bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Airway Progenitor Homeostasis and Cell Composition by Tight Junction Protein Claudin‐18
Authors: Castaldi Alessandra , Arias Nataly , Pinson-Rose William , Samimi Kayla , Allen Alexa , Horie Masafumi , Li Changgong , Minoo Parviz , Zhou Beiyun , Stripp Barry , Ryan Amy L. , Borok Zea 
Published: April 2020

RationaleClaudin‐18 (Cldn18) is comprised of two isoforms, Cldn 18.1 and Cldn18.2. The lung‐specific isoform, Cldn18.1, regulates distal lung epithelial progenitor cell homeostasis. Its role in airway epithelium has not been elucidated, although Cldn18 expression is decreased in lungs of patients with chronic airway diseases (e.g., asthma and chronic obstructive pulmonary disease (COPD)). The airway epithelium is comprised of three progenitor populations, basal cells (BC), pulmonary neuroendocrine cells (PNEC) and club cells, and two non‐progenitor cell populations, namely goblet and ciliated cells. Club cells are secretory cells that are susceptible to naphthalene (NAP) injury. Club cell depletion occurs by day 3 post‐NAP, with spreading of ciliated cells to cover the denuded epithelial surface, and full reconstitution of the airway epithelium by day 14–21. Here we investigated the role of Cldn18.1 in airway epithelium using a previously developed global Cldn18 knockout (KO) mouse model.MethodsWild‐type (WT) and Cldn18 KO mice were injected intraperitoneally with NAP (225–275 mg/kg, corn oil as control). Lungs were harvested at baseline and 3‐days (maximal injury), 7‐days (beginning of regeneration) and 21‐days (full regeneration) post‐NAP, inflated with 4% paraformaldehyde and fixed overnight prior to embedding and sectioning. Hematoxylin and eosin, Periodic Acid‐Schiff (PAS) and immunofluorescence staining were performed.ResultsIn WT mice, Cldn18.1 was expressed in a small subset of airway ciliated cells. Cldn18 KO mice showed increased numbers of pulmonary neuroendocrine cells and neuroendocrine bodies, as well as ectopic localization of club cells and localization of keratin 5‐positive cells within the distal parenchyma. At day 3 post‐NAP in WT mice, Cldn18.1 was transiently expressed in all the ciliated cells covering the denuded epithelium, returning to being expressed in only a subset of cells by day 7. Cldn18 KO mice subjected to injury unexpectedly developed marked goblet cell hyperplasia at day 14–21 post‐NAP, with cells expressing markers for both club (CC‐10) and goblet (MUC5AC) cells.ConclusionsOur findings demonstrate an unexpected role for Cldn18.1 in regulating airway progenitor homeostasis and cell composition, implicating Cldn18.1 in regulation of downstream signaling pathways that inhibit goblet cell differentiation. A transient increase in the number of Cldn18.1‐positive ciliated cells in WT mice in the initial stages post‐injury suggests paracrine regulation of these inhibitory effects. Modulation of Cldn18 expression may be beneficial in the treatment of airway diseases characterized by goblet cell metaplasia.Support or Funding InformationFunding: American Lung Association, Hastings Foundation, NIH
Induced Pluripotent Stem Cell‐Derived Neuroprogenitor Cells: A Model for Studying Cannabinoid Induced Cell Signaling
Authors: Ryan Gabriella 
Published: April 2020

Chronic pain is a growing epidemic and public health issue that continue to affect the aging population in the US. Managing pain is particularly difficult with patients who suffer from cancer‐associated pain, neuropathic pain, as well as central pain states. These conditions are typically treated inadequately with opioids, antidepressants, and anticonvulsive drugs. There is growing evidence that cannabinoids natural products are beneficial for a range of clinical conditions, including pain and inflammations. The use of cannabinoids instead of highly regulated prescription drugs is fast approaching and the development of such cannabinoid drugs requires well planned medical trials as well as very stringent guidelines for contaminants such as heavy metals, pesticides, mycotoxins, etc. However, federal guidelines have not kept up with this emerging industry. Currently, each state is left to manage their own set of regulations, resulting in an industry that has great uncertainty and chaos as it attempts to satisfy the growing demand for its products. A low‐cost cellular model to study the neurological effects of cannabinoids is challenging. Here we show that Neuroprognitor cells (NPs) derived from induced pluripotent stem cells (iPSCs) can be used as a model for studying pathways associated with pain and cannabinoids. NPs derived from iPSCs can be differentiated into multiple lineages of neurons and glial cells. These cells have the Cannabinoid Receptor 1 (CB1) present which, when activated by cannabinoids, inhibits the inflammatory and pain stimulation of Cyclooxygenase 2 (COX2). We show expression and activation of CB1 in correlation to Cannabinoids treatment. Likewise we show how, in iPSC‐derived NPs Cannabinoid treatment leads to the inhibition of Cox‐2, therefore theoretically lessoning the potential for inflammation and pain.Support or Funding InformationOmni International, Inc935 Cobb Place BlvdKennesaw Ga 30144
A Renal Potassium‐Switch Prioritizes Dietary Potassium Over Sodium, Driving Salt‐Sensitive Hypertension
Authors: Grimm P. Richard , Delpire Eric , Welling Paul A. 
Published: April 2020

Reducing dietary salt (NaCl) is well appreciated to lower blood pressure (BP), but a growing body of evidence indicates that increasing dietary potassium (K+) intake plays an equally important role. A ‘renal potassium switch’ that turns on the thiazide‐sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) in response to low dietary potassium intake and off in response to high potassium intake has been implicated.Here we test the idea in genetically engineered mice, containing a DCT1‐specific, conditionally active SPAK (CA‐SPAK mice) which phospho‐activates NCC, and locks the switch “on.” BP responses to small, physiologic changes in plasma [K+] (P[K+]) in CA‐SPAK mice were compared to control mice. Dietary potassium content was varied to acutely titrate P[K+] over a physiologic range (3.7mM (LK), 4.4mM (MK), and 5.1mM (HK)). Blood pressure was monitored by telemetry at each P[K+] level in mice consuming control or high salt diet (HNa). At the end of each dietary treatment, the BP response to hydrochlorothiazide (HCTZ) was measured to assess the contribution of NCC to BP.BP decreased by ~10 mmHg when P[K+] increased from 3.7 to 5.1 mM in control mice, coincident with inactivation of NCC. When the switch was on (LK and MK groups), HNa significantly elevated BP but had no effect when the switch was inactivated by HK. HCTZ significantly reduced BP in the LK/HNa and MK/HNa groups but had no effect on BP in the HK/HNa group, further supporting the idea that low potassium‐dependent activation NCC exacerbates the effects of sodium. Studies in CA‐SPAK mice reveal a causal relationship between switch activation and BP responses to sodium and potassium. In contrast to control mice, increasing P[K+] in CA‐SPAK mice had no effect on BP under control salt conditions and failed to blunt the significant hypertensive effects of HNa. HCTZ significantly decreased BP in all CA‐SPAK groups to near control levels, consistent with NCC‐driven salt reabsorption. Thus, locking on the potassium switch prevents the anti‐hypertensive effects of HK. No sex differences were found.In summary, low K+ consumption, common in modern diets, presses the switch pathway to turn on to conserve K+ at the expense of increasing sodium retention, even in the face of high dietary sodium, and this elevates BP. Thus, switch activation can drive salt‐sensitive hypertension.Support or Funding InformationNIH
Rational design, synthesis and evaluation of “smart”, uncharged bis‐oxime antidotes of OP inhibited human acetylcholinesterase
Authors: Radic Zoran , Gorecki Lukas , Gerlits Oksana , Kong Xiaotian , Cheng Xiaolin , Blumenthal Donald , Taylor Palmer , Ballatore Carlo , Kovalevsky Andrey 
Published: April 2020

Organophosphate (OP) intoxications from nerve agent and OP pesticide exposure are treated by outdated, questionably efficient pyridinium aldoxime‐based therapies. Pyridinium cations hamper central nervous system (CNS) uptake, while structural diversity of OPs prevents efficient reactivation of OP inhibited acetylcholinesterase (AChE). Improvements must include much better reactivation of AChE in the CNS. We present here seven novel uncharged bis‐oximes created from our X‐ray structures of an uncharged monoxime, RS194B, reversibly bound to human AChE (hAChE). Bis‐oxime protonation results in equilibration between up to sixteen distinct ionization forms, including uncharged forms capable of diffusing into CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions acting as structural variants of the compound significantly improved in vitro reactivation of diverse OP‐hAChEs. The enhanced reactivation orientation for one of the bis‐oximes was confirmed by X‐ray structure. Our novel, CNS directed bis‐oximes represent a promising class of “smart”, accelerated antidotes against OP intoxication.Support or Funding InformationThis research was supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke (NINDS), [Grant Numbers U01 NS083451 and R21 NS098998] and by the UCSD Academic Senate grant BG084144.
Localization and changes of tyrosine phosphorylated protein expressions in female reproductive tracts of PCOS rats
Authors: Bunsueb Sudtida , Arun Supatcharee , Iamsaard Sitthichai 
Published: April 2020

Polycystic ovary syndrome (PCOS) is a cause of female infertility and its mechanism is still unclear. Tyrosine phosphorylated (TyrPho) proteins have been shown to be important for male infertility. However, the biological functions of TyrPho proteins involved in PCOS condition have never been reported. This study aimed to localize and investigate the TyrPho protein expression in PCOS rats. Twenty female Sprague‐Dawley rats were divided into control and PCOS (induced by letrozole, 1 mg/KgBW, for 21 consecutive days) groups. The estrus cycle of animals was confirmed by vaginal smear daily. At the end of experiment, ovary, oviduct, and uterus tissues of both groups were localized for TyrPho proteins by immunohistochemistry. In addition, the expression of TyrPho proteins in reproductive tissues were investigated and quantified. The results showed that TyrPho proteins were localized in the developing ovarian follicles and epithelial cells of oviduct and uterus. Moreover, TyrPho proteins were significantly overexpressed in ovary (68, 52, 42 and 28 kDas), oviduct (56 and 42 kDa), and uterus (170, 53 and 42 kDas) of PCOS group as compared to control. We concluded that female reproductive tracts have tyrosine phosphorylation essential for female fertility.Support or Funding InformationKhon Kaen University
Brown adipose tissue mediates cardiac function via release of 12,13‐diHOME
Authors: Stanford Kristin , Pinckard Kelsey , Shettigar Vikram , Baer Lisa , Ziolo Mark 
Published: April 2020

Brown adipose tissue (BAT) increases energy expenditure and decreases plasma glucose and lipids, making it a potential target to decrease the risks of obesity, type 2 diabetes, and cardiovascular disease (CVD). BAT functions in an endocrine and autocrine manner to improve whole‐body metabolism, however the role of BAT in the cardiovascular system is unknown. Here, we determined that transplantation of BAT (+BAT) increases in vivo cardiac hemodynamics. Lipidomics analysis revealed that the lipokine most up‐regulated in +BAT mice was 12,13‐diHOME. 12,13‐diHOME acutely increases cardiac hemodynamics via direct effects on the cardiomyocyte. Furthermore, incubation of cardiomyocytes with 12,13‐diHOME increased mitochondrial respiration via the ryanodine receptor. The effects of 12,13‐diHOME were absent in NOS1−/− mice and cardiomyocytes. We found that 12,13‐diHOME is decreased in human patients with heart disease, and this was correlated with decreased ejection fraction. Our results suggest an endocrine role for BAT to enhance cardiac function that is mediated by regulation of calcium cycling via 12,13‐diHOME and NOS1.Support or Funding InformationR01‐HL138738, R01‐AG060542
Downregulation of HMGB1 is required for the protective role of Nrf2 in EMT‐mediated PF
Authors: Gao Jian 
Published: April 2020

AimPulmonary fibrosis (PF) is an interstitial lung disease characterized by the activation of accumulated myofibroblasts and deposition of extracellular matrix (ECM). Epithelial‐mesenchymal transition (EMT) is considered to be one of the major hypotheses in the formation of PF. High mobility group box1 (HMGB1) plays an important biological role in infection, inflammation, and immune responses. Nuclear factor E2‐related factor 2(Nrf2) is an important transcription factor for the regulation of oxidative stress. However, there are no direct evidences regarding the relationship between Nrf2 and HMGB1 in PF.MethodsPF animal model was established by intratracheal injection of bleomycin. Histological morphology of the lungs were reflected by H&E staining and Masson staining. The expressions of Nrf2, HMGB1 and EMT associated markers were determined by Western blot or immunohistochemistry. In vitro, the effect of Nrf2, HMGB1 and the relationship between them in TGF‐β1‐induced EMT were determined by western blot , coupled using respective pharmacological activators or transfection with siRNA.ResultsEMT was aggravated and the expression of HMGB1 was significantly elevated in Nrf2−/− mice compared with WT mice exposed to BLM. Moreover, the uptake of exogenous Nrf2 or ablation of HMGB1 ameliorated TGF‐β1‐induced EMT in rat type II alveolar epithelial cell line (RLE‐6TN) and human alveolar epithelial cell line(A549). In contrast, Nrf2 deficiency or HMGB1 activation aggravated TGF‐β1‐induced EMT. Furthermore, inhibition of HMGB1 diminished the protective effect of Nrf2 on EMT.ConclusionThese findings suggest that the inhibitory effect of HMGB1 on EMT in PF is regulated by Nrf2 and provides a new strategy of clinical treatment for PF.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China( No.: 8197141254, 81274172, 81473267, 81503330 and 81503129 ).
Analyzing Nitrogenase’s Regulator ‐ NifA’s Interaction with DNA
Authors: Standke Heidi , Kim Lois , Medina Michael , Owens Cedric 
Published: April 2020

The bacterium Gluconacetobacter diazotrophicus contains an enzyme called nitrogenase which converts dinitrogen gas to ammonia ‐ an essential plant nutrient. The breakdown of dinitrogen has a high energy demand, utilizing 16 ATPs and 16 reducing equivalents. For this reason, nitrogenase is highly regulated. Regulation of nitrogenase is controlled by the protein NifA, a sigma‐54‐activator. NifA is a transcriptional activator that binds to DNA through its DNA binding domain (DBD) to activate nitrogen fixation genes when G. diazotrophicus needs ammonia. NifA also regulates nitrogen fixation depending on whether sufficient redox equivalents are present to support nitrogenase. However, while the role of NifA is known, its mechanism is not fully understood. It is not known if NifA senses oxygen or if it directly senses reducing equivalents. There are two cysteines at the start of NifA’s DNA binding domain. Cysteines are redox active and oxidize to form disulfide bonds which are known to affect protein activity. It is unknown if these cysteines sense oxygen and/or redox conditions and if this potential sensing mechanism affects NifA’s DNA binding. Here, we determine the functional importance of the two cysteines by investigating their redox and O2 sensing activity, and how the redox state of the cysteines influences NifA’s interaction with DNA. To test the hypothesis that NifA’s DNA binding is regulated by the redox state of the cysteines, two constructs of NifA’s DBD were made: one truncated version containing neither cysteine (No‐Cys) and another containing both cysteines (2‐Cys). Both the No‐Cys and 2‐Cys constructs were cloned into expression plasmids, expressed heterologously in E. coli, purified, and functionally examined through DNA binding assays and biophysical methods. We hypothesized that if NifA is a redox/O2 sensor, it will only be active when both cysteines are reduced. Our current results indicate that oxidized conditions may be required for proper DNA binding, resulting in Kd values in the lower micromolar range. Experiments are currently underway to conclusively determine the redox state of the cysteines and the structure of NifA’s DNA binding domain.Support or Funding InformationChapman University's Undergraduate Student Scholarly Research/Creative GrantChapman University's Summer Undergraduate Research Fellowship (SURF)
Calcium Homeostasis Modulator (CALHM1/2) and Pulmonary Arterial Hypertension
Authors: Rodriguez Marisela , Babicheva Aleksandra , Xiong Mingmei , Jain Pritesh P. , Zhao Tengteng , Lai Ning , Rahimi Shamin , Balistrieri Francesca , Hernandez Moises , Thistlethwaite Patricia A. , Makino Ayako , Yuan Jason X.-J. 
Published: April 2020

RATIONALEConcentric pulmonary vascular remodeling is a major cause for the elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in patients with pulmonary arterial hypertension (PAH). Excessive PASMC proliferation and inhibited PASMC apoptosis have been implicated in the development and progression of pulmonary vascular wall thickening in PAH. Transition from the contractile phenotype to the synthetic or proliferative phenotype of PASMC is thus an important pathogenic process that promotes vascular remodeling. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is not only a trigger for PASMC contraction (and pulmonary vasoconstriction), but also an important stimulus for PASMC proliferation. Calcium homeostasis modulators (CALHM), CALHM1/2, are membrane Ca2+ channels that are allosterically regulated by voltage and extracellular Ca2+. CALHM1/2 are closed at the resting membrane potential but can be opened by strong membrane depolarization. Reduction of extracellular [Ca2+] increases CALHM open probability, which allows the channel to be activated at negative potential. We aimed to study whether CALHM1/2 are involved in the phenotypical transition of PASMC and whether CALHM1/2 are upregulated in highly proliferative PASMC from patients with PAH.METHODSFreshly isolated pulmonary artery with the endothelium removed (as contractile PASMC) and primary cultured PASMC (as synthetic or proliferative PASMC) from rats were used for this study. Western blot analysis was used to compare protein expression levels of CALHM1 and CALHM2 in fresh PA tissue and cultured PASMC derived from the same rats. In addition, we compared protein expression levels of CALHM1/2 in PASMC from normal subjects and PAH patients.RESULTSThe protein expression level of CALHM2 in proliferative PASMC (the primary cultured PASMC in growth factor‐containing medium) was significantly higher than in contractile PASMC (the freshly isolated PA tissue). The increased CALHM2 in proliferative PASMC was indeed associated with a significantly increased expression of PCNA, a cell proliferation marker, and a significantly decreased expression of MHC and TGLN4, markers for contractile phenotype of SMC. Furthermore, we also observed that mRNA expression level of CALHM2 was significantly higher in PASMC isolated from PAH patients than in PASMC from normal subjects.CONCLUSIONUpregulation of CALHM2 may play an important role in the phenotypical transition of PASMC from the contractile phenotype to the synthetic or proliferative phenotype. Ca2+ influx through voltage‐ and extracellular Ca2+‐regulated CALHM2 in the plasma membrane of PASMC is an important trigger for PASMC proliferation and may play an important role in stimulating PASMC proliferation in patients with PAH.Support or Funding InformationNIH
Deciphering the role of a unique methyltransferase in Mycobacterial infection
Authors: Rani Anshu , Tripathi Prajna , Manjunath P. , Hasnain Seyed Ehtesham , Ehtesham Nasreen Z. 
Published: April 2020

Tuberculosis is a life threatening disease caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), resulting in around two million deaths annually worldwide. Mtb is known to have acquired certain features that help in establishing infection and persisting for longer durations in the host. Recent studies have revealed the acquisition of certain unique methyltransferases by Mtb as a function of phylogenetic evolution which points towards their potential involvement in mycobacterial pathogenesis. In the present study, we identified one such hypothetical methyltransferase (Rvxxxx) gene unique to Mtb complex i.e., absent in non‐pathogenic Mycobacteria. Our in‐silico analysis revealed that Rvxxxx gene possesses an S‐adenosylmethionine binding site and a methyltransferase domain, which was further confirmed through in vitro studies. The introduction of Rvxxxx in the genome of Mycobacterium smegmatis lead to significant perturbations in the physiological and morphological features of the bacilli as well as markedly enhanced its ability to resist and adapt to hostile conditions. Furthermore, recombinant M. smegmatis showed increase in infectivity and survival within host macrophages ex vivo. In addition, Rvxxxx was also found to modulate the proteome profile of recombinant M. smegmatis. Cumulatively, the results of this study indicate that the identified hypothetical methyltransferase Rvxxxx may play a significant role in Mtb pathogenesis and niche adaptation.Support or Funding InformationThe Council of Scientific and Industrial Research, Govt. of India
Mechanisms of Transcriptional Regulation by an ER Stress‐Responsive Enhancer
Authors: He Yanfeng , Tomomori-Sato Chieri , Sato Shigeo , Conaway Joan W , Conaway Ronald C 
Published: April 2020

ATF6α is a transcription factor that functions as a master regulator of many ER stress‐responsive target genes. HSPA5 (also known as Grp78) is an ATF6α‐targeted ER‐stress responsive genes. It contains 3 tandemly repeated ER stress response elements (ERSE). The ERSE enhancer‐like sites are very close to the core promoter, making it an excellent model for studies of enhancer function using a natural promoter with its regulatory regions. Previous studies have shown that a collection of transcription factors contribute to transcriptional regulation via the HSPA5 ERSE region; however, little is known about how these transcription factors collaborate to drive activation. To gain insight into this question, we have established an in vitro system that recapitulates ATF6α‐dependent transcriptional activation with purified RNA polymerase II, Mediator, and transcription factors. An unexpected finding from this work is our discovery that Elongin, best known as an RNA polymerase II elongation factor, plays an important role in ATF6α and NF‐Y dependent activation of the HSPA5 promoter. Mechanistic studies suggest that Elongin functions with TFIID and Mediator at an early stage in assembly of active initiation complexes. These results provide a foundation for future efforts to dissect the interplay of ERSE‐binding transcription factors and coregulators in HSPA5 activation.Support or Funding InformationThis work was supported in part by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.