bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hypoventilation in the Mdx Mouse Model of Duchenne Muscular Dystrophy
Authors: Burns David P , Arijit Roy , McDonald Fiona , Wilson Richard J , O'Halloran Ken D 
Published: November 2023

Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by a lack of the structural protein dystrophin. Patients have severe muscle wasting and chronic respiratory insufficiency. Hypoventilation and sleep disordered breathing are both common features of the disease and are known to culminate in periods of hypoxaemia and hypercapnia. There is a paucity of information regarding the control of breathing in animal models of DMD. We measured ventilation, oxygen consumption (VO2) and carbon dioxide (VCO2) production in freely behaving mdx (dystrophin deficient; n=11) and wild‐type (WT; n=10) mice at 8 weeks of age. qRT‐PCR was used to examine inflammation and hypoxia associated gene expression in diaphragm muscle from WT and mdx mice. Minute ventilation (VE; whole‐body plethysmography) was significantly reduced in mdx mice compared with WT controls (30% reduction). There was no significant difference in VO2 and VCO2 when expressed in absolute terms. Carotid sinus nerve unitary discharge was significantly depressed during normoxia in mdx (n=6) compared with WT (n=6) ex vivo perfused preparations. NF‐kB and TNF‐α mRNA expression were both significantly increased in mdx diaphragm compared with WT. HIF‐1α was significantly increased, while HIF‐2α was significantly decreased in mdx diaphragm muscle. Young mdx mice hypoventilate during air breathing (decreased VE/VCO2), which likely relates to reduced carotid body discharge during normoxia. Mdx diaphragm muscle is in a pro‐inflammatory state and there is differential expression of hypoxia associated genes. We reveal that respiratory control is altered in the mdx mouse model of DMD at a young age.Support or Funding InformationFunded by the Department of Physiology, School of Medicine, University College Cork and Muscular Dystrophy Ireland.
Rewiring of spinal respiratory neural network via cervical glutamatergic interneurons preserves respiratory function in progressive cervical spinal cord compression injury (CSM)
Authors: Satkunendrarajah Kajana , Karadimas Spyridon K , Khazaei Mohamad , Samson Patricia , Fehlings Michael G 
Published: November 2023

Cervical spinal cord injury (SCI) disrupts the spinal respiratory neural circuitry leading to the loss of diaphragmatic and intercostal muscle function. While acute traumatic SCI leads to significant ventilatory impairment, CSM, in which the cervical spinal cord is progressively compressed over time, results in milder respiratory dysfunction despite the significant disruption of the cervical neural network. The chronic and progressive nature of this disease modifies the respiratory neural network to limit respiratory insufficiency.Using a unique mouse animal model of CSM, we found a significant loss of PMNs (retrograde labeled with CTb) that innervate the main inspiratory muscle, yet these animals do not exhibit severe respiratory insufficiency similar to what is observed in human CSM patients. Progressively increased Vglut2 positive boutons on the preserved PMNs indicates that despite the significant loss in the number of PMNs, respiratory motor output is maintained via compensatory and progressive increases in glutamatergic input onto preserved PMNs. While the PMNs were decreased following CSM there was an increase in the number of prephrenic cervical interneurons labeled with pseudorabies virus‐152 (PRV‐152). We set out to specifically delineate the extent of glutamatergic presynaptic inputs directly arising from rVRG neurons and those that are relayed via prephrenic cervical interneurons in CSM. To assess direct monosynaptic excitatory glutamatergic projections arising directly from rVRG and cervical spinal cord onto PMNs, a modified rabies virus (Rb) missing the glycoprotein but carrying a GFP protein (Rb‐ΔG‐GFP) with adeno associated virus expressing the Rabies‐Glycoprotein (RG, essential for subsequent trans‐synaptic transfection), was injected into the diaphragm of CSM and sham Vglut2::cre;tdtomato mice. We observe significantly altered input from glutamatergic monosynaptic inputs from rVRG neurons and those from prephrenic cervical interneurons onto PMNs during CSM. In conclusion, this study provides novel insights into the alterations in spinal respiratory networks that occur in the setting of CSM and a greater understanding of the neural control of breathing and the compensatory changes that occur in neural circuits with chronic, graduated compression.Support or Funding InformationPVA
The Hairpin‐Hinge Model of Tat Protein Transport
Authors: Hamsanathan Shruthi , Bageshwar Umesh , Musser Siegfried 
Published: November 2023

The bacterial twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane. Biochemical evidence indicates that the TatBC receptor complex contains the signal peptide binding site and the interaction of the signal peptide with this complex triggers the assembly of a translocation channel. However, it is unclear how the signal peptide interacts with the receptor complex and how it enables the movement of cargo through the channel. Here we show using fluorescence quenching and disulfide crosslinking that TatBC complex inserts pre‐SufI as a hairpin that extends about half‐way across the membrane. Photo crosslinking studies indicated that the c‐terminal of the hairpin interacts with TatB. Furthermore, computational analysis of ~500 Tat signal peptides and docking simulations of TatBC and pre‐SufI revealed 3 common features of the hairpin structure: 1) a helical structure; 2) a helix destabilizing residue; and 3) a relatively disordered domain. A hinge motion at the hairpin bend would allow the signal peptide to be bound to TatC surface via double arginines (n‐terminal) and allow the cleavage site (c‐terminal) to migrate to the periplasmic side. Disulfide crosslinking of n‐ and c‐terminal hairpin ends inhibited the transport of the cargo suggesting a novel Hairpin‐Hinge Translocation for Tat pathwaySupport or Funding InformationNIH Grant GMO65534
OMENTAL HIGH ENDOTHELIAL VENULES AS CRITICAL NEUTROPHIL EXIT SITE IN PERITONITIS
Authors: Buscher Konrad , Wang Huiyu , Zhang Xueli , Ley Klaus , Sorokin Lydia , Song Jian 
Published: November 2023

Neutrophil recruitment from the blood into the peritoneal cavity is critical in acute peritonitis but precise mechanisms of extravasation are largely unknown. In this study we identified the greater omentum as major exit site in early phases of mono‐ and polymicrobial peritoneal inflammation. A novel intravital microscopy technique was developed to image the inflamed omentum showing that high endothelial venules, known for their role in constitutive lymphocyte homing, are the preferred vascular bed for neutrophil recruitment with a unique requirement of adhesion receptors. Specific inhibition of the omental pathway in mice leads to delayed neutrophil deployment in the peritoneum, increased bacterial load, and aggravates septic progression of bacterial peritonitis.Support or Funding Information‐ German Research Foundation grant to LS (CRC1009/A02, So285/9‐1, EXE1003)‐ German Research Foundation grant to KB (BU 3247/1)‐ Cells‐in‐Motion Cluster of Excellence Flexible Funds grant (FF‐2014‐07) to JS and KB‐ Grant of the Interdisciplinary Center for Clinical Research (IZKF) of the Medical Faculty of the University of Muenster to KB (SEED 04/12)
The Role of BCAR1 and FYN on CD133+ GBM stem cells
Authors: Yukselten Yunus , Bal Merve Gulsen , Ozkanca Seyma , Yildiz Dilara Akcora , Bunsuz Merve , Ugur Hasan Caglar , Sunguroglu Asuman 
Published: November 2023

OBJECTIVEGlioblastoma Multiforme (GBM) is the most prevalent and aggressive primary brain tumor. Glioblastoma stem cells (GSCs) are thought to play important roles in tumor initiation, establishment, and dynamic evolution. CD133 (prominin‐1), a transmembrane glycoprotein, has been shown to mark stem like cells in colon, prostate and GBM. The integrin signaling adaptors including BCAR1 (p130 Crk‐associated substrate; p130CAS and FYN (FYN Proto‐Oncogene, Src Family Tyrosine Kinase) are important players in cell invasion and migration. Moreover, BCAR1, p130CAS and FYN promotes cell proliferation, survival and motility, indicating a connection with tumorigenesis. In this study, we investigated the gene expression levels of cell adhesion molecules in both CD133+ GSCs and CD133− GBM cells.MATERIAL METHODSGBM primary cells were freshly isolated from glioblastoma tissue samples and cultured in DMEM supplemented with 10% fetal calf serum and 1% penicillin‐streptomycin at 37 °C in 5% CO2‐humidified incubator. We isolated CD133+ and CD133− cells from 10 GBM primary cells using MACS system. Following RNA isolation from those positive and negative cells, cDNA synthesis was performed. Finally, according to microarray protocol, cell adhesion array was applied. Gene expression levels were analyzed using the ‐delta delta Ct method. One‐sample t‐test was used to determine the mean fold changes (increase/decrease) of genes of interest between CD133+ and CD133− cells. Statistical analysis was performed using SPSS software for windows version 13.0.RESULTS & CONCLUSIONAccording to our results, compared to CD133‐GBM cells BCAR1 and FYN gene expression levels were found to be 3.96 and 6.05‐fold increased respectively in CD133+ GSCs (p<0.05).In summary, we found increased transcript levels of BCAR1 and FYN genes in CD133+ GSCs compared with CD133− cells. A deeper understanding of the gene expression signature of CD133+ GCSs might be helpful to improve the therapies against this tumor.This research has been supported by The Scientific and Technological Research Council of Turkey (No:114S189).
Biocompatibility Assessment of Green Synthesized Cu2O Decorated Graphene Nanosheets for Therapeutic Applications
Authors: Alshatwi Ali A , Athinarayanan Jegan , Periasamy Vaiyapuri Subbarayan , Lei David KY 
Published: November 2023

Nanotechnology offers incredible prospective for variety of future applications; specifically, several kind of nanostructures have been broadly applied for biomedical applications owing to their unique physical, chemical and biological properties. Currently, graphene‐based nanohybrids have received a considerable in diagnosis and therapeutic applications due to their tremendous aqueous processability, easy to cellular internalization, biocompatibility and facile surface functionalizability. In this present study, a facile synthetic route to fabricate Cu2O/graphene nanocomposites (Cu2O/G) was developed using green chemistry approach. The copper ions anchored surface of graphene oxide to Cu(OH)2/graphene oxide was prepared using NaOH. Subsequently, Cu(OH)2/graphene oxide was reduced to Cu2O/G using date palm fruit syrup. This Cu2O/G was analyzed adopting field emission scanning electron microscopy, Fourier transform infrared spectroscopy, X‐ray diffraction, and energy dispersive X‐ray spectroscopy. From a typical FESEM images, it is observed that Cu2O nanoparticles with diameters of about 50–70 nm were anchored on the wrinkled G sheets. The XRD pattern of Cu2O/G nanocomposites diffraction peaks can be exclusively indexed to the cubic phase Cu2O on G sheets. The Cu2O/G nanosheets show biocompatibility in human mesenchymal stem cells (hMSCs) up to 100 μg/mL. There is no major changes were observed in cellular and nuclear morphology of nanocomposite treated hMSCs. The Cu2O/G exhibits biocompatibility, and it can be a promising material for biomedical applications.Support or Funding InformationWe gratefully acknowledge the financial support of the Deanship of Scientific Research, King Saud University, Saudi Arabia (Grant No.: RGP‐VPP‐276)
Defining the Regulatory Role of Lipid Raft Microdomains During T cell Receptor Activation
Authors: Osigwe Chinweoke Crystal , Levine Alan D 
Published: November 2023

We have previously concluded that the heterogeneity of the lipid composition of the plasma membrane plays a critical role in the T cell receptor (TCR) activation signaling pathway. Our lab has presented that highly‐ordered, densely packed lipids in raft‐like microdomains are required for the activation of TCR complex. In particular, we have also shown that the early, enzymatic events associated with TCR activation are dependent on the lipid raft micro‐domains. While our results support the importance of these lipid raft micro‐domains in TCR signaling, no evidence is available to describe how these rafts are assembled and organized within the plasma membrane. The objective of our study is to define the three dimensional changes on the plasma membrane during TCR signaling pathway from the perspective of lipid rafts. The following techniques have been employed to investigate this objective. From whole blood, peripheral mononuclear cells (PBMCs) were isolated by Ficol‐hypaque density separation. Purified T cell lymphoblast were expanded from PBMC culture by a seven day feeding with interleukin‐2. Both purified T‐cell lymphoblast and Jurkat T cells were activated by cross‐linking the TCR with anti‐CD3/anti‐CD28 co‐stimulatory antibodies. Propagation of the signal was analyzed through immunoblot analysis through a phospho‐ERK, phospho‐LAT, and phospho‐Lck response. Lipid raft composition and structure will be probed with fluorescently labelled phospholipids that selectively associate with or exclude distinct regions of the plasma membrane, thus labeling either a raft or non‐raft domain. Localization and assembly of these fluorescently labelled phospholipids, balanced against the localization of protein components of the TCR signaling complex using fluorescently tagged antibodies, will be analyzed with confocal and deconvolution microscopy. The study's biological implication will address how TCR engagement within the context of a lipid raft may lead to an immune response or immune tolerance.Support or Funding InformationAmerican Physiological Society STRIDE Fellowship
Modulation of Lemur Tyrosine Kinase 2 (LMTK2) Expression by TPA Response Elements
Authors: Dey Isha A , Bradbury Neil A. 
Published: November 2023

Lemur Tyrosine Kinase 2 (LMTK2), a membrane anchored Serine/Threonine kinase, has been shown to be involved in a number of diseases, including cystic fibrosis and prostate cancer, as well as neuronal development and intracellular endocytic trafficking. Our previous studies have shown that LMTK2 is downregulated in prostate cancer, and that modulation of LMTK2 affects CFTR‐mediated chloride conductance at the plasma membrane of polarized epithelial cells. Moreover, CFTR, the Cl− selective anion channel mutated in cystic fibrosis has been found to a substrate for LMTK2 kinase activity. Based on these observations, we propose that LMTK2 to be a novel therapeutic target for both cystic fibrosis and prostate cancer. However, the precise mechanism to modulate the expression or activity of LMTK2 is not understood.In our current study, we have investigated the molecular mechanisms regulating LMTK2 gene expression. Since the promoter of a gene regulates its expression, we analyzed the region upstream of LMTK2 start codon for possible regulatory elements. We identified a putative TPA response element (TRE). TPA, a phorbol ester, is a synthetic activator of the classical and novel isoforms of Protein Kinase C, which in turn initiates a plethora of signaling cascades in a cell. We observed that TPA regulates LMTK2 expression in a dose‐ and time‐dependent manner, both at the protein and the mRNA levels. This effect is seen in multiple cell lines. However, TPA does not affect the stability of the LMTK2 mRNA transcript. Moreover, TPA increases the promoter activity of LMTK2. We further deduced that the TPA response on LMTK2 expression is mediated by direct binding of the activator Protein‐1 (AP‐1) complex on LMTK2 promoter. Overall, we have shown that LMTK2 is a TPA‐responsive element‐containing gene, and a novel gene upregulated downstream of PKC activation. Future studies will identify the cellular and extracellular signaling elements causing PKC activation and increased LMTK2 expression.Support or Funding InformationR01HL102208 to NAB
Oxytocin is involved in cellular signaling within the retina
Authors: York Nathaniel Walter , Halbach Patrick , Chiu Michelle , Bird Ian , Pillers De‐Ann , Pattnaik Bikash 
Published: November 2023

BackgroundOxytocin (OXT) is a neuropeptide that activates the oxytocin receptor (OXTR), a rhodopsin family G‐protein coupled receptor. We have recently localized OXTR to the retinal pigment epithelium (RPE) and OXT to the adjacent cone photoreceptors.ObjectiveWe sought to characterize the signaling mechanism of OXTR in the RPE and explore the downstream effects of OXT, focusing on the regulation of inwardly rectifying K+ channel Kir7.1.MethodsCa2+ response to OXT was measured in cultured human fetal RPE cells (hfRPE) using Fura‐2 AM in the presence of 2‐APB and nifedipine, pharmacological inhibitors of Ca2+ signaling pathways. HEK‐293 cells where used for the stable expression of human OXTR. Cellular signaling was visualized using live cell fluorescence imaging following transient expression of PH‐GFP and PKC‐GFP, monitors of GPCR metabolites PIP2 and DAG. Whole cell patch clamp electrophysiology was performed on HEK‐OXTR cells transfected with GFP‐fused Kir7.1 as well as freshly isolated mouse RPE cells to study Kir7.1 channel function.ResultsOXT treatment of RPE cells in culture resulted in a transient increase in cytoplasmic Ca2+ that was reduced by 95% in the presence of the IP3R antagonist, 2‐APB (P < 0.0001). Upon bathing the cells in Ca2+‐free extracellular solution or nifedipine, the Ca2+ response to OXT was not inhibited, although amplitude was reduced. Time to the rise in [Ca2+]i was not altered but recovery was slowed down with time constants (τ) of 0.35 ± 0.02 (r2 =.99), 0.34 ± 0.03 (r2 =.98), and 0.20 ± 0.005 (r2=.99) min for Ca2+‐free, nifedipine, and Ringer's solution, respectively. We also demonstrate that OXTR activation blunted Kir7.1 channel current. In isolated mouse RPE, we observed an average 61.81 ± 4.77 % decrease in K+ inward current amplitude at ‐160mV and an average 11.4 ± 3.2 mV depolarization in resting membrane potential by OXT.ConclusionWe propose that OXTR stimulates a mobilization of intracellular Ca2+, independent of extracellular Ca2+, through 2nd messengers coupled to OXTR/G‐protein in the RPE. This OXT‐OXTR signaling in the RPE cell also integrated the parallel modulation of the Kir7.1 channel. Kir7.1 channel is responsible for controlling RPE resting membrane potential and can influence retinal health and vision. We suggest that novel OXT‐OXTR signaling pathways in the outer retina will be of fundamental importance for eye development, health and visual function.Support or Funding InformationNIH EY024995, UnityPoint Health Meriter Foundation, Pediatrics, Graduate School, School of Medicine and Public Health
MALIGNANT HYPERTHERMIA: The motor unit potential (MUP) predicts susceptibility to developing the MH syndrome.
Authors: Williams Charles H 
Published: November 2023

Mating MH positive pigs to MH positive pigs produces an F1generation that is highly stress susceptible. Recording the MUP shows MH + pigs have a higher u voltage than control pigs. Older MH+ pigs have an even higher u voltage than control pigs. The duration of the voltage spike is also increased in MH+ pigs versus control pigs and older MH+ pigs have even a longer duration of the voltage spike.We can assume that by concentrating the MH genetic defect in the F1 generatioon that the population of defective sodium channels in the acetylcholine receptor was present at a high concentration. Since the acetylcholine receptors are spatially located under the footpiece of the myoneural junction which makes them a bank of receptors that are readily accessible when acetylcholine is released by the nerve, and the action of acetylcholine is very rapid. Therefore, the electromyographic data reflects the genetically defective sodium channels as the major functional component when we recorded the data.The sodium channels can be likened to a low voltage switch in a telephone circuit that is used to route telephone calls.We would suggest that the sodium channel at the acetyl choline receptor has been adapted to produce heat as well as muscle contraction and that the ability to produce copious amounts of heat is the biological mechanism that differentiates warm blooded animals from cold blooded animals.
CALCIUM OXALATE (CaOx) CRYSTAL ADHESION/AGGLOMERATION DEPENDENCE OF CLC‐5 EXPRESSION IN RENAL EPITHELIAL CELLS
Authors: Lorsung Rebecca , Harris Peter C , Lieske John C , Chang Minhwang 
Published: November 2023

Dent disease I is a rare X‐linked recessive chronic kidney disorder caused by mutations of the CLCN5 gene, which encodes the electrogenic Cl−/H+ exchanger (transporter) ClC‐5. CLC‐5 protein has been localized to the apical membrane and endosomal vesicles of the kidney in the proximal tubule as well as other tubule segments. Clinical manifestations of patients with Dent disease include low molecular weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, bone disease (rickets), and often progressive renal failure. While it is known that the CLC‐5 protein functions in the formation and maturation of endosomes, the etiology of the nephrocalcinosis and kidney stones often observed in Dent disease patients is not known. Therefore, in this project we investigated the effects of several CLC‐5 mutations identified in Dent disease patients, including S244L, Q629X, and R345W, on cellular processing of calcium oxalate (CaOx) crystals. Monolayers of renal proximal tubular cells (LLC‐PK1) and renal cortical tubular epithelial cells (RCTE) transfected with GFP tagged WT and mutated CLC‐5 were subjected to a CaOx crystal adhesion/agglomeration assay for thirty minutes then imaged by DIC and epifluorescence microscopy. The total area of CaOx crystal formation around each transfected cell within a 35, 55, and 75 μm diameter concentric circle was analyzed using ImageJ, and compared to adjacent un‐transfected cells which served as an internal control. A significant increase in CaOx aggregation was observed around cells expressing CLC‐5 mutants compared to wild type cells, especially for S244L. In LLC‐PK1 cells crystal adhesion/agglomeration decreased in this order: S224L > R345W > Q629X = WT; while in RCTE it decreased in this order: S224L = Q629X > R345W = WT. These findings support the hypothesis that mutated CLC‐5 proteins alter the apical surface of epithelial nephron cells and encourage CaOx aggregation/adhesion. It is also possible that decreased internalization and dissolution of CaOx crystals which form on the cell surface plays a role, as the greatest crystal adhesion/agglomeration was found within the 35 μm diameter circle around cells transfected with mutated CLC‐5 (35 μm > 55 μm > 75 μm). Further investigation into the impact of specific CLC‐5 mutation of protein structure and function, including protein‐protein interactions, is needed.Support or Funding InformationSupported by O'Brien Urology Research Center U54‐DK100227 summer student supplement and the Rare Kidney Stone Consortium (U54‐DK083908)