bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Characterization of the First Known Mutation of the Human SLC12A2 (NKCC1) Gene
Authors: Flores Bianca , Schornak Cara , Wolfe Lynne , Adams David , Delpire Eric 
Published: November 2023

An 8 year old girl with multiple health problems that began at the age of 6 months visited the NIH Undiagnosed Disease Program in 2010. At the time of her visit, she suffered from obstructive apnea, episodes of vomiting and dehydration; decrease energy and fatigue, exercise intolerance, dilated cardiomyopathy (left ventricle dilation) and seizure‐like episodes. In early 2015, she was found to have a de novo mutation in SLC12A2, the gene encoding the Na‐K‐2Cl cotransporter, NKCC1. Using genomic DNA isolated from the patient, we confirmed an 11‐bp deletion in exon 22 leading to protein truncation. We created the mutant cDNA for expression in Xenopus laevis oocytes. In contrast to wild‐type cotransporter which demonstrated significant bumetanide‐sensitive and hypertonic‐stimulated K+ uptake, the mutant cotransporter was non‐functional. Western blot analysis of oocytes expressing of a c‐myc tagged mutant cotransporter revealed a molecular size consistent with a dimer, in contrast to oocytes expressing wild‐type cotransporters that showed all three forms of the cotransporter: core, glycosylated, and dimer. Co‐expression of mutant NKCC1 with wild‐type NKCC1 did not result in dominant‐negative effect of mutant on wild‐type transporter. K+ influx measurements in fibroblasts isolated from the patient revealed that 50% of the K+ influx was ouabain‐sensitive or mediated by the Na+/K+ pump, whereas 41% of the flux was bumetanide‐sensitive or mediated by NKCC1. Addition of ouabain and bumetanide resulted in 87% reduction in K+ influx. These data indicate that the mutation leads to a non‐functional allele without deleterious effect on the function of the cotransporter derived from the wild‐type allele. Whether the mutant protein exerts deleterious effects on other cellular functions still need to be explored.Support or Funding InformationSupported by NIH grant DK093501
KCC3 activity contributes to the elongated spindle‐shaped morphology of invasive gliomas
Authors: Gagnon Kenneth Bradley 
Published: November 2023

Salt and obligatory water efflux across the cell membrane contributes to the elongated spindle‐shaped morphology commonly observed with invasive glioma cells. Immunofluorescent localization of the K‐Cl cotransporter isoform 3 (KCC3) to migrating extensions of rat glioblastoma (C6) cells suggests that its activity may be responsible for this salt and water efflux and thus play a significant role in the motility of these tumor cells through the tight extracellular spaces in the brain. Unfortunately, the ubiquitous expression of KCC3 in other tissues makes it an unlikely target for novel anti‐cancer therapeutics. K‐Cl cotransporters are activated by dephosphorylation of specific threonine residues located in their intracellular carboxy‐terminal domain. Western blot analysis of protein lysates from several different immortalized tumor cell lines demonstrates an abundant expression of protein phosphatase 1 (PP1). In a series of wound healing experiments, Calyculin A inhibition of PP1 activity prevented C6 cells from moving into the wound area without varying the viability of cells outside the wound area. Furthermore, C6 cells incubated with Calyculin A no longer exhibited an elongated spindle‐shape, but rather a truncated rounder form. Interestingly, this same truncated form was observed in C6 cells where KCC3 was stably knocked down with short hairpin RNA, confirming that KCC3 activity plays a role in maintaining the elongated spindle‐shape associated with invasive motility. These results suggest that targeting the regulatory pathway of K‐Cl cotransport in glioma cells might be an effective way of slowing the spread of these malignant brain tumors.Support or Funding InformationSupported by a Special Projects Grant from the University of Saskatchewan College of Medicine to Kenneth B. Gagnon.
Hydronephrosis and Urinary Bladder Hypertrophy in ROMK Bartter's Mouse
Authors: Kim Jun‐Mo , Guo Xiaoyun , Hu Haiyan , Xu Shuhua , Wang Tong 
Published: November 2023

The renal outer medullary potassium channel (ROMK; Kir1.1) plays an important role in Na+ and K+ homeostasis. ROMK knockout mice (KO) show a similar phenotype to Bartter's syndrome of salt wasting and dehydration due to reduced Na‐2Cl‐K‐cotransporter activity. ROMK KO mice also show hydronephrosis; however, the mechanism of this phenotype has not been understood. We investigated ROMK expression and regulation by high and low K intake in mouse bladder, by Western blot and immunofluorescence (IF) staining. We also compared the bladder weight, bladder capacity, and thickness of urothelium and smooth muscle layer between WT and ROMK KO mice. Western blotting detected ROMK expression at 45~48 kDA, and IF staining showed that the luminal membrane of umbrella cells were strongly stained in WT compared to KO mice. The expression of ROMK in the luminal membrane disappeared and signal intensity of the detrusor muscle was decreased by low potassium diet (LK) in WT mice. In contrast, there was no significant change in ROMK intensity, but the thickness of urothelium was increased after mice were treated with HK diet. The thickness of urothelium was 168.1±25.8 μm in normal K (NK) and was 300.7±83.6 μm (p<0.001) in the HK treated group compared with NK in WT mice at the same age. The bladder weight, bladder capacity and the thickness of urothelium in ROMK KO showed average increases 2~4 fold greater than WT at both 2 months and 12 months of age. The thickness of the urothelium was 648.8±33.2 μm vs 302.7±16.5 μm (p<0.001) and the detrusor muscle 1940.7±98.9 μm vs 1308.2±102.1 μm (p=0.013) respectively in 12‐month ROMK KO mice. In conclusion, ROMK expressed in the urinary bladder can be regulated by K+ intake; significant enlargement and hypertrophy of the bladder may contribute to hydronephrosis in ROMK KO mice.Support or Funding InformationNIH RO1 DK099284 and PO1 DK 17433
Chromogranin B forms the chloride channel required for regulated secretion
Authors: Jiang Qiu‐Xing , Yadav Gaya , Zheng Hui , Yang Qing 
Published: November 2023

Endocrine cells use regulated secretory pathway to process and package small proteins and organic molecules into granular secretory vesicles that are released in a finely controlled manner (1–3). Acidification of secretory granules is essential for cargo maturation inside them. Vesicular H+‐ATPase pumps protons into secretory granules(4). But identity of the accompanying anion channel to neutralize charge accumulation in granules remains uncertain (5–7). We discover that chromogranin B (CHGB) is both sufficient and necessary to serve the highly selective anion conductance required for acidification and cargo maturation in insulin‐secreting cells. We find that CHGB uses different domains for channel formation and granular biogenesis, allowing the separation of these two essential functions. Our results establish that CHGBs constitute a new type of pH and calcium‐regulated chloride channel specific to regulated secretion.Support or Funding InformationNIH R01GM093271Welch Foundation I‐1684CPRIT RP120474AHA 12IRG9400019NIH R01GM088745
An Investigational Anti‐Cancer Agent Synergizes with VX‐809 in F508del Correction
Authors: Ambrosetti Adam , Fan Yifei , Kirby Emily F. , Riggs Robert M. , Wang X. Robert 
Published: November 2023

Cystic Fibrosis (CF) is caused by functional deficiency of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel located on the plasma membrane of the epithelia lining the lungs, pancreatic ductules, sweat glands, and other organs. Over 2,000 CFTR mutations have been identified to cause CF with the majority of patients carrying at least one copy of the CFTR gene that has the deletion of a phenylalanine at residue 508 (F508del). This mutation causes improper folding of the nascent CFTR protein and its impaired trafficking to the cell surface. While there are a number of ways to treat the symptoms, current evidence posits that small‐molecule combination therapy is an effective method to correct the underlying defects that cause CF. B6, an investigational anti‐cancer agent, was found to alter the level of an endoplasmic reticulum (ER) chaperone. The objective of the current study is to evaluate the impact of B6 on the steady‐state level of F508del CFTR both in the ER and at the cell periphery. HEK293 cells stably expressing F508del CFTR were incubated with increasing concentrations of B6, and the steady‐state level of CFTR in both the ER core‐glycosylated (band B) and Golgi complex‐glycosylated (band C) forms was determined using quantitative immunoblotting. B6 increases the level of F508del band B in a dose‐dependent manner with proportional increase in band C. To further explore the potential usefulness of B6 in F508del rescue, B6 was combined with F508del investigational corrector VX‐809. Combination of 1 μM VX‐809 with B6 at 10 and 40 μM increases the level of F508del band C by 57% and 171%, respectively, as compared with VX‐809 alone. Similar experiments were performed on CFBE‐ΔF, a CF airway epithelial cell line stably expressing F508del CFTR. Addition of 40 μM B6 to 5 μM VX‐809 increased the level of F508del band C by 119%. Our data suggest a synergistic enhancement of F508del correction by the combination of B6 and VX‐809. These data suggest B6 operates synergistically with VX‐809 to promote maturation of F508del CFTR. A better understanding of the underlying mechanism might lead to the development of novel small‐molecule compounds that dramatically enhance the efficacy of existing F508del correctors.Support or Funding InformationThis work was supported by NIDDK/NIH grant P30DK072482 and the Cystic Fibrosis Foundation grant WANG12P0. A.A. and E.F.K. were supported by Samford University McWhorter School of Pharmacy Pharmaceutical Sciences Summer Internship Program.
Sarcolipin Deletion in mdx Mice Worsens Dystrophic Pathology by Impairing Calcineurin Signaling and Reducing Utrophin Expression
Authors: Fajardo Val Andrew , Gamu Daniel , Rietze Bradley A , Kwon Frenk , Chambers Paige J , Quadrilatero Joe , Tupling A. Russell 
Published: November 2023

Duchenne's muscular dystrophy (DMD) is the most severe form of muscular dystrophy affecting 1 in 3500 live male births. There is no cure for DMD and males typically succumb to early mortality induced by respiratory and cardiac insufficiency. Thus, finding viable therapeutic targets is of utmost importance. Sarcolipin (SLN), a sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pump inhibitor, is highly upregulated in dystrophic muscles from mdx mice, a mouse model for DMD. To date, the role of SLN in muscular dystrophy remains unknown, and therefore, in this study we crossed SlnKO mice with mdx mice to generate the mdx/SlnKO double knockout mutant mouse. We hypothesized that Sln deletion would worsen dystrophic myopathy based on unpublished results from our laboratory and previous studies showing that SLN can stimulate calcineurin – a Ca2+‐dependent serine/threonine phosphatase shown to counteract muscular dystrophy. Our findings show that mdx/SlnKO myofibers from soleus (P = 0.0001) and diaphragm (P < 0.0001) were significantly smaller than those from mdx. Furthermore, mdx/SlnKO soleus and diaphragm muscles displayed a slow‐to‐fast fiber type shift, which was associated with a significant reduction in utrophin (P = 0.004). Utrophin, a dystrophin homolog that confers compensatory membrane stability, is largely controlled by calcineurin, suggesting that SLN in mdx mice stimulates calcineurin signaling. In addition, the significant reductions in utrophin expression in the mdx/SlnKO mice can account for their worsened dystrophic pathology revealed by greater serum creatine kinase (P = 0.01) and greater variability in myofiber size (minimal Feret's diameter, P = 0.01) compared with mdx. Finally, lower central nuclei counts in muscles from mdx/SlnKO mice compared with mdx (P = 0.04) suggests that genetic deletion of Sln also potentially impairs the muscles’ regenerative capacity. Taken together, these results, for the first time clearly demonstrate SLN's vital role in countering muscular dystrophy.Support or Funding InformationThis work was supported by the Canadian Institutes of Health Research (CIHR; MOP 86618 and MOP 47296 to A.R.T).
Transforming Growth Factor‐beta Promotes Glioblastoma Multiforme to Establish Radiation Resistance by Mesenchymal Differentiation
Authors: Liu Chan‐Chuan , Yeh I‐Chun , Wu Sheng‐Nan , Sze Chun‐I 
Published: November 2023

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. Current standard therapy is surgery combined with radio‐therapy and/or chemo‐therapy. Due to the existence of resistance, GBM is one of the most difficult‐to‐treat tumor. It has been shown radio‐therapy may induce mesenchymal differentiation (MD). Transforming Growth Factor‐beta (TGF‐β) is a modifier of radiation response, and plays an important role in cell transformation. Therefore, we hypothesized that TGF‐β pathway might be involved in the development of radiation resistance via regulating MD.Thus, we explored whether TGF‐β regulates MD in radiation resistance in GBM. We first examined the effect of radiation doses on cell viability by Trypan blue assay, and evaluated cell viability and morphology. The cells showed morphology change were defined as transformed cells. We used the radiation dose of 50% cell viability to establish consecutive‐irradiation‐induced resistance. The radiation dose of the cell viability greater than 50% was established high‐dose‐irradiation‐induced resistance positive control. When viability of cells recovered from consecutive‐irradiation exposures which were higher than positive control, the cells were defined as consecutive‐irradiation‐induced resistance. To develop radiation resistance, 3.5Gy exposure for 6 times were required for 1306MG, and 2Gy exposure for 4 times were needed for U87MG. The numbers of transformed cells increased in radiation resistant cells population. Moreover, immunocytochemistry staining showed expression of certain MD markers on transformed cells were different from on non‐transformed cells. Immunoblotting results showed increased mesenchymal marker N‐cadherin and decreased epithelial marker beta‐catenin in radiation resistance. Finally, we used TGF‐β and TGF‐β receptor inhibitor LY364947 to determine if TGF‐β/Smad pathway regulates radiation‐induced resistance. The immunoblotting results revealed that inhibition of TGF‐β pathway reversed MD in consecutive‐irradiation‐induced resistant cell lines via increasing the expression of β‐catenin and reducing the expression of N‐cadherin and Fibronectin. We concluded that TGF‐β promotes GBM to establish radiation resistance by MD.
The effect of dehydration on the metabolic rate and locomotor performance of two species of tree frogs found in Southern California (Pseudacris regilla and P. cadaverina)
Authors: Alvarez Chelsea , Contreras Heidy L. 
Published: November 2023

Pacific tree frogs (Psedacris regilla) and California tree frogs (P. cadaverina), native to Southern California, have adapted to frequent periods of drought. These tree frogs show both behavioral and physiological adaptations that help diminish water loss to their environment. However, despite these adaptations, tree frogs may still experience periods of dehydration in their natural habitat. Dehydration significantly decreases aerobic scope in amphibians; frogs being more susceptible than toads. When dehydrated, semi‐aquatic frogs rely heavily on anaerobic metabolism, which has been shown to induce hyperglycemia and decreased organ ATP levels. In this study we are interested in evaluating the effects of dehydration on the aerobic scope of P. regilla and P. cadaverina, two highly terrestrial treefrogs. Furthermore we will investigate the effects of dehydration on the respiratory quotient of these frogs. Evaporative water loss in frogs can lead to a loss of 6–9% of initial body mass per day on land. In this study 8 frogs from each species were dehydrated to 5, 10, 15% of their initial body weight and then placed in a respiratory chamber so that standard metabolic rates were measured. After metabolic rates were recorded, hoping speed and the time it took for animals to right themselves were measured. After an exercise bout, the metabolic rate of all frogs was once again measured in all animals to determine the effect of dehydration on aerobic scope. Previously we have reported that P. regilla drinks water opportunistically whereas P. cadaverina must reach at least 18% dehydration before water absorption behaviors are consistently employed. Since there are significant differences in the levels of dehydration that each frog can endure before rehydration is normally established we expect to see differences in both aerobic scope, locomotion performance and the time it takes for frogs to switch to carbohydrates as the primary fuel for metabolism.Support or Funding InformationTitle V STEM Grant
Sex differences in the effects of environmental enrichment after maternal separation
Authors: Doreste‐Mendez Raura J. , Morales‐Bernecer Dayanira , Rivera‐Lopez Leslie , Torres‐Reveron Annelyn 
Published: November 2023

It is well known that early stressful experience in life affect neurochemical, cognitive and behavioral development. Maternal separation in early life is a stressor which can cause depression and anxiety behaviors later in life. On the other hand, environmental enrichment can improve behavioral response and neurochemical markers. The aim in this study was compare the effect of early environmental enrichment in anxiety and depressive‐like behaviors and assess synaptic plasticity markers after maternal separation in both males and females. Our hypothesis is that there will be a sex‐specific response to environmental enrichment in animals that have undergone an early life stressor. Male and female Sprague‐Dawley rat pups (n=46) were separated from their dam for 3 hours daily during postnatal days (PND) 1 to 21. At PND 22 rats were divided into enriched (toys, larger cage, larger social group) and standard environment until PND 77. Open field test was used to measure anxiety and forced swim test to measure depressive‐like behaviors. After this, rats were sacrificed and their brains removed and processed for later immunohistochemical studies of the synaptic vesicle protein, synaptophysin in CA3 and dentate gyrus (DG) of the hippocampus. We found that female rats showed higher anxiety and depression compared to males. Male rats from the environmental enrichment group have reduced labeling of synaptophysin in the DG than males from the standard housing. This difference was not observed in the females. The increase in synaptophysin in control males can be a compensatory change in response to an impoverished environment. Environmental enrichment seems to have more effect in improving behavioral disorders in male rats than females after an early life stressor. For future studies, why this compensation is occurring in males but not in females remains to be elucidated.Support or Funding InformationFunding: 1K07AT008027 and MD007579.
GABA Transporters (GATs) within the nucleus Tractus Solitarii (nTS) Modulate GABAergic Signaling, Neuronal Activity and Cardiorespiratory Function
Authors: Matott Michael P , Hasser Eileen M , Kline David D 
Published: November 2023

GABAergic neurons and their signaling are the major inhibitory influence within the nTS and provide both tonic and activity‐dependent modulation of nTS circuits, cardiorespiratory function and visceral reflexes. Altered GABAergic function is implicated in cardiorespiratory pathologies such as hypertension. Thus, maintaining GABA concentration within the synaptic and extrasynaptic space is critical to maintaining the efficacy of the nTS synapse and the system as a whole. GABA is removed from the extracellular milieu by GATs found on neurons and astrocytes. Despite the importance of GABAergic signaling in the nTS, the role of nTS GATs on synaptic and neuronal modulation and cardiorespiratory function is unknown. We hypothesized GABA transporters tonically restrain inhibitory synaptic activity to 2nd order nTS neurons to influence cardiovascular function. RT‐PCR of nTS tissue demonstrated GAT3 mRNA was expressed nearly 2‐fold greater than GAT1. Immunohistochemistry revealed robust GAT3 immunoreactivity (‐IR) whereas GAT1‐IR was weak. GAT3‐IR surrounded GAD67‐IR synaptic terminals and colabeled with the astrocyte marker S‐100B. Given this expression pattern, we focused primarily on the functional role of GAT3. Blockade of GAT3 in nTS slices with (S)‐SNAP 5114 (SNAP) produced a significant hyperpolarizing current in nTS neurons. In the majority of neurons, GAT3 blockade significantly decreased spontaneous inhibitory postsynaptic current (IPSC) frequency but did not affect sIPSC amplitude. Stimulation of the medial nTS (0.5 Hz) evoked nTS‐IPSCs which were not altered by GAT3 blockade. Bilateral microinjection of SNAP into the caudal nTS of anesthetized rats significantly increased mean arterial pressure (MAP), heart rate (HR), sympathetic nerve activity (SNA), and minute phrenic (Phr) nerve activity. The latter was due primarily to augmentation of Phr amplitude. These responses mimicked the cardiorespiratory effects of exogenous GABA microinjection. Furthermore, SNAP pretreatment enhanced the effects of GABA microinjection on MAP, HR, and SNA. Together these results demonstrate that inhibitory tone under basal conditions in nTS is significantly affected by GAT activity and modulates basal cardiorespiratory function. GATs therefore are likely to be important in cardiorespiratory reflex modulation under physiological and pathophysiological conditions.Support or Funding Information15GRNT25710159 (AHA) and HL098602 (NIH)
Hypothalamic Activated Microglia Augment Blood Pressure Elevation during Early Developing Phase of Hypertension in Stroke‐prone Spontaneously Hypertensive Rats
Authors: Takesue Ko , Kishi Takuya , Hirooka Yoshitaka 
Published: November 2023

BackgroundInflammation within the paraventricular nucleus of the hypothalamus (PVN) mediated by microglia with microglial morphological change is an important pathology of sympathoexcitation. Although previous reports showed activation of microglia within the PVN of angiotensin II‐induced hypertensive or spontaneously hypertensive rats (SHR), we did not confirmed that deactivation of microglia within the PVN could decrease blood pressure in hypertensive rats. Our previous data indicated that PVN microglia were much more activated at earlier stage of hypertension in stroke‐prone SHR (SHRSP) compared to normotensive Wistar‐Kyoto (WKY) rats. Therefore, We examined whether deactivation of microglia within the PVN could attenuate blood pressure elevation during early developing phase of hypertension in genetically hypertensive rats.Methods and resultsSix week‐old SHRSP and age‐matched normotensive WKY were intracerebroventriculary (ICV) administered minocycline (5 μg/h), microglial deactivator, for 4 weeks. ICV infusion of minocycline significantly attenuated systolic blood pressure elevation in SHRSP over 4 weeks (at the end of experiments; 203.2±2.2 mm Hg vs. 215.9±2.7 mm Hg, n=8–9, P<0.05) but not in WKY. 24‐h urinary norepinephrine excretion tended to be lowered by minocycline infusion in SHRSP. Activation of microglia within the PVN, identified by microglia specific ionized calcium‐binding adaptor molecule 1 (Iba‐1) immunoreactivity, was evaluated by the roundness and perimeter of microglia. Minocycline significantly decreased roundness and increased perimeter of microglia, indicating deactivation of microglia, within the PVN of SHRSP.ConclusionHypothalamic activated microglia with morphological alteration accelerate blood pressure elevation probably by sympathoexcitation during early developing phase of hypertension in SHRSP.Support or Funding InformationThis study was supported by a Grant‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24390198 to Dr. Hirooka and 22790709 to Dr. Kishi).