation will induce antiviral responses in the placenta and fetal brain, and these responses will differ between male and female fetuses.MethodsTo test this hypothesis, pregnant Sprague‐Dawley rats were injected intravenously with 4 mg/kg PolyI:C (N = 5) or saline (N = 4) on GD18.5. Five hours after injection, placentas and fetal brains were collected. Quantitative RT‐PCR was used to measure expression of various genes associated with antiviral responses (e.g., Ifna, Il6, Il1a, Il1b, Tnfa, Ido1, Cxcl10, Rsad2, Cxcl11, Ccl5, and Tlr3) in the placenta. A Student’s t‐test was used to compare differences between two means, and a two‐way analysis of variance, followed by Sidak’s post‐hoc analysis was used to compare differences between treatments and sexes. Means were considered statistically significant when P ≤ 0.05.ResultsPolyI:C‐treated male placentas (n = 9) showed significantly higher levels of Rsad2(2.4‐fold), Ifna (2.8‐fold), Mx1(9.4‐fold), and Cxcl10(23.3‐fold) compared to saline‐treated male placentas (n = 8; P ≤ 0.05). PolyI:C‐treated female placentas (n = 10) showed a significant upregulation of Cxcl10(25.4‐fold) and Mx1 (11.4‐fold), as well as of Cxcl11(19.8‐fold) and Tnfa(2.3‐fold) compared to saline‐treated female placentas (n = 8; P ≤ 0.05). Among PolyI:C‐treated samples, male placentas showed significantly increased expression of Ifna when compared to female placentas (2.8‐fold, P ≤ 0.05).ConclusionOur results show that PolyI:C‐induced MIA at late gestation stimulates a robust antiviral response in the placenta, including higher levels of Ifna in male placentas than female placentas.SignificanceFuture work will characterize PolyI:C‐induced antiviral and sex‐dependent responses in fetal brains. Overall, this research will provide further insight into MIA‐induced susceptibility to pregnancy complications and neurodevelopmental outcomes.
Sympathetic Neurocirculatory Responses to Chemoreflex Activation in Young Women and Men
Authors: Sayegh Ana Luiza C. , Fan Jui‐Lin , Dawes Mathew , Paton Julian F. , Fisher James P. 
Published: May 2022

Sex‐differences in the ventilatory responses to central and peripheral chemoreflex activation have been extensively examined. Along with the control of respiration, chemoreflexes play a key role in sympathetic regulation. Herein, we determined whether central, peripheral, and combined central and peripheral chemoreflex activation evoke differing sympathetic neurocirculatory responses in young women and men.In ten women (29±6 years, 23±2kg/m2: mean±SD) and ten men (30±7 years, 25±3kg/m2), we recorded muscle sympathetic nerve activity (MSNA), minute ventilation (V̇E) and mean arterial pressure (MAP) during 5‐min trials of breathing room‐air (eucapnia), isocapnic hypoxia (10% oxygen [O2]; peripheral chemoreflex activation), hypercapnic hyperoxia (7% carbon dioxide [CO2], 50% O2; central chemoreflex activation) and hypercapnic hypoxia (7% CO2, 10% O2). Trials were randomized. Women were studied in the early follicular phase of their menstrual cycle.During eucapnia and isocapnic hypoxia, no sex‐differences were observed in MSNA, V̇E and MAP (P>0.05). During hypercapnic hyperoxia (∆278±215 vs. ∆158±107%, P=0.05) and hypercapnic hypoxia (∆507±290 vs. ∆262±268%, P=0.05) MSNA responses were augmented in women compared to men, however V̇E responses were attenuated (hypercapnic hyperoxia ∆15.2±6.7 vs. ∆25.6±6.7L/min, P=0.007; hypercapnic hypoxia ∆28.8±9.3 vs. ∆39.9±12.5L/min, P=0.004). MAP responses were similar in women and men (hypercapnic hyperoxia ∆10±2 vs. ∆23±2mmHg, P=0.35; hypercapnic hypoxia ∆16±3 vs. ∆25±3mmHg, P=0.35).In summary, young women have greater MSNA response to central chemoreflex activation than young men, which explains their augmented sympathetic response to combined central and peripheral chemoreflex activation. Future studies should determine the effect of menopause on these sex‐differences in the sympathetic neurocirculatory responses to chemoreflex activation.
Fibroblast Senescence: A Risk Factor for Remodeling, Inflammation, and Arrhythmias in the Post‐MI Heart
Authors: Sengun Elif , Baggett Brett , Turan Nilufer , Mi Eric , Kun Bianca , Sedivy John , Koren Gideon 
Published: May 2022

ObjectivesCellular senescence is a stress response involving permanent replicative arrest. It is accompanied by a complex senescence‐associated secretory phenotype (SASP), which is characterized by the presence of pro‐inflammatory cytokines and chemokines, growth factors, and tissue‐remodeling metalloproteinases. Little is known regarding the role of senescence and the SASP in the context of arrhythmias post myocardial infarction (MI). Here, we characterized the arrhythmogenic cardiac consequences and molecular mechanisms underlying tissue remodeling changes in impaired or accelerated cardiac myofibroblast (CMF) senescence and the pro‐inflammatory SASP during post‐MI wound healing process in genetically engineered mice.Material‐MethodsImpaired or accelerated CMF‐specific senescence was achieved by using cre‐lox mouse models to delete p53 or MDM2 genes. MI surgery via direct ligation of the left anterior descending artery (LAD) was performed in 4‐month‐old wild‐type (WT), Pstn‐Cre(+/‐) p53(fl/fl), Pstn‐MerCreMer(+/‐) MDM2(fl/fl), and respective littermate control (LMC) mice of both sexes. Changes in cardiac structure and function post‐MI were assessed by echocardiography. Senescence‐associated β‐galactosidase (SA‐βgal) staining was applied to detect and quantify senescent cells in three zones: infarct zone (IZ), infarct border zone (IBZ), and remote zone (RZ). Immunofluorescence staining identified the types of senescent cells. Masson’s trichrome staining was used for fibrosis assessment. RT‐qPCR was performed to quantitate the expression of known senescence genes and canonical SASP factors.ResultsPstn‐Cre(+/‐) p53(fl/fl) mice displayed significantly decreased CMF senescence two weeks post‐MI in the scar compared to littermate control p53(fl/fl) and WT mice. We noticed a similar trend in the IBZ, but this did not reach statistical significance. Pstn‐MCM(+/‐) MDM2(fl/fl) mice showed significantly increased CMF senescence in the IZ compared littermate control MDM2(fl/fl) mice. We observed a similar trend in the IBZ, but this did not reach statistical significance. In all mice, we observed essentially no senescence in the RZ. Our data did not show any significant difference in fibrosis between any genotype in any zone. We did not observe any overall trend in differential expression in senescence and SASP genes between p53(fl/fl) and Pstn‐Cre(+/‐) p53(fl/fl) mice in any zone. There was no evidence of any sex‐specific differences in aforementioned results.ConclusionsA significant increase and persistence of CMF senescence likely contributes to pathological tissue remodeling during the post‐MI wound healing process. This is likely due to SASP‐induced pro‐arrhythmogenic chronic inflammation or via direct cell‐cell interactions between senescent CMFs and myocytes.
The lysine demethylase KDM6A regulates pancreatic β cells function
Authors: Zhang Bo , Wang Shirley , Sun Zhongjie 
Published: May 2022

Background and ObjectiveDiabetes mellitus refers to a group of metabolic disorders characterized by impaired glucose homeostasis. Both type 1 diabetes (T1D) and type 2 diabetes (T2D) are characterized by insulin insufficiency. β cell failure in diabetes are often due to posttranslational modifications of histones and transcription factors. Lysine demethylase 6A (KDM6A) belongs to the KDM6 family of histone H3 lysine 27 (H3K27) demethylases which is important for development of various organs. The objective of this study is to investigate whether KDM6A regulates pancreatic β cell function using targeted β cell‐specific deletion of KDM6Agene.Methods and ResultsBriefly, inducible insulin 1‐cre and KDM6A‐loxp mice were bed to generate cre‐loxp mice. Injection of tamoxifen induced deletion of KDM6A gene in β cells which led to increased H3K27me3 levels. KDM6A‐KO mice exhibited reduced baseline insulin levels compared with wild‐type mice. Interestingly, despite low baseline insulin levels, KDM6A‐KO mice demonstrated enhanced glucose tolerance. Insulin release was increased in KDM6A‐KO mice vs WT mice at 15 min following glucose challenge. Furthermore, ex vivo glucose‐induced insulin secretion was increased in cultured islets isolated from KDM6A‐KO mice. In cultured Min6 β cells, knockdown of KDM6A using KDM6A shRNA largely enhanced glucose‐induced insulin release. When challenged with high‐fat diet, KDM6A‐KO mice displayed similar weight gain vs wild‐type mice. However, the insulin sensitivity test showed that KDM6A‐KO enhanced peripheral insulin sensitivity as evidenced by the delayed recovery of glucose level in KDM6A‐KO mice fed with high‐fat diet.ConclusionOur results suggest a novel role of KDM6A in the regulation of pancreatic β cell function. KDM6A may be a potential therapeutic target for β cell dysfunction.
Investigating Susceptibility of ꞵ‐catenin‐mutated Hepatocellular Carcinoma to Checkpoint Inhibitors
Authors: Delgado Evan R. , Tao Junyan , Bell Aaron W. , McGaughey Jackson , Ganesh Jai , Poddar Minakshi , Singh Sucha , Monga Satdarshan P. 
Published: May 2022

Hepatocellular Carcinoma (HCC) is the 5th most common cause of cancer‐related death in the United States with an estimated 32,000 annual deaths. The standard of care for HCC as established by the IMBrave150 trial is a combination of the PD‐L1 inhibitor, Atezolizumab, and an antiangiogenic, Bevacizumab. While this combination extends overall survival to 19.2 months in unresectable HCC, only 52% of patients survive to 18 months. Furthermore, objective response rates are around 32% and hence clarifying underlying mechanisms regulating the immune response in HCC oncogenesis might strengthen immunotherapy efficacy. Especially important is to identify response rates of these agents in specific molecular subclasses of HCC may help in better patient selection. The oncogene ꞵ‐catenin is consistently mutated and activated among 30% of HCC patients. The Wnt/ꞵ‐catenin pathway has been approached as a target for precision medicine in HCC. We recently modeled HCCs with ꞵ‐catenin mutations in mice up to 69% genetic similarity. Here, we combine hydrodynamic tail vein injections with the Sleeping Beauty Transposase which is a non‐viral method of expressing genes of interest. Our model co‐expresses an S45Y point mutant of ꞵ‐catenin with the tyrosine kinase receptor MET (B+M). This combination represents ~10% of patient HCCs and provides a clinically relevant tool for the investigation of personalized HCC therapeutics.A recent study demonstrated HCCs driven by a Δ90‐truncation mutant of ꞵ‐catenin in combination with the Myc oncogene are shown to lack sufficient immune cell surveillance. These HCCs have poor cytotoxic T cell and dendritic cell recruitment while responding poorly to checkpoint inhibitors. Since activating point mutations in CTNNB1 gene are common in patient HCCs, we asked whether poor immune surveillance extends to HCC models harboring point mutations. We first checked and found the presence of CD45+, F4/80+, and CD11b+ cells both 2 and 4 weeks after B+M tumor induction. This indicates the presence of both adaptive and innate immune cells in the B+M tumor microenvironment. We next asked whether checkpoint inhibition will effectively reduce tumor burden in the B+M model. Therefore, we treated B+M mice with either IgG or anti‐PD1 monoclonal antibodies 4 weeks after tumor induction. Tissues were harvested 3.5 weeks after treatment. We found a modest 20% reduction in liver weight to body weight ratio, a measure of tumor burden, in B+M mice treated with anti‐PD1 compared to IgG controls. Our results demonstrate that HCCs with missense ꞵ‐catenin mutations do respond albeit marginally to checkpoint inhibitors. In the future, combination of checkpoint inhibitors with other inhibitors will be tested in these models to determine their efficacy in treating ꞵ‐catenin‐mutated HCCs.
A New Method to Analyse Myocardial Substrate Metabolism and Mitochondrial Function in Intact Cardiac Tissue Slices
Authors: Heinen Andre , Bottermann Katharina , Spychala Andre , Eliacik Asena , Amin Ehsan , Moussavi‐Torshizi Seyed‐Erfan , Klöcker Nikolaj , Gödecke Axel 
Published: May 2022

The progression of cardiac diseases is often accompanied by disturbances in substrate metabolism. Extracellular flux analysis has become a standard tool to investigate metabolic alterations in cell lines. However, the enzymatic digestion of the heart to isolate adult cardiomyocytes as well as the cultivation procedure that is required for cell attachment to the cell culture plates might affect metabolism. Therefore, we developed a flux analyser‐based method to measure substrate metabolism of intact cardiac tissue slices. Furthermore, we tested this method in a proof‐of‐principle approach in remote myocardium after myocardial infarction.To yield cardiac tissue pieces of comparable size, mouse cardiac tissue was sliced (150 µm) using a vibratome, and tissue pieces (diameter 1.9 mm) of these slices were punched out. Using “islet capture plates” in a Seahorse XFe 24 analyser, oxygen consumption rates (OCR) were measured at baseline and after FCCP‐induced uncoupling in palmitate, glucose (Glc) and glutamine (Gln) enriched medium. To determine long‐chain fatty acid metabolism, CPT1 was inhibited by etomoxir, and Glc/Gln metabolism by inhibition of mitochondrial pyruvate carrier (MPC) and glutaminase (Gls) with UK5099/BPTES. Optical mapping of membrane potential was used to assess action potentials in tissue slices as indicator of cellular integrity. Finally, the developed method was used to analyse substrate metabolism in the remote myocardium at day 3 after myocardial ischemia and reperfusion (n=7) in comparison to sham mice (n=8). Data are mean±SD; unpaired two‐sample t‐test.Basal OCR was 53±8 pmol/min, and FCCP increased OCR to 92±18 pmol/min. Both etomoxir and UK5099/BPTES reduced OCR indicating that both palmitate and Glc/Gln are metabolised. Optical mapping of tissue slices showed regular action potential characteristics and propagation. After myocardial infarction, CPT1 inhibition caused a smaller reduction of uncoupled mitochondrial OCR in I/R animals compared to sham (40±13 vs. 52±4%, p<0.05). This effect was caused by an increased metabolism of Glc/Glu (37±13 vs. 24±4 pmol/min, p<0.05), whilst the effect of CPT1 was not different.Here, we describe a new method to analyse cardiac metabolism using cardiac tissue slices that metabolise fatty acids as well as glucose, and show high functional integrity. Therefore, this method has the potential to expand the methodological alternatives to investigate cardiac substrate metabolism. In a proof‐of‐principle approach, the analysis of cardiac substrate metabolism of the remote myocardium after I/R showed an augmented glucose/glutamine metabolism.