bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studying KLF6 and PEPD‐gene Promoter by Chromatin Immunoprecipitation Assay
Authors: Lanaghan Zeljka Miletic , Eni-Aganga Ireti , Balasubramaniam Muthukumar , Dash Chandravanu , Pandhare Jui 
Published: April 2020

Prolidase (PEPD) is a ubiquitously expressed cytosolic dipeptidase that has the unique ability to cleave proline or hydroxyproline on the C‐terminus of imidodipeptides and imidotripeptides. The reaction is vital for collagen degradation. Since collagen is the most abundant protein in the body, our objective is to get a better understanding of the enzymes involved in its turnover is crucial. In silico analysis on prolidase promoter showed that putative transcription factor of interest is KLF6. To test the hypothesis that the expression of PEPD gene may be regulated by the binding of the transcription factor KLF6 to candidate binding sites we used chromatin immunoprecipitation (ChIP) assay. HEK293T cells were transfected with FLAG‐hKLF6 plasmid and the cellular lysates were obtained to measure the KLF6 protein expression by immunoblotting. After that, purified DNA from the ChIP assays were used in end‐point PCR with primers targeting candidate KLF6‐binding sites in PEPD promoter. The primers used were expected to yield a 615 bp amplicon. PCR products were resolved on 1% agarose/TAE gel. The second round of PCR was performed in which endpoint PCR products from the first round of PCR were used as a template for nested PCR with primers designed to amplify 164 bp. Nevertheless, our results showed no detectable PEPD promoter‐specific signals in the PCR analysis of the ChIP products. However, due to the inherent caveats in the experimental design, choice of reagents, and analysis method, it would be premature to conclude at this time point that PEPD expression is not regulated by KLF6. Accordingly, the design of future experiments will effectively address the limitations of this current work and will help reveal the identity of transcription factors regulating PEPD expression.Support or Funding InformationSupported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐Aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).
Kv3 channels Modulate Epithelial Mesenchymal Transition through Akt pathway
Authors: Sim Hun Ju , Lee So Yeong 
Published: April 2020

Voltage‐gated potassium (Kv) channels, especially Kv3.1 and Kv3.4, have been suggested that they are closely related to cell migration and invasion. This process is associated with epithelial mesenchymal transition (EMT) which is important to cancer metastasis.In the present study, we used HeLa and canine breast cancer cells, CHMp, to investigate the involvement of Kv3 channels in the EMT processes. We found that blood depressing substance‐II (BDS), which is a specific Kv3 subfamily blocker, decreased the protein expressions of vimentin and N‐cadherin, mesenchymal cell markers. In addition, BDS inhibited cancer cell migration. Blocking Kv3 subfamily by BDS down‐regulated of phospho‐Akt S473 and up‐regulated of PTEN. These results suggest that blockade of Kv3 channels using BDS induced reversed EMT through Akt pathway in HeLa and CHMp cells. Further study is needed to explain the detailed mechanisms of controlling the relationship between Kv3 channels and EMT.Support or Funding InformationThis research was supported by the Bio and Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF‐2016M3A9B6026771).
In‐frame Genetic Disruption of SPECC1L Microtubule‐Interaction Domain Causes Embryonic Tissue Movement and Fusion Defects
Authors: Saadi Irfan , Wenger Luke , Hall Everett , Goering Jeremy , Isai Dona Greta , Umar Zaid , Moedritzer Michael , Paroya Shahnawaz , Czirok Andras 
Published: April 2020

Movement and fusion events are critical during embryonic development; defects in these events lead to common birth anomalies. For example, failure of neural tube, secondary palate and ventral body wall closure leads to exencephaly, cleft palate, and omphalocele, respectively. Autosomal‐dominant SPECC1L mutations have been identified in patients with syndromic malformations, including hypertelorism, cleft palate and omphalocele. These mutations cluster in the second coiled‐coil (CCD2) and calponin homology (CHD) domains, which interact with microtubule and actin, respectively. To study SPECC1L function in mice, we first generated genomic deletions that resulted in out‐of‐frame truncations. Homozygous mutants for these truncations, including those lacking CCD2 and CHD, died shortly after birth without cleft palate or omphalocele. We then generated a series of targeted in‐frame deletions in CCD2, homozygotes for which also exhibited perinatal lethality but now with ~50% exencephaly, ~50% cleft palate, and ~50% omphalocele. While the omphalocele phenotype segregated equally with the other two phenotypes, exencephaly and cleft palate were never observed in the same embryo, suggesting antagonistic tissue mechanics between neural tube and palate closure. Consistently, we found that the oral cavity was narrower in mutant embryos with exencephaly, allowing for the palatal shelves to close eventually. We have shown previously that SPECC1L deficiency leads to impaired cell speed and directionality. We now show that subcellular SPECC1L‐ΔCCD2 protein distribution was abnormal, accumulating asymmetrically and associating almost exclusively with filamentous actin. Thus, loss of SPECC1L interaction with microtubules – while maintaining actin association – was more detrimental to cell and tissue movement than loss of SPECC1L protein entirely. Taken together, our results showed that in‐frame perturbations of CCD2 in SPECC1L protein specifically affect embryonic closure of several tissues, suggesting that human SPECC1L CCD2 missense mutations are gain‐of‐function. Our data also revealed a novel role for SPECC1L cross‐linking of actin and microtubule cytoskeletons in embryonic tissue movement and fusion events.Support or Funding InformationThis project was supported in part by the National Institutes of Health grants DE026172 (I.S.). Core support and pilot funding was also provided by National Institutes of Health Center of Biomedical Research Excellence (COBRE) grant (P20 GM104936), Kansas IDeA Network for Biomedical Research Excellence (INBRE) grant (P20 GM103418), and Kansas Intellectual and Developmental Disabilities Research Center (IDDRC) grant (P30 HD 002528).
Sex‐differences in AMPK activity and kidney function parameters post uninephrectomy
Authors: Saitta Biagio , Li Hui , Pham Jessica , Mancino Valeria , Sepehr Saman , Hallows Kenneth R. , Pastor-Soler Nuria M. 
Published: April 2020

BackgroundLiving donor (LD) kidney transplant is the preferred therapy for end‐stage kidney disease. LDs have generally excellent outcomes, yet in the first year post‐uninephrectomy (UNX) there is an increased risk of hypertension and microalbuminuria. In some chronic kidney disease (CKD) models activity of the metabolic sensor AMP‐activated kinase (AMPK) is lower compared to healthy kidneys. We hypothesized that low AMPK activity has a detrimental effect on kidney function post‐UNX when combined with a high‐Na diet in both male and female mice. Our findings could lead to protective interventions for LDs with AMPK activation.MethodsWe used adult male and female mice with double‐floxed AMPK alpha subunits (AMPKfl) and ± tamoxifen‐driven (Tam) expression of CAG‐Cre recombinase (Cre+ vs. Cre‐AMPKfl). Mice underwent UNX 5–6 wks post‐Tam (vs. vehicle corn oil injection). We also performed control Sham surgeries. Post surgery (Sham or UNX), mice were placed on a high‐Na (HNa) diet. Euthanasia was performed at 22 weeks. We measured GFR, urine albumin and plasma complete blood count electrolytes pre‐ and post intervention. Explant and remnant kidneys were examined by immunoblot and qPCR.ResultsCre+AMPKfl males and females kidneys had significant AMPK knockdown (KD) compared to Cre‐AMPKfl mice after Tam. There was a significant decrease in AMPK protein expression in mice receiving Tam compared to vehicle. AMPK‐KD females had significant albuminuria compared to AMPK‐sufficient females, although we did not detect such change in males. In both males and females, AMPK‐KD mice was associated with worse anemia as compared with AMPK‐sufficient mice. Interestingly, GFR increased after UNX+HNa in AMPK‐sufficient females, yet this increase was not evident in the AMPK‐KD female mice. GFR changes were not observed in males. In male mice, UNX+HNa had a detrimental effect on BUN that was independent of AMPK status.ConclusionsAMPK KD in female mice worsens kidney injury in a model of kidney donation. Male mice did not achieve the same degree of AMPK KD as females, which may explain the lack of AMPK dependence of kidney injury post‐UNX+HNa in males. These findings implicate AMPK as an important target for potential pharmacological interventions to prevent CKD in men and women LDs.Support or Funding InformationKeck School of Medicine of USC Dean’s Pilot. University Kidney Research Organization (UKRO).
Hormonal Regulation of Novel Glycogen Metabolic Proteins Hari Priya Vemana, Ehab M. Abo‐Ali, John L. Croft, Shraddha Bhutkar, Michael Woolford, Vikas V. DukhandeDepartment of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, USA
Authors: Vemana Hari Priya , Abo-Ali Ehab M. , Croft John L. , Bhutkar Shradda , Woolford Michael , Dukhande Vikas V. 
Published: April 2020

Lafora disease (LD) is a rare autosomal recessive disorder manifesting as a progressive myoclonic epilepsy. LD is characterized by the formation of Lafora bodies, insoluble deposits containing abnormal glycogen‐like material in many tissues. LD is caused by mutations in the genes encoding dual‐specificity phosphatase laforin (EPM2A) or the E3 ubiquitin ligase malin (EPM2B / NHLRC1). Several research studies on LD have shown that laforin functions as a phosphatase for glycogen. Malin forms a functional complex with laforin and either malin alone or the laforin‐malin complex is known to ubiquitinate multiple proteins involved in glycogen metabolism. It is well established that hormones such as insulin regulates glycogen metabolism but hormonal regulation of laforin and malin is not completely elucidated. In type‐2 diabetes, defects in insulin responsiveness leads to a state called insulin resistance. Our objective is to study the effect of hormones and nutrients on novel glycogen metabolic proteins laforin and malin in physiology and diabetes pathophysiology. We used liver cells in culture and a rodent model of diabetes using high‐fat diet and low dose streptozotocin (HF‐STZ). We measured gene expression, protein expression, protein posttranslational modifications, and functional activities of laforin and malin. Our preliminary data indicates that both diabetic conditions and insulin modulate the expression of LD proteins. Our ongoing studies are directed towards delineating the molecular mechanisms of these alterations. This study can serve to find novel potential targets for the pharmacotherapy of type‐2 diabetes.Support or Funding InformationThis study is supported by an award from National Institute of General Medical Sciences of the National Institute of Health, Award number: SC2GM125550.
Effects of Social Defeat on Hippocampal CA1 Neuronal and Sympathetic Nerve Activities in Conscious Rats
Authors: Yaguchi Kana , Ikegame Shizuka , Yoshimoto Misa , Miki Kenju 
Published: April 2020

Social defeat stress has been linked to depression, autonomic disorders, and cardiovascular disorders. The hippocampus has been implicated in the pathogenesis of depression, and hippocampal neuronal activity is presumed to be involved in autonomic disorders induced by social defeat stress. Potential linkages among hippocampal neuronal activities, sympathetic nerve activities, and cardiovascular function have received less attention. In the present study, we measured time courses of changes in hippocampal CA1 neuronal activity (CA1NA), renal sympathetic neuronal activity (RSNA), and lumbar sympathetic nerve activity (LSNA), arterial pressure (AP), and heart rate (HR) in response to social defeat stress in rats. Wistar rats were chronically implanted with electrodes for the measurement of CA1NA, RSNA, LSNA; electroencephalography, cervical electromyography, and diaphragm electromyography were performed, and a catheter was used to measure AP. Social defeat was induced by introduction of a Long–Evans rat, which was heavier and more aggressive than a Wistar rat, into the Wistar rat’s home cage for 30 minutes. The intrusion of the Long–Evans rat sometimes resulted in fighting between the Long–Evans and Wistar rats. Following defeat, Wistar rats displayed a submissive posture and exhibited immobilization for several minutes. Fighting caused immediate reduction of CA1NA, which remained suppressed during the period of submissive posture and immobilized behavior. However, the AP, HR, RSNA, and LSNA increased in transient fashion during immobilized behavior. These findings showed that the defeat stress considerably influenced CA1NA, whereas it marginally influenced RSNA, LSNA, AP, and HR. Therefore, it is likely that social defeat stress suppressed hippocampal function in tonic fashion, which could not be measured on the basis of sympathetic nerve activity and cardiovascular function.Support or Funding InformationJSPS Grant
Isolating Group A Streptococcus Infected Phagosomes
Authors: Young Kyrlia , Johnson Mitchell , Yuen Isabelle , Okumura Cheryl Y.M. 
Published: April 2020

The number of Group A Streptococcus (GAS) infections has dramatically risen in the last several decades and is an increasing public health concern worldwide. Normally, the body’s innate immune system responds to pathogens by phagocytosing and degrading the bacteria within the phagolysosome by a number of different molecular processes, including the oxidative burst response and lysosomal acidification. However, it has been shown by our lab and others that GAS has a number of different mechanisms to subvert immune defenses, including evasion of digestion in the phagolysosome. Because it is unusual for pathogens to reside in phagolysosomes, we want to develop a proteomic profile of GAS‐infected phagosomes. Because isolation of phagosomes is technically difficult, we set out to develop a method to isolate and purify GAS‐infected phagosomes using magnetic particles. We coupled magnetic particles to live GAS and demonstrated that we can effectively isolate viable coupled bacteria. Following infection of THP‐1 macrophages, we employ a magnetic separation protocol that yields a high concentration of GAS‐infected phagosomes largely absent of other organellar contamination. With these magnetically‐isolated GAS phagolysosomes, we aim to conduct a proteomics survey using mass spectrometry to characterize GAS phagosome modification to elucidate its mechanisms of survival. Our work will help identify novel therapeutic targets and strategies to treat GAS infections to enhance normal immune processes aimed at combating GAS infections, and thus limiting the use of antibiotics.Support or Funding InformationAmerican Heart Association (17GRNT3410851), Occidental College Undergraduate Research Center
Roles of nNOS and eNOS in rat heart ; comparison between the left and right ventricle myocytes
Authors: Kwon Jaewon 
Published: April 2020

Right ventricle (RV) has physiological as well as anatomical differences compared with left ventricle (LV). There are constitutive NO synthase isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS) in myocardium, regulating the calcium dynamics and contractility. Here we compared the roles of nNOS and eNOS in the RV and LV myocytes from S‐D rats. After isolating the myocytes using Langendorff perfusion system, their sarcomere shortening and [Ca2+]i were simultaneously measured using the Ionoptix system while paced by 2 Hz field stimulation. nNOS‐specific inhibitor, S‐methyl‐L‐thiocitrulline (SMTC) and the non‐specific NOS inhibitor, L‐NG‐Nitroarginine methyl ester (L‐NAME) were used to pharmacologically dissect the roles of NOS isoforms.According to the immunoblot assay, the protein amounts of nNOS and its phosphorylated form were not different between RV and LV. Although the amount of eNOS was lower in RV than LV, its phosphorylated form was similar. RV myocytes showed slower contraction (longer TPeak) and slower relaxation (longer Trelax) than LV myocytes. The treatment with SMTC or L‐NAME shortened TPeak and Trelax of RV myocytes, abolishing the kinetic differences of ΔSL. It was notable that the Trelax of LV myocytes became slightly longer by SMTC. Paradoxically, the length of sarcomere shortening (ΔSL) was increased while the amplitude of calcium transient (Δ[Ca2+]i) was decreased by SMTC in RV myocytes. However, SMTC did not affect the ΔSL and Δ[Ca2+]i of LV myocytes. Our study suggest that the slower contraction and relaxation of RV myocytes might be partly due to differential responses to the nNOS‐dependent intracellular signaling. The opposite direction of changes in the amplitudes of ΔSL and Δ[Ca2+]i by SMTC suggest that the nNOS in RV myocytes is responsible for negative inotropy despite the positive effects on the calcium transient, which is not evident in LV myocytes.
Afferent Nerve Fibers of the Skin ‐ Influence of salt intake
Authors: Ditting Tilmann , Rodionova Kristina , Reeh Peter , Eberhard Miriam , Veelken Roland Albert 
Published: April 2020

Sodium can be accumulated without commensurate water retention in the skin (non‐osmotic sodium storage). Macrophages play a pivotal role in this context and their depletion can induce salt‐sensitive hypertension (shin). On the other hand renal peptidergic nerves are also involved the development of ssHTN. Since the skin is densely innervated by peptidergic sensory nerves we hypothesized that high salt diet might enhance the release of neuropeptides from these nerve fibers.In a cross‐over design, two groups of rats (n=4, each) were fed either low salt diet (LS; 0.2%) with free access to tab water for 14 days or high salt diet (HS; 8%) with free access to 0.9% saline as drinking water. After 14 days a skin sample (3×3mm) of the groin area was excised, and the diet was switched for another 14 days. Then a contralateral skin sample was taken. Tissue analyzed in an organ‐bath and CGRP content in the supernatant was measured with ELISA. After two baseline measurements within 5 min, the tissue was superfused with hypertonic saline (4.5%) for 5 min, and three further samples of the supernatant were taken every 5 min.Baseline CGRP release was similar with both diets (LS 11.9±1.5 vs HS 13.6±1.5ng/g skin). Maximum release was higher with HS diet (LS 17.5±1.8 vs HS 29.8±1.3ng/g skin; P<0.05). After diet switch the results were similar: baseline LS 9.4±1.2 vs HS 10.1±, however, in rats that were on HS diet before max release tended to be higher with LS diet, Nevertheless with HS diet the release was higher again (LS 19.7±2.1 vs HS 29.3±1.7; P<0.05).High sodium diet sensitizes neuropeptide release from peptidergic sensory nerves in the skin. Hence peptidergic afferent nerves might be an integrated body‐wide system involved in sodium handling in very different target areas like skin and kidney. Putative peptidergic mechanisms (vasoregulation, chemotaxis) remain to be determined.
Defective membrane insertion of mutant Na,K‐ATPase, a cause of fatal epilepsy
Authors: Howard Rebecca , Ygberg Sofia , Andersson Magnus , Akkuratov Evgeny , Jans Daniel , Brismar Hjalmar , Lindstrand Anna , Aperia Anita 
Published: April 2020

Given its critical role in establishing ionic gradients in neurons and other excitable cells, functional disruption of the Na/K‐ATPase is expected to substantially compromise neurological homeostasis. However, to date only a few examples of spontaneous Na/K‐ATPase mutations have been identified in human patients. Here we describe a de novo Trp‐Arg mutation in the ATP1A1 gene, encoding the Na/K‐ATPase catalytic α1 subunit, in an infant who died at 10 months from therapy‐resistant epilepsy. Located in a conserved transmembrane helical region of the α1 subunit, the mutation was associated primarily with neurological symptoms and a low capacity to retain magnesium. Confocal imaging of the mutant Na/K‐ATPase α1 subunit expressed in rat hippocampal neurons revealed decreased but detectable membrane expression. Expressed in Xenopus oocytes, the mutant Na/K‐ATPase catalytic α1 subunit was associated with nonselective, ouabain‐resistant leak currents, consistent with a sizable, continuous hydration defect in the transmembrane domain. Indeed, molecular dynamics simulations in both POPC and mixed‐lipid bilayers showed accumulation of water at the protein periphery, particularly in the vicinity of crystallographic Na‐binding sites. These results provide evidence, from clinical diagnosis to atomistic simulation, for a mechanism of ion‐gradient disruption that could account for leak currents now observed in multiple pathological Na/K‐ATPase variants. Our finding of a pathological interaction between the mutated ion transporter and the membrane lipids has implications for development of new treatment strategy in many cases of genetic epilepsy.Support or Funding InformationSwedish Research Council to AA and to HB
Effect of MED28 on glucose metabolism in human non‐small cell lung cancer (NSCLC) cells
Authors: Lin Yi-Chang , Cheng An-Chin , Lee Tzu-Yi , Lee Ming-Fen 
Published: April 2020

Cancer cells can undergo “Warburg effect” for energy production to support cell growth by increasing glucose uptake and glycolysis. Aberrant Wnt/β‐catenin signaling in cancer may not only increase cell proliferation but also upregulate glycolytic capacity. Our laboratory has previously reported that MED28, a Mediator subunit for transcriptional activation, modulates metalloproteinase 2 (MMP2)‐dependent migration in human non‐small cell lung cancer (NSCLC) cells. We have also found that MED28 mediates Wnt/β‐catenin signaling in human colorectal cancer cells. The objective of the current study is to investigate if MED28 also plays a role in glucose metabolism in NSCLC besides MMP2‐dependent migration. Suppression of MED28 in A549 human NSCLC cells decreased the expression levels of Akt/Wnt/β‐catenin signaling‐related molecules including p‐Akt (Ser473), p‐GSK3β (Ser9), β‐catenin, and c‐Myc as shown by RNAi technology and protein immunoblotting. Meanwhile, siMED28 downregulated glycolysis by reducing the expression of relevant proteins such as glucose transporter 1 and hexokinase 2. Our data indicate a role of MED28 in regulating glucose metabolism, partially through Akt/Wnt/β‐catenin signaling, in human NSCLC cells. The underlying mechanism is currently under investigation.Support or Funding InformationThis work was supported by the grants MOST 106‐2320‐B‐039‐062‐MY3 and CMU108‐MF‐13 to M‐F Lee.