bluuebunny/crossref_metadata_2025_split huggingface dataset:
Loss of Ca2+ spark‐to‐BK channel coupling impairs cerebral autoregulation in hypertension
Authors: Pritchard Harry A. , Taylor Jade , Nelson Mark T. , Greenstein Adam S. 
Published: May 2022

IntroductionA reduction in blood flow to the brain in hypertension leads to vascular dementia, but what drives this is unknown. In vascular smooth muscle cells (VSMCs) the close spatial coupling (~20 nm) between Ca2+ release events from the sarcoplasmic reticulum (SR) (Ca2+ sparks) and the vasodilatory large‐conductance Ca2+ activated K+ (BK) channels, buffers pressure induced constriction and ensures correct cerebral perfusion.HypothesisImpaired BK channel activation was responsible for the impaired cerebral autoregulation in hypertension‐induced dementia.MethodsMale hypertensive mice (BPH/2) and the normotensive control strain (BPN/3) were euthanized in adhered to the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. Experiments were carried out using pressure myography, confocal microscopy and electrophysiology techniques.ResultsPressure‐induced contraction was studied at a range of intraluminal pressures (10‐120 mmHg), and cerebral arteries from BPH/2 mice showed a more contractile phenotype compared to BPN/3 controls, confirming impaired autoregulation. BK channel activation by Ca2+ sparks was recorded using perforated patch electrophysiology to record spontaneous transient outward currents (STOCs). STOCs recorded from VSMCs from BPH/2 cerebral arteries, showed a significant reduction in both frequency and amplitude. To determine the mechanism behind this loss of BK activity, we first studied Ca2+ sparks in pressurised cerebral arteries loaded with Fluro‐4‐AM and found no difference between BPH/2 and BPN/3 mice. Next, we looked at BK alpha subunit expression, by recording whole cell BK currents with a fixed [Ca2+] in the pipette solution. Paradoxically, there was an increase in BK current density in the VSMC from the BPH/2 mice compare to BPN/3 mice. We then looked at the Ca2+ sensitivity of the BK channel using single channel electrophysiology, but again there were no differences between the BPH/2 and BPN/3 groups. Subsequently we used live‐cell fluorescent membrane dyes to look at the coupling between the SR (ER‐Tracker) and the plasma membrane (CellMask) in freshly isolated VSMCs. 3D reconstruction of the deconvolved images showed a reduction in both the coupling site volume and frequency in cells isolated from BPH/2 mice.ConclusionsThis data suggests that an increased spatial distance between the SR and the plasma membrane reduces [Ca2+] around the BK channel resulting in increased constriction of the arteries. Opportunities to restore the spark‐to‐BK coupling could be a novel approach to prevent vascular dementia caused by hypertension.
Interplay between TPR nucleoporin and TREX‐2 complex in mRNA export
Authors: Aksenova Vasilisa , Esnault Caroline , Chen Shane , Regmi Saroj , Arnaoutov Alexei , Dasso Mary 
Published: May 2022

Nuclear pore complexes (NPCs) are conduits between the cytoplasm and the nucleus. NPCs consist of roughly 35 distinct nucleoporins with filaments extending from both the nuclear and cytoplasmic faces of the pore. Nucleoporins regulate many processes, including nucleocytoplasmic trafficking, protein modification, chromatin remodeling and transcription, as well as mRNA processing and export. Our current research is focused on basket nucleoporin TPR, which works as a docking platform for other proteins, including transport receptors and subunits of the TRanscription and EXport 2 (TREX‐2) complex. We found that Auxin Inducible Degron (AID)‐mediated TPR elimination specifically disrupts TREX‐2 complex recruitment to NPCs, and results in rapid and profound changes in transcriptomic profiles of TREX‐2‐dependent RNAs and retention of those RNAs within the nucleus. The molecular mechanism through which TPR controls TREX‐2‐dependent gene expression is not clear.We are currently working in two ways to understand TREX‐2‐dependent RNA export and how TPR facilitates this pathway. First, we are examining the components of TREX‐2. GANP is a major scaffold subunit of the TREX‐2 complex, and loss of GANP disrupts the association of TREX‐2 complex subunits to the NPC. We have AID‐tagged other TREX‐2 complex members (PCID2, ENY2, CETN2) and are analyzing how their loss impacts TREX‐2 targeting, transcriptomic profiles and RNA retention. Our findings suggest that GANP or PCID2 depletion rapidly leads to polyA RNA retention inside the nucleus, but this was not the case after depletion of ENY2 or CETN2. Further comparison of TREX‐2 complex subunit profiles to each other should elucidate the sequence of their actions during mRNP maturation.Second, we are investigating the point(s) at which TPR controls TREX‐2‐dependent gene expression: TPR may help to remodel mRNP particles within the nucleus, to recruit ready‐to‐export mRNP particles to the nuclear envelope, or it may perform both of these functions. To distinguish between these possibilities, we have analyzed a TPR mutant that lacks an NPC recruitment domain, to see whether it can rescue any part of TPR’s function in this pathway without recruiting TREX‐2 to the NPC. We found that this TPR mutant resides inside the nucleus and can not restore normal export of polyA RNA. We speculate that TPR preferentially acts as a landing platform for TREX‐2‐bound mRNAs at the nuclear pore and may help with quality control of the transcripts. To explore if TPR‐dependent transcripts have unique signatures, we are analyzing mRNA length, GC content and intron context in other AID‐tagged nucleoporins and comparing transcriptomic profiles after their loss in comparison to TPR.Alltogether, these data will help us to unfold the role of TREX2 complex subunits in targeting GANP‐TPR dependent transcripts to the pore and to gain a better understanding of how TPR controls gene expression.
Long‐term Anesthesia in the Mexican Axolotl Salamander
Authors: Andersson Sofie A. , Dittrich Anita , Lauridsen Henrik 
Published: May 2022

Research animals are routinely anesthetized for varying durations of time and using different anesthetics. However, the anesthesia is usually limited to the duration of surgical procedures or other interventions usually lasting no more than a few hours.Establishing a long‐term anesthetic model in regenerative competent species, such as the Mexican axolotl, would allow for better estimations of physiological parameters during regeneration as the behavioral aspect is detached. As such, we aim for an anesthetic duration of 60 days, a period of time compatible with regeneration of limb amputations as well as heart and spinal cord injury. In this study, axolotls were anesthetized using propofol for increasing periods of time starting with 24 hours and gradually increasing to 7, 12 and 26 days.During this study, parameters such as oxygen consumption, echocardiography and body mass was assessed. After anesthesia, activity level, response to tactile and nociceptive stimuli, and recovery time was examined to ensure no lasting effect of long‐term anesthesia. Long‐term anesthesia was associated with a significant, but reversible, reduction in oxygen consumption (approximately 50%) and a significant increase in body mass despite fasting. Anesthetic periods exceeding 7 days in hypoosmotic medium like tap water were associated with complications including severe swelling and even death, but these complications were alleviated using isosmotic medium. Tactile and nociceptive stimulation was scored on a scale from 1 to 3 prior to anesthesia. No significant change in response was detected following anesthesia demonstrating that no permanent nerve damage had occurred. Axolotls showed a reversible decrease in activity levels following an anesthetic period with levels returning to baseline within a week of recovery.In conclusion, this study shows that axolotls can be anesthetized for extended periods of at least 26 days presenting new possibilities for future experiments in which long‐term anesthesia or immobilization is preferable. Additionally, stable long‐term anesthesia paves the way for non‐recovery experiments and more severe interventions as animals can be kept anesthetized for the duration.
Effects of Aging on Proteome Dynamics in Mice Brain Microvessels: ROS Scavengers, mRNA/Protein Stability, Glycolytic Enzymes, Mitochondrial Complexes, and Basement Membrane Components
Authors: Chandra Partha K. , Cikic Sinisa , Rutkai Ibolya , Guidry Jessie J. , Katakam Prasad V. G. , Mostany Ricardo , Busija David W. 
Published: May 2022

BackgroundAging is an unavoidable stress with ever‐increasing detrimental effects on the brain microvasculature, which affects neuronal health/function and adds vulnerability to strokes and neurological diseases. In this study, we performed an extensive examination of proteins involved in the structure and function of mouse brain cortical microvessels (MVs) using a discovery‐based quantitative proteomic approach.MethodsCortical MVs were isolated from age‐matched, male and female, young (4–6 months), middle‐aged (12–14 months), and old (20–21 months) mice obtained from Jackson Laboratory [Tg(Thy1‐EGFP)MJrs/J] (Jax No. 007788) and bred in a C57B16J background. The presence of end‐arterioles, capillaries, and venules in MVs was confirmed by light microscopy and by alkaline phosphate staining. Proteomics analysis was performed using liquid chromatography/mass spectrometry.ResultsMost differentially expressed (DE) proteins (> 90%) showed no significant disparities between the sexes; however, some significant DE proteins showing sexual differences in MVs decreased from percentage in young (8.3%), to middle‐aged (3.7%), to old (0.5%) mice. Therefore, we combined male and female data for age‐dependent comparisons but noted sex differences for examination. Key proteins involved in the oxidative stress response, mRNA or protein stability, basement membrane (BM) composition, aerobic glycolysis, and mitochondrial function were significantly altered with aging. Relative abundance of superoxide dismutase‐1/‐2, catalase, and thioredoxin were reduced with aging. Proteins participating in either mRNA degradation or pre‐mRNA splicing were significantly increased in old mice MVs, whereas protein stabilizing proteins decreased. Glycolytic proteins were not affected in middle age, but the relative abundance decreased in MVs of old mice. Although most of the 41 examined proteins composing mitochondrial Complexes I‐V were reduced in old mice, six of these proteins showed a significant reduction in middle‐aged mice, but the relative abundance increased in fourteen proteins. Nidogen, collagen, and laminin family members as well as perlecan showed differing patterns during aging, indicating BM reorganization starting in middle age.ConclusionsWe suggest that increased oxidative stress during aging leads to adverse protein profile changes of brain cortical MVs that affect mRNA/protein stability, BM integrity, and ATP synthesis capacity.
Differential Regulation of Retinoic Acid Signaling in Fanconi Anemia
Authors: Blaize Justin L. , Henrikson Kathryn A. , Noori Bahaa M. , Ardito Alan A. , Hunter Kelsey P. , Atoyan Janet A. , Howlett Niall G. 
Published: May 2022

Fanconi anemia (FA) is a rare genetic disease characterized by heterogeneous congenital defects, and increased risk for early‐onset bone marrow failure and cancer. Biallelic mutations in any one of 23 genes leads to FA. In vitro experiments have established a coordinated role for the FA gene network in the repair of DNA interstrand crosslinks (ICLs). However, the endogenous source(s) of DNA damage and chromosome instability in FA patients remain largely unknown. Recent studies have suggested that the FA proteins may play an important role in the repair of DNA damage caused by genotoxic reactive aldehydes produced during cellular metabolism, e.g., acetaldehyde and formaldehyde.To discover novel metabolic pathways linked to FA, we recently performed RNA‐seq analysis on telomerase‐immortalized mutant and functionally complemented FA‐D2 (FANCD2‐/‐) patient cells. Gene ontology enrichment analysis uncovered dysregulation of retinoid metabolism and retinoic acid signaling in mutant FA‐D2 (FANCD2‐/‐) cells. For example, we observed increased expression of the retinoid metabolism genes RDH10, ALDH1A1, and CYP26B1, in the absence of FANCD2. RDH10 converts retinol to retinaldehyde; ALDH1A1 converts retinaldehyde to retinoic acid; CYP26B1 degrades excess retinoic acid. In addition, we observed altered expression of multiple retinoic acid regulated genes including RARA, EGR1, and TGM2, and several members of the HOX, PAX, and SOX gene families. Using immunoblotting and qPCR, we have validated differential expression of many of these genes. We have also observed altered retinoid metabolism in Fancd2‐/‐ mouse cells. Furthermore, in preliminary flow cytometry experiments, we have determined that FA‐D2 (FANCD2‐/‐) patient cells exhibit increased aldehyde dehydrogenase activity.These findings are potentially highly significant and suggest that dysregulated retinoid metabolism and retinoic acid signaling ‐ as well as retinaldehyde‐associated genotoxicity ‐ might be key molecular features of FA. Further characterization of this newly discovered link could pave the way for the discovery of new therapeutic targets for FA patients.
High Throughput Screening of Protein Arginine N‐Methyltransferases – Filter Binding and Phosphor Screening (FBAPS) assay
Authors: Rowley Michael J. , Taylor Shona A. , Frankel Adam 
Published: May 2022

Study ObjectiveWe seek to develop a cost‐effective assay to screen protein arginine N‐methyltransferase (PRMT) enzyme activity in a high throughput manner by combining P81 filter binding with phosphor imaging.HypothesisA combination of the P81 filter binding assay and phosphor screening (FBAPS) will provide improved throughput for testing inhibitors against PRMTs while eliminating the economic and environmental costs incurred with current PRMT assays.Statement of methodsRecombinantly‐expressed PRMT1 and coactivator‐associated arginine methyltransferase 1 (CARM1) were used to develop the FBAPS assay using GST fusions of polyA binding protein 1 (PABP1(437‐488)) and glycine‐ and arginine‐rich (GAR) protein as substrates, respectively, and radiolabelled S‐adenosyl‐L‐[methyl‐14C]‐methionine as cofactor. Methylation reactions were spotted onto P81 filter paper in a dot blot apparatus and radioactive signal was measured both by phosphor imaging and liquid scintillation counting. Kinetic parameters (KM, kcat) for enzymes and substrates were determined, and IC50 values were obtained for well‐characterized inhibitors S‐adenosyl‐L‐homocysteine (SAH), MS023, EPZ020411, and Diamine 12 using FBAPS.Summary of ResultsFBAPS yielded kinetic parameters with no statistically significant differences to what was obtained using liquid scintillation counting. The IC50 values obtained by the FBAPS assay for PRMT1 and CARM1 were comparable to values reported in literature.Statement of ConclusionsThe FBAPS assay is a modification to the P81 filter binding assay with a dot blot apparatus that allows for parallel processing of samples in a multi‐well format, dramatically increasing throughput. Signal detection by phosphor imaging offers a cost effective and quantitative method that can be used to screen several inhibitors simultaneously against PRMT enzymes with high accuracy.
Direct effects of anti‐inflammatory medication on barrier function in intestinal epithelial cells
Authors: Richter Konstantin , Kollmann Catherine , Burkard Natalie , Germer Christoph‐Thomas , Schlegel Nicolas , Meir Michael 
Published: May 2022

An impaired intestinal epithelial barrier (IEB) is a hallmark in the pathogenesis of inflammatory bowel diseases (IBD), including ulcerative colitis and Crohn’s disease. Most of the IBD patients are treated with anti‐inflammatory reagents including glucocorticoids, anti TNFα (tumor necrosis factor alpha) antibodies and mesasalazine. While the Immune‐modulating effects of those therapies are well documented, we hypothesized whether there are direct effects of those reagents on enterocytes and the IEB.To address this hypothesis we used Caco2 cells as well as 2D and 3D murine organoids and performed analysis of intestinal epithelial barrier function. Inflammation‐induced alterations were mimicked using cytomix for 24h (TNFα, Interleukin 1 beta and Interferon gamma). The enterocytes were treated with 100ng/ml anti‐TNFα antibody Infliximab, 1mM prednisolone or 5mM mesasalazine, respectively following the challenge with cytomix. Epithelial permeability as revealed by measurements of 4kDa FITC Dextran flux and of transepithelial electric resistance showed that Infliximab and a pre‐incubation with prednisolone for 24h attenuated inflammation‐induced breakdown of the IEB. This was not the case after application of mesasalazine or a simultaneous incubation of prednisolone. The functional changes in intestinal epithelial barrier function following incubation with Cytomix were paralleled by alterations in the expression of “leaky” tight junction protein Claudin2, as revealed by Western blots. Furthermore, immunostainings demonstrated an altered distribution of barrier sealing proteins such as Claudin1, Desmoglein2 and E‐Cadherin. All of these effects were blocked by infliximab and prednisolone.In summary, our data indicate that infliximab and prednisolone have direct effects on the IEB that may contribute to the efficacy in the treatment of IBD.
Design and Synthesis of Bi‐aryl Methylated Lactam Derivatives to Inhibit the BRD7 Bromodomain Function in Prostate Cancer
Authors: Ordonez‐Rubiano Sandra Carolina , Maschinot Chad , Dykhuizen Emily 
Published: May 2022

Targeting bromodomain‐containing proteins has emerged as a promising therapeutic strategy and multiple clinical studies indicate that bromodomain (BD) inhibitors can act as anticancer and anti‐inflammatory agents. Bromodomain‐containing protein 7 (BRD7) is a subunit of the SWI/SNF chromatin remodeling complex, which is involved in the fine‐tuning of gene expression through changes in the organization of DNA. It regulates the progression of different types of cancers such as nasopharyngeal carcinoma and hormone‐dependent breast and ovarian cancers. BRD7 is also implicated in progression of prostate cancer, another hormone‐dependent malignancy; however, the role of BRD7 in this type of cancer is undefined. (Figure 1) We have characterized BRD7 as an oncogenic driver across a variety of prostate cancer cell lines, implicating BRD7 inhibitors as a potential therapeutic strategy to combat therapy resistant cancers.To develop inhibitors of BRD7 we are developing small molecules that bind selectively to the bromodomain of BRD7. The main challenge when developing BRD7 BD inhibitors is the high similarity to the BRD9 BD, its closest homolog. Several dual inhibitors of BRD7 and BRD9 have been developed, all of them being more selective for the BRD9 BD over the BRD7 BD. Using the X‐ray crystal structure of both BDs bound to a BRD9‐selective inhibitor, we identified an accessible hydrophobic region in the binding pocket of BRD7 that is not present in BRD9, which we hypothesized could be exploited to develop BRD7‐selective inhibitors. Based on this structural difference in the BRD7 binding pocket, we designed a library of compounds that share a bi‐aryl methylated lactam core. (Figure 2)From in silicomolecular docking simulations and in vitro affinity screenings, we have identified several ligands selective for BRD7. These compounds also display potent anti‐proliferative effects in prostate cancer cell lines, providing further evidence for proposing BRD7 as a therapeutic target in prostate cancer. Our on‐going work focuses on optimizing the potency and solubility properties of BRD7 inhibitors, and characterizing their selectivity using cell‐based models.
Mapping and specific viral targeting of peripheral pancreatic innervation
Authors: Jimenez Gonzalez Maria , Li Rosemary , Pomeranz Lisa E. , Alvarsson Alexandra , Marongiu Roberta , Hampton Rollie F. , Vasavada Rupangi C. , Schwartz Gary J. , Stanley Sarah A. 
Published: May 2022

Neuronal inputs from central nervous system (CNS) are essential in regulating pancreatic endocrine function. It has been shown that perturbation of CNS activity alters pancreatic hormone release. CNS and peripheral organs constantly communicate through the peripheral nervous system (PNS), which can be divided into autonomic efferent (parasympathetic and sympathetic) and afferent sensory nervous systems. While we know that the pancreas is densely innervated by the PNS, we lack a comprehensive map of peripheral ganglia providing inputs to the pancreas. These data could provide crucial information about pancreatic function. Published studies suggest parasympathetic inputs to the pancreas lower blood glucose by enhancing insulin secretion. On the other hand, sympathetic action results in increased blood glucose levels by stimulating glucagon secretion and inhibiting insulin. However, these studies are based on electrical stimulation or transection of peripheral splachnic and vagal nerves that may affect the activity of other metabolically active organs (liver, stomach, intestine,). Highly targeted modulation of specific pancreatic nerve activity in health and pancreatic diseases requires new approaches.In the present project, we provide a detailed map of the distribution of peripheral pancreas‐innervating neurons using retrograde tracing, tissue clearing and 3D imaging techniques (Idisco+). We show that a substantial pancreatic innervation (Fig 1b) with significant neuronal populations in sympathetic, parasympathetic and sensory ganglia projecting to pancreatic tissue (Fig 1 c,d). In addition, we achieved targeted gene delivery to specific pancreatic efferent and afferent pathways by optimizing adeno‐associated viral serotypes (AAV), promoters, titers and delivery (Fig2 a‐d). By utilizing our optimized approach, we show that highly targeted chemogenetic neural activation of intrapancreatic parasympathetic cholinergic neurons increased plasma insulin and significantly improved glucose tolerance in male mice (Fig 2 e,f). Additionally, using a dual viral strategy to specifically target pancreas‐projecting sympathetic neurons, we demonstrated that targeted activation of pancreas‐projecting sympathetic neurons impaired glucose tolerance in male mice (Fig 2g,h).Our innovative viral approaches allow highly targeted gene expression and neuromodulation of defined pancreatic nerves. These methodologies will allow examination of functional roles of pancreatic parasympathetic and sympathetic innervation in glucose metabolism as well as in digestion (exocrine function). Our work also allow for future studies to determine the specific contribution of pancreatic innervation to metabolic diseases, pancreatic inflammation and cancer.