bluuebunny/crossref_metadata_2025 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

Introduction

A 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.

Hypothesis

Impaired BK channel activation was responsible for the impaired cerebral autoregulation in hypertension‐induced dementia.

Methods

Male 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.

Results

Pressure‐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.

Conclusions

This 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.
Role of Transcription Factor Pdc2 in the Thiamine Signal Transduction Pathway in Candida glabrataand Saccharomyces cerevisiae
Authors: Wykoff Dennis , Iosue Christine , Lang Emma , Loshnowsky Anita , Ugras Julia , Dottor Cory
Published: May 2022

Pdc2 is critical for the transcriptional response to thiamine starvation in S. cerevisiae and C. glabrata. In this study, we determined how Pdc2 drives transcription of thiamine regulated (THI) promoters using genetic analyses, RNA‐sequencing, chromatin immunoprecipitation (ChIP)‐sequencing, and promoter‐YFP reporter constructs. We hypothesized that Pdc2 binds to THI promoters because the upregulation of THI genes requires Pdc2, but the Pdc2 binding site was unknown. Previously, we identified conserved sequences that are necessary for transcriptional upregulation in low thiamine conditions. Here, we determine that while PDC2 is essential in standard medium in S. cerevisiae, PDC2 in C. glabrata is not, but the essentiality of ScPDC2 is suppressible by overexpression of PDC1. We demonstrate that CgPDC2 is unable to suppress the Scpdc2Δ lethality, indicating that the two Pdc2 proteins have different specificities. We perform RNA‐seq on both species where is PDC2 is deleted, with either ScPDC2 or CgPDC2 supplied on a plasmid, and observe that Pdc2 regulates a relatively small subset of genes related to thiamine biosynthesis (THI genes), and both Proteins are partially functional in either species. Performing ChIP, we find that ScPdc2 binds weakly to DNA, whereas CgPdc2 strongly enriches at some THI promoters. Using ChIP‐seq, we demonstrate that CgPdc2 binds in the same location as a conserved sequence necessary for upregulation. With promoter‐YFP reporter constructs, we identify ~100 bp regions in the CgPMU3, CgTHI20, and ScPDC5promoters that are sufficient to confer regulation to a non‐thiamine regulated promoter in both species, and identify a DNA sequence that likely binds Pdc2. The sequence is repeated twice in the ScPDC5promoter and is a 20/22 bp duplicate containing a palindromic site. Finally, using an electrophoretic mobility shift assay (EMSA), we demonstrate that the DNA binding domain of Pdc2 from either species binds this sequence in the promoters of both species. This work defines the role of Pdc2 in transcription and opens up new questions. Specifically, it is unclear why the two species’ Pdc2 proteins have different affinities for DNA in vivo, and how CgPdc2 upregulates the CgPMU3 promoter. CgPMU3 is a recently evolved thiamine‐regulated gene which is PDC2 dependent, but ChIP‐seq data indicates that it is not closely bound by Pdc2. These results suggest that there are many ways for Pdc2 to regulate transcription.
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

Background

Aging 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.

Methods

Cortical 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.

Results

Most 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.

Conclusions

We 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.
The Epithelial‐Specific ER Stress Sensor IRE1β Enables Host‐Microbiota Crosstalk to Affect Colon Goblet Cell Development
Authors: Grey Michael J. , De Luca Heidi , McCormick Beth A. , Turner Jerrold R. , Lencer Wayne I.
Published: May 2022

Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express IRE1β (Ern2), a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor IRE1α. How IRE1β functions at the host‐environment interface and why a second IRE1 paralogue evolved remain incompletely understood. Using conventionally raised and germ‐free Ern2‐/‐ mice, we found that IRE1β was required for microbiota‐induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced IRE1β expression. IRE1β acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although IRE1α can also splice Xbp1 mRNA, it did not act redundantly to IRE1β in this context. By regulating assembly of the colon mucus layer, IRE1β further shaped the composition of the gut microbiota. Mice lacking IRE1β had a dysbiotic microbial community that failed to induce goblet cell development when transferred into germ‐free wild type mice. These results show that IRE1β evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.
Investigating the Application of Size‐Exclusion Chromatography to Purify Bacterial Extracellular Vesicles
Authors: Gaborski Thomas , Guernsey Cara , Henretta Sara , Hosseinifakhr Atiyeh , Torabian Pantea , Kasper Anna , Michel Lea
Published: May 2022

Sepsis is a leading cause of death as well as the most expensive condition to treat in U.S. Hospitals. Sepsis occurs when host proinflammatory response is unusually elevated, in part due to bacterial molecules secreted in bacterial extracellular vesicles (EVs). Due to their abundance, robustness, and unique characteristics between bacterial species, these bacterial EVs can potentially serve as a molecular diagnostic biomarker for Sepsis. Thus, considering the importance of diagnosing Sepsis accurately and early, a high‐purity purification technique is necessary for isolating bacterial EVs for further characterization. We propose that size exclusion chromatography (SEC) can be used to isolate bacterial EVs from the sera of sepsis patients. Additionally, we hypothesize that SEC columns packed with CL‐4B Sepharose beads will isolate bacterial EVs from heterogeneous samples with a finer resolution than columns packed with CL‐2B Sepharose beads. Previous literature has shown that CL‐2B SEC columns successfully separate human EVs from smaller particles according to size in heterogeneous samples. We have extended this work to investigate bacterial EV separation and CL‐4B Sepharose beads using three different systems: fluorescent beads and rhodamine, simulated sera, and bacterial EVs. Together, these preliminary experiments have shown that both CL‐2B and CL‐4B successfully separate bacterial EVs from smaller particles in heterogeneous samples. Future work will likely show that CL‐4B SEC columns separate bacterial EVs from other particles in patient samples with a higher resolution compared to CL‐2B beads due to their smaller pore sizes. Additional future studies will combine the use of these SEC approaches with ultrathin membranes in a single‐pass microfluidic device.
Rotavirus infection elicits host responses and amplifies viral replication via P2Y1 purinergic signaling
Authors: Engevik Kristen , Kawagishi Takahiro , Greenberg Harry , Hyser Joseph
Published: May 2022

Rotavirus causes life‐threatening diarrhea in children, resulting in ~130,000 deaths each year. Rotavirus infects a limited number of cells at the tips of the villi in the small intestine. Yet, rotavirus dysregulates cells far away from the site of infection. We recently identified, using simian (SA11) and rhesus rotavirus (RRV), that infected cells release the purinergic signaling molecule ADP, which binds to the P2Y1 receptor on nearby uninfected cells. Furthermore, using the in vivo mouse model, mild rotavirus diarrhea in mouse pups was alleviated by daily treatment with a P2Y1 inhibitor. To elucidate the role of purinergic signaling via P2Y1 receptors during rotavirus infection, we used the mouse‐like rotavirus (D6/2) to investigate the effects of purinergic signaling in the context of severe rotavirus infection in vivo. C57Black6 mouse pups were orally gavaged D6/2 rotavirus at day 4‐6 of age and assessed over the course of 5 days. Beginning at day 1 post infection, infected pups were treated daily by oral gavage with saline or 4mg/kg MRS2500, a selective P2Y1 antagonist. Stool was collected and scored for diarrhea daily prior to each treatment. Pups were euthanized and small intestine tissue was collected at 3 and 5 days post infection for immunostaining, qRT‐PCR and luminal contents. Similar to mild rotavirus infection, treatment of D6/2‐infected mouse pups with MRS2500 results in decreased severity and incidence of diarrhea. MRS2500 treated pups also exhibit decreased luminal serotonin and chloride content compared to control infected pups. Together, these results confirm that P2Y1 signaling is also involved in the pathogenesis of a homologous murine rotavirus strain. Viral stool shedding, assessed by qRT‐PCR for rotavirus gene 11 levels, revealed that MRS2500 treated pups had significantly lower viral shedding starting at day 4 post infection compared to saline treated pups, which suggests P2Y1 signaling may amplify rotavirus replication. Collectively these findings point to the conserved role of purinergic signaling in the pathophysiology of rotavirus infection, and indicate P2Y1 is a new candidate for host‐targeted therapeutics that could have both antiviral and antidiarrheal effects against rotavirus pathophysiology.
An Antagonist of KSR1‐Driven Adaptive Resistance to Clinical RAS‐MAPK Inhibitors
Authors: Chow Arthur , Yerabolu Jayasudhan R. , Reyes Kyna , Scopton Alex , Dar Arvin C.
Published: May 2022

Kinase Suppressor of RAS (KSR), RAF and MEK function downstream of RAS and upstream of ERK to mediate growth factor and receptor tyrosine kinase (RTK) signaling. Significant clinical breakthroughs have occurred in targeting mutant oncogenes and vulnerabilities in the RAS‐MAPK pathway, including against KRAS‐G12C, BRAF‐V600E, and MEK1/2. However, these drugs have revealed that the inhibition of individual RAS‐MAPK signaling molecules can lead to relief of negative feedback signaling and paradoxically amplify RAS‐MAPK output over time. Indeed this mechanism, sometimes referred to as ‘rebound signaling’, has arose as a key driver of adaptive drug resistance to several classes of therapies. We hypothesize that KSR1, an isoform implicated in several Ras‐driven cancers, may function in the resistance mechanisms to Ras‐MAPK inhibition therapy. To test this hypothesis, we evaluate KSR1 as a driver of resistance to RAS‐MAPK inhibition and developed a selective KSR1 antagonist, K‐X1. We discovered strong synergy between K‐X1 and MEK inhibition in models of adaptive resistance. In vivo target engagement assays suggest that K‐X1 operates through alterations in the stability and interactions of KSR1‐mediated complexes. Our work highlights a potential strategy to overcome adaptive resistance to therapies targeting the MAPK pathway via KSR1.
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 Objective

We 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.

Hypothesis

A 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 methods

Recombinantly‐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 Results

FBAPS 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 Conclusions

The 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.
Transient local secondary structure in the intrinsically disordered C‐term of the Albino3 insertase
Authors: Okoto Patience S. , Furr Mercede , Baucom Dustin , Kumar Vivek , Thallapuranam Suresh K. , Heyes Collin , Mahmoud Moradi , Henry Ralph
Published: May 2022

Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light‐harvesting complex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma‐exposed C‐terminus (Alb3‐Cterm) that is responsible for binding the LHC‐loaded transit complex before LHC membrane insertion. Alb3‐Cterm has been reported to be intrinsically disordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, and the functional roles of its four different motifs have been debated. Using a novel combination of experimental and computational techniques such as single‐molecule fluorescence resonance energy transfer, circular dichroism with deconvolution analysis, site‐directed mutagenesis, trypsin digestion assays, and all‐atom molecular dynamics simulations in conjunction with enhanced sampling techniques, we show that Alb3‐Cterm contains transient secondary structure in motifs I and II. The excellent agreement between the experimental and computational data provides a quantitatively consistent picture and allows us to identify a heterogeneous structural ensemble that highlights the local and transient nature of the secondary structure. This structural ensemble was used to predict both the inter‐residue distance distributions of single molecules and the apparent unfolding free energy of the transient secondary structure, which were both in excellent agreement with those determined experimentally. We hypothesize that this transient local secondary structure may play an important role in the recognition of Alb3‐Cterm for the LHC‐loaded transit complex, and these results should provide a framework to better understand protein targeting by the Alb3‐Oxa1‐YidC family of insertases.
Rapid Intravenous Injection of Fentanyl (FNT) Causes a Vagal‐Mediated Lethal Sudden Ventilatory Arrest in Anesthetized Rats
Authors: Zhuang Jianguo , Shi Shan , Xu Fadi
Published: May 2022

Opioids have been used for analgesia and anesthesia mainly via activation of central opioid mu‐receptors (MORs). It also causes overdose deaths due to ventilatory failure, in which FNT is the deadest. Rapid intravenous (IV) injection of overdose FNT triggers a sudden death within a few minutes or even seconds, but the relevant mechanism remains unclear. Because bolus IV injection of FNT at a low dose produced a vagal‐mediated brief apnea (Zhuang et al, AJP 2012), This study was aimed to characterize the cardiorespiratory disorders responsible for the sudden death and determine the vagal role in generating the lethal ventilatory arrest. EMGs of external and internal intercostal (EMGEI and EMGII), thyroarytenoid and superior pharyngeal constrictor muscles (EMGTA and EMGSPC) were simultaneously recorded before and after IV injection of FNT in the anesthetized vagal intact and vagotomized rats. The optical fiber scope of an oral video camera was applied to visualize the vocal and pharyngeal responses. Immunohistochemical approach was used to define the presence of MOR expression in the pulmonary vagal sensory neurons retrogradely labeled by DiI. FNT induced an apnea or a lethal ventilatory arrest in a dose‐dependent manner associated with hypotension and bradycardia. The apnea/ventilatory arrest was characterized by silence of EMGEI with tonic discharges of EMGII, EMGTA, and EMGSPC, i.e., the central apnea (expiratory) coupled with vocal closure and pharyngeal constriction/collapse and chest wall rigidity (triple‐apnea). Vagotomy abolished the evoked cardiorespiratory responses. MOR expression in vagal pulmonary neurons, especially C‐neurons of the nodose and jugular ganglia. Our results suggest that rapid IV injection of FNT at a high dose uniquely causes a vagal‐mediated triple‐apnea, leading to the sudden death in anesthetized rats.
Target Discovery Platform Identifies Cardioprotective Genes Using Hibernation Omics Data
Authors: Sajdak Benjamin , Altay Gokmen , Song Rui , Galeas Jacqueline , Tran Evelyn , Zehnder Ashley , Goodman Linda , Grabek Katharine
Published: May 2022

Over the course of their evolution, many mammals have naturally solved a number of human biomedical problems. The genes underlying their protective strategies are likely neither novel nor species‐specific, but rather shared among all mammals, including humans. Identifying and validating these genes could therefore lead to new therapeutic treatments for human clinical indications. To this end, we developed a target discovery platform which mines multi‐omics data from mammals resistant to human pathologies in order to identify novel therapeutic genetic targets. More specifically, our platform is built upon transcriptomic and epigenomic data from bio‐banked 13‐lined ground squirrel (13‐LGS) tissues collected at precise seasonal and physiological time‐points across their annual cycle of hibernation. Our platform also incorporates myriad genomic, transcriptomic, and proteomic datasets publicly available for this and other disease‐resistant species, as well as human genomics and transcriptomics data. Here we sought to identify novel genetic targets protective against cardiac fibrosis. While the hearts of 13‐LGS experience repeated bouts of ischemia‐reperfusion during hibernation, long‐term fibrotic scarring resulting from this type of injury is not detected. To identify protective targets, we first generated RNASeq data from hearts of 13‐LGS across 11 different seasonal and physiological groups (n≥5 per group). After obtaining transcript abundance counts for each sample library, we performed weighted correlation gene network analysis (WGCNA) to identify modules of transcripts co‐expressed among the different physiological groups. One resulting WGCNA module contained TGFB1 (transforming growth factor beta 1), a known central mediator of fibrogenesis. Knockdown (KD) of this module’s hub gene, designated here as FAUN211G, as well as the second top‐ranking gene, designated FAUNA212G, significantly decreased collagen deposition in an in vitro 2D model of cardiac fibrosis using primary human cardiac fibroblasts. We next perturbed these genes in an in vitro 3D spheroid model of human cardiac fibrosis and detected improved parameters of cardiac function. Finally, we tested small molecule inhibitors on FAUNA211G in both 2D and 3D in vitro models; we identified significant reductions in myofibroblast activation, proliferation and extracellular matrix deposition. Both FAUNA211G and FAUNA212G were previously unknown to be involved in cardiac fibrosis. In sum, we demonstrate that network analysis of the hibernator’s transcriptome is a strategy to identify novel target genes for treatment of human clinical indications.
Role of ConE’s ATPase Motifs in Protein‐Protein Interactions within the Conjugation Machinery of Bacillus subtilis
Authors: Toyoda Hunter C. , Chen Sirui , Berkmen Melanie B.
Published: May 2022

Conjugation is the transfer of DNA from one bacterial cell to another. Conjugation is a major form of horizontal gene transfer, resulting in the spread of genes that play roles in antibiotic resistance, virulence, symbiosis and metabolism. ICEBs1 is an integrative and conjugative element of Bacillus subtilis. The conjugation machinery that transfers DNA is a Type 4 Secretion System (T4SS). The ICEBs1 T4SS includes the proteins ConB, ConC, ConD, ConE, ConG, ConQ, and CwlT. The focus of our work is the ConE peripheral membrane protein, which belongs to the VirB4 family of ATPases. We analyzed whether ConE interacts with any of the other ICEBs1 T4SS proteins using bacterial two hybrid (BACTH). We found that ConE interacts with itself, ConB, and ConQ. We used site‐directed mutagenesis of the conE gene to make alanine substitutions within five conserved residues within ATPase motifs of the ConE protein that have previously been shown to be critical for mating. We used BACTH to determine if these mutations affect ConE’s protein interactions. We discovered that interaction of ConE with itself or ConB is unaffected by mutations in any of the ATPase residues tested, but that the ConE‐ConQ interaction depends on two conserved residues within the ATPase motifs. Our research sheds new light on the role of conserved residues within the ATPase motifs of ConE.
Increased Fatty Acid Synthesis and Catabolism Supports Metastatic Breast Cancer Cell Migration
Authors: Andolino Chaylen J. , Teegarden Dorothy
Published: May 2022

Lipid accumulation is positively associated with breast cancer aggressiveness and poorer clinical outcomes. Despite this observation, mechanisms underlying the increase and role of stored lipid in breast cancer progression remain incompletely understood. The accumulation of triacylglycerol (TAG) is dependent on the balance of uptake, synthesis, and utilization of fatty acids (FAs). In the current study, we utilized non‐metastatic MCF10A‐ras and metastatic MCF10CA1a human breast cancer cells to determine differences in TAG storage and catabolism for sustaining migration—a critical step in the metastatic cascade. Our results demonstrate that MCF10CA1a cells have 90% more TAG than MCF10A‐ras (TAG Assay and Transmission Electron Microscopy), although no significant difference in 14C‐palmitate uptake. MCF10CA1a cells have greater FA synthesis as shown by greater incorporation of 13C‐acetate (60%), 13C‐glucose (65%), and 13C‐glutamine (50%) into palmitate compared to the MCF10A‐ras cells (LC‐MS/MS), as well as higher protein levels of FA synthase (FASN). Additionally, MCF10CA1a cells display greater flux of uniformly‐labeled 13C‐glucose and 13C‐glutamine to the FA synthesis precursor, citrate, and lower intracellular citrate pool size compared to MCF10A‐ras. In addition to increased FA anabolism, MCF10CA1a cells rely on FA oxidation (FAO) for cellular migration compared to MCF10A‐ras cells, as determined by inhibiting the rate‐limiting enzyme of FAO, carnitine palmitoyltransferase 1, with etomoxir (transwell and wound‐healing assays). Pretreatment with the FASN inhibitor TVB‐3166 (3 d) significantly reduced TAG levels and subsequent migration by 67% compared to vehicle‐treated cells. Similarly, pretreatment with inhibitors of the TAG‐synthesizing enzymes diacylglycerol O‐acyltransferase 1 and 2 reduced TAG accumulation and subsequent migration (75%) of MCF10CA1a cells compared to vehicle. Together, the data suggest that FA synthesis may contribute to TAG storage necessary to sustain breast cancer migration. To test this hypothesis, we inhibited the initial enzyme of the TAG lipolysis pathway, adipose triacylglycerol lipase (ATGL; inhibitor ATGListatin), and measured migration. Inhibition of ATGL alone reduced MCF10CA1a cell migration (20% decrease compared to vehicle), and addition of the FAO inhibitor (etomoxir) provided no further effect. Our study indicates that metastatic MCF10CA1a cells accumulate FAs by increasing de novo lipogenesis, store them as TAG, and that catabolism of these stores drives FAO‐dependent migration.
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.
Characterizing Subsynaptic Myonuclei in Aged Muscle
Authors: Ruiz Lloyd
Published: May 2022

Introduction

During aging, the neuromuscular junction (NMJ) undergoes structural fragmentation and functional deficits that alter neuromuscular communication, impair muscle contraction, and contribute to muscle atrophy. At the NMJ, myonuclei accumulate and localize in a fashion that juxtaposes three‐to‐six myonuclei in the postsynaptic region. Although the entire role of these so‐called subsynaptic myonuclei remains enigmatic, previous literature suggests they contribute to gene regulation of synapse‐specific proteins required for maintaining the structural integrity of the synaptic architecture. Interestingly, a recent report find that NMJs in muscles of aged mice contain fewer subsynaptic myonuclei compared with muscles of adult mice in parallel with degenerating or degenerated endplates. Furthermore, disturbance of the nuclear envelope by in‐vivo knock‐out of Lmna appears to accelerate NMJ degeneration and reduces the number of myonuclei confined to the endplate. Therefore, with the observed reductions in subsynaptic myonuclei in degenerated NMJs, our study aims to further examine and characterize subsynaptic myonuclei and their role in NMJ degeneration.

Methods

Gastrocnemius muscles were harvested from Young (6 months) and Aged (28 months) wild type mice and 6‐ and 12‐month‐old Sod1KO mice (N=3/group) and briefly fixed in 4% PFA for 10 minutes. Myofiber bundles were mechanically teased under a dissecting microscope and subjected to immunohistochemistry for visualization of motor neurons (neurofilament‐NF and synaptic vesicle glycoprotein 2‐SV2), motor end plates (alpha‐bungarotoxin), and myonuclei (nesprin 1 and 4’,6‐diamidino‐2‐phenylindole‐DAPI). Myofiber bundles were then mounted onto a slide and imaged via High‐sensitivity confocal microscopy. Captured images were then 3D projected in FIJI for assessment and quantification of subsynaptic myonuclei, innervation status, and post synaptic area assessed via aNMJ morph.

Results

In young mice with primarily fully innervated endplates, the number of myonuclei associated with the endplate was lower than the number of synaptic myonuclei in muscles of aged and Sod1KO mice, both of which contain greater numbers of partially and non‐innervated endplates.

Conclusion

There may be a greater number of subsynaptic myonuclei localized to degenerating or degenerated endplates, although whether the change in nuclear number is a consequence of NMJ degeneration or contributes to the denervation cannot be established from this study.