bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of Tryptophan‐Nicotinamide (TRP‐NAM) Pathway in Malnutrition Induced Liver Dysfunction
Authors: Hu Guanlan , Ling Catriona , Chi Lijun , Furse Samuel , Koulman Albert , Bandsma Robert 
Published: April 2020

BackgroundMalnutrition contributes to 45% of the death of children under 5 years of age. Mortality in severe malnutrition is often related to metabolic disturbances, including hypoglycemia, indicating hepatic metabolic dysfunction. However, the underlying pathophysiology is poorly understood. Children with complicated severe malnutrition have been found to have significantly lower circulating tryptophan levels in their blood. Disturbances in the tryptophan‐nicotinamide pathway have been implicated in liver and other organ diseases, potentially mediated by NAD‐dependent deacetylase sirtuin‐1 (SIRT1). However, there have been no studies that have evaluated the role of tryptophan‐nicotinamide pathway in severe malnutrition.ObjectivesTo evaluate the effect of modulating the tryptophan‐nicotinamide pathway on hepatic metabolism dysfunction in a mouse model of severe malnutrition.MethodsWeanling male C57BL/6J mice were randomized into different groups fed with a control diet (18% protein), or a malnutrition‐inducing diet (1% protein) with or without supplementation of modulators of the tryptophan‐nicotinamide pathway (nicotinamide, nicotinamide‐riboside and tryptophan). In addition, SIRT1 modulators treatment including resveratrol, EX‐527 and vehicle were tested. Comprehensive metabolic, histological and molecular analyses were performed.ResultAfter diet treatment, the mice in the 1% protein diet group had significantly lower body weight and length than in the 18% protein group, without any effect of modulators of the tryptophan‐nicotinamide pathway. Feeding mice with a 1% diet led to severe hepatic steatosis, mitochondrial structural alterations, lower ATP levels, disrupted TRP‐NAM pathway metabolites, and a decrease of phospholipids to triglycerides ratio. Nicotinamide, nicotinamide‐riboside and tryptophan treatment all led to a partial reversal of the hepatic steatosis in the 1% protein‐fed mice. ATP levels, mtDNA copy number, and PPARα target genes in the β‐oxidation pathway were also recovered in treatment group. We postulate that the effect is mediated through changes in levels of nicotinamide adenine dinucleotide (NAD+), which is critical in redox reactions and is a substrate for SIRT1, important for mitochondrial function.SignificanceModulating TRP‐NAM pathway can partially improve liver metabolic function in a mouse model of severe malnutrition. This study will improve our understanding of the pathophysiology of severe malnutrition. The results of this project could lead to the development of new interventions aimed at improving metabolic disturbances in children with severe malnutrition, which could then be taken to clinical trials.Support or Funding InformationBill & Melinda Gates Foundation
Modeling Blood‐Testis Barrier Characteristics with Novel CRISPR/Cas9 Functional ENT1 and ENT2 Knockout HeLa Cell Lines
Authors: Miller Siennah R. , Zhang Xiaohong Rebecca , Hau Raymond K. , Jennings Erin Q. , Galligan James J. , Wright Stephen H. , Cherrington Nathan J. 
Published: April 2020

Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood‐testis barrier. ENT1 is located on the basal membrane while ENT2 is located on the apical membrane of human Sertoli cells, creating a transepithelial transport pathway across the blood‐testis barrier. Improving drug disposition to the testis with drugs that use transepithlelial transport pathways may more effectively treat viral infections and cancer within the male genital tract. Here, we characterized uridine transport in two novel ENT cell lines and assessed the impact of nucleoside reverse transcriptase inhibitors (NRTIs) on uridine uptake to gain further insight on ENT function and substrate selectivity. ENT1 or ENT2 functional knockout HeLa cell lines were generated using CRISPR/Cas9 to determine the kinetics and selectivity of each ENT individually. We demonstrate the functional loss of each transporter through quantitation of [3H]uridine uptake in the presence of the ENT specific inhibitor S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR). At low concentrations (100 nM), NBMPR inhibits ENT1; conversely, at high concentrations (100 μM) NBMPR inhibits both ENT1 and ENT2. In ENT2 knockouts, >97% of [3H]uridine uptake was inhibited by 100nM NBMPR, indicating ENT1 function is predominant. In ENT1 knockouts, only 5% of [3H]uridine uptake was inhibited by 100 nM NBMPR and 90% was inhibited by 100 μM NBMPR, indicating ENT2 function is predominant. The Jmax for ENT1 is 23.4 pmol cm−2 min−1 and for ENT2 is 34 pmol cm−2 min−1. The Kt for ENT1 is 10 μM and for ENT2 is 110 μM. We next evaluated the impact of nine NRTIs (abacavir, entecavir, zidovudine, tenofovir, lamivudine, emtricitabine, zalcitabine, didanosine and stavudine) on [3H]uridine uptake, revealing abacavir as the most potent inhibitor of [3H]uridine uptake, with an IC50 of 72 μM for ENT1 and 350 μM for ENT2. Zidovudine had an IC50 of 1.4 mM for ENT1 and 1.2 mM for ENT2. Entecavir had an IC50 of 2.6 mM for ENT1 and 1.08 mM for ENT2. This suggests that abacavir has higher affinity for ENT1, and entecavir has a higher affinity for ENT2. Based on previous results using wild‐type HeLa cells and NBMPR, we believe that abacavir is an inhibitor of the ENTs, entecavir is a substrate, and zidovudine is a low‐affinity substrate. These functional knockout cell lines will allow further screening of therapeutic compounds to establish the substrate selectivity between ENT1 and ENT2, which can aid in the development of future compounds that are able to circumvent the blood‐testis barrier via the ENT1‐ENT2 transepithelial transport pathway.Support or Funding InformationNIGMS R01GM123643, 5P30ES006694, 2T32ES007091‐36A1
Mutations in GET4 disrupt the transmembrane domain recognition complex pathway
Authors: Tambe Mitali A. , Ng Bobby G. , Shimada Shino , Wolfe Lynne A. , Adams David , Gahl William A. , Malicdan May C.V. , Freeze Hudson H. 
Published: April 2020

The transmembrane domain recognition complex (TRC) targets cytoplasmic C‐terminal tail‐anchored (TA) proteins to their respective membranes in the endoplasmic reticulum (ER), Golgi and mitochondria. It is composed of three proteins, GET4, BAG6 and GET5. We identified an individual with compound heterozygous missense changes (p.Arg122His, p.Ile279Met) in GET4 that reduced all three TRC proteins by 70–90% in his fibroblasts, suggesting a possible defect in TA protein targeting. He presented with global developmental delay, intellectual disabilities, seizures, facial dysmorphism and delayed bone age. Indeed, we found the TA protein, syntaxin 5, is poorly targeted to Golgi membranes compared to normal controls. Since GET4 regulates ER to Golgi transport, we hypothesized it would be disrupted in his fibroblasts, and indeed retrograde (but not anterograde) transport is significantly reduced. Despite reduction in these TRC proteins their mRNA levels are unchanged, suggesting increased degradation in patient fibroblasts. Treating fibroblasts with the FDA‐approved proteasome inhibitor Bortezomib (10nM) restored syntaxin 5 localization and nearly normalized the levels of all three TRC proteins. Our study identifies the first individual with GET4 mutations and suggests a potential therapy.Support or Funding InformationThis work was supported by grant R01DK99551 and The Rocket Fund; the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director; and the NHGRI Intramural Research Program of the National Institutes of Health.
Molecular Mechanisms of P311 on White Adipogenesis
Authors: Badri Kameswara Rao , White Donald A. 
Published: April 2020

BackgroundThe obesity epidemic is a constant and chronic detriment to US as well as global health outcomes. Two‐thirds of the US population is either overweight or obese, and obesity can be linked to many chronic diseases, including diabetes mellitus, cardiovascular diseases, stroke and cancer. Diet and exercise remains the primary therapeutic approaches, but still obesity (40% of US adult population) incidence and prevalence continues to rise, prompting the development of new interventions. P311 is an 8 kDa protein shown to induce adipogenesis in an in vitro 3T3‐L1 cell culture model (Nunez et al. 2019). In the current study, we investigated the molecular mechanisms of P311 in white adipocyte biology.MethodsBoth male and female C57BL/6 control mice and P311 knockout (KO) mice were used for this study. Adipocytes were isolated from individual white adipose tissue depots and adipocyte number and size were quantified using LipidTox staining followed by Guava EasyCyte analysis. Additionally adipocyte cellular functions of P311 like apoptosis, lipophagy and adipose tissue macrophage numbers were verified in P311 KOs and compared with wild type control mice. Real‐time PCR, western blot and immunohistology studies were conducted as described by Badri et al. (2013) and Nunez et al. (2019)ResultsP311 was expressed in murine gonadal and subcutaneous white adipose tissue. White adipocytes isolated from 4‐month‐old mice had significantly higher levels of P311 compared to adipocytes isolated from the white subcutaneous adipose tissue of 1‐month‐old mice. However, progenitor cells (stromal vascular fraction) showed no (basal) expression of P311. P311 KO mice showed decreased PPARγ2 expression. P311 KO mice did not differ from wild type mice in subcutaneous or gonadal white adipose tissue weights, but they had fewer and enlarged adipocytes. Further, P311 KOs also showed decreased apoptosis and lipophagy along with decreased adipocyte macrophage numbers and browning of white adipose tissue. In addition, adult human visceral adipose tissue also showed the expression of P311 in adipocytes implicating a translational role for P311 in adipogenesis and obesity.ConclusionsP311 plays a key role in white adipogenesis and browning of WAT partly through modulating PPARγ2 and also by regulating adipocyte cellular processes including adipocyte apoptosis, lipophagy, browning and macrophage numbers.Support or Funding Information• 2019 Medical Student Summer Research and Public Health Experience (Morehouse School of Medicine)• NIMHD/NIH grant funding through S21MD000101 (to Morehouse School of Medicine)• U54MD008621 (Hampton University, Hampton, VA)
The nucleus accumbens core and shell sub‐regions differentially regulate conditioned fear expression and extinction
Authors: Dutta Sohini , Beaver Jasmin , Gilman T. Lee , Jasnow Aaron M. 
Published: April 2020

The nucleus accumbens (NAcc), consisting of core (NAcC) and shell (NAcS) sub‐regions, has primarily been studied as a locus mediating the effects of drug reward and addiction. This is due to the region’s role of ascribing valence and salience to internal and external cues. However, there is emerging evidence that this region is also involved in regulating aversive responses, but its exact role is not yet clear. Further, the two sub‐regions differ in their connectivity, neurochemical and physiological properties, and each sub‐regions specific involvement in fear processing has not been determined. In our studies, we sought to elucidate the differential role of the two sub‐regions on cued fear expression and extinction. Using adult male C57BL/6 mice, we stereotaxically implanted bilateral cannulae directed at either the NAcC or NAcS. Twenty‐four hours following fear training to an auditory tone, we infused lidocaine, a sodium channel blocker, into the targeted subregion to temporarily inactivate the subregion 5 mins prior to a fear expression test which also served as extinction training. Within the NAcS, fear expression was not influenced by lidocaine infusions, however, when the mice were tested 24 h later for extinction retention, mice that had their shell subregion inactivated exhibited enhanced fear, indicating that this subregion is important for fear extinction consolidation. Mice that had their NAcC inactivated showed an attenuated fear response during expression testing, suggesting that the core sub‐region is necessary for the expression of fear. This effect was not due to alterations in the ability to produce freezing behavior. In addition, there was no effect on extinction consolidation. We next examined whether activation of ERK signaling, downstream of mGluR1, in the nucleus accumbens shell is necessary for extinction consolidation. Thus, we infused the MEK inhibitor, U0126, prior to extinction training and observed a facilitation of extinction consolidation during the retention test. However, with pre‐extinction infusions we also observed within‐session changes in fear behavior during extinction training. To control for this we conducted post‐extinction infusions. Here, mice that were infused with the MEK inhibitor exhibited enhanced freezing during the retention test suggesting poor extinction consolidation and that this pathway is necessary for appropriate extinction consolidation. These findings suggest that the NAc may be considered a critical region involved in regulating both fear expression and its extinction. It’s dual role in appetitive and aversive regulation likely underlies the high co‐morbidity between anxiety disorders and substance use disorders.Support or Funding InformationThis work was supported by funds from grant NIMH1R15MH118705‐01 to Dr. Aaron M Jasnow.
Repeated remote ischemic preconditioning enhances post‐ischemic myocardial STAT5A and STAT3 but not STAT5B to confer cardioprotection in diabetic rats
Authors: Xiaodong YE , Cai Yin , Wang Tingting , Li Xia , Wang Sheng , Wu Qingping , Liu Danyong , Irwin Michael Garnet , Xia Zhengyuan 
Published: April 2020

BackgroundRemote ischemic preconditioning (RIPC) protects the hearts from ischemia/reperfusion (I/R) injury experimentally. In patients undergoing cardiac surgery, RIPC exhibits cardioprotection, while in studies that included diabetic patients, RIPC lost effectiveness in cardioprotection. In order to confer cardioprotection in diabetes, the threshold for RIPC has to be increased. In diabetic rats, repeated RIPC (rRIPC) for three days reduces myocardial infarction. In a recent study that included diabetic patients, rRIPC conferred beneficial effects (Balin M. et al. Cardiovasc Ther. 2019; doi: 10.1155/2019/9592378), but the mechanism is unclear. RIPC when applied well in advance of cardiac surgery conferred cardioptection that was associated increases of both cardiac STAT3 and STAT5. We hypothesized that both STAT5A and STAT5B are needed for rRIPC cardioprotection.MethodsEight‐week STZ‐induced diabetic rats received rRIPC (three episodes of 5 min occlusion of the left femoral artery followed by 5 min of reperfusion for 3 consecutive days) before being subjected to myocardial I/R (30 minutes of coronary artery ligation and 2 hours of reperfusion). In vitro, cardiac myoblast H9c2 cell hypoxia/reoxygenation (H/R) injury model was established with 6 hours of hypoxia followed by 12 hours of normoxia reoxygenation. The cells in the conditioning group were exposed to 3 cycles of 10 minutes of alternating nitrogen‐flushed hypoxia and reoxygenation as stimulated RIPC (sRIPC) 24 hours before inducing prolonged H/R.ResultsrRIPC reduced post‐ischemic myocardial infarct size and apoptotic cardiomyocyte death in diabetic rats (p <0.05) that was concomitant with significantly increases of myocardial p‐STAT3, p‐STAT5A, but not p‐STAT5B. In cultured H9C2 cells, sRIPC significantly attenuated the H/R‐induced increases in LDH release and the reduction in cell viability (all p<0.05, sRIPC+H/R vs. H/R). However, sRIPC mediated cellular protection was cancelled respectively by the Janus Kinase 2 Inhibitor AG490, by gene knock‐down of either STAT3 or STAT5A, but not by STAT5B gene knockdown.ConclusionsOur findings suggest that STAT5A and STAT3 but not STAT5B are critical for repeated RIPC to confer cardioprotection.Support or Funding InformationThis study is supported by Health and Medical Research Fund (05161826) and NSFC grants(81770824 and 81670770)
Histone H1 Remains Bound to the Nucleosome Dyad During TF Binding within a Nucleosome
Authors: Burge Nathaniel L. , Thuma Jenna L. , Poirier Michael G. 
Published: April 2020

H1 is a lysine rich histone that binds nucleosomes and alters nucleosome/chromatin dynamics. Chromatin dynamics, such as chromatin compaction and nucleosome unwrapping, are integral for regulation of fundamental cellular processes including transcription, DNA replication, and DNA repair. Nucleosomal DNA in the entry/exit region spontaneously dissociates from the histone octamer allowing transcription factors (TFs) to bind to DNA in the region when the nucleosome is in an unwrapped state. Instead of blocking wrapping, H1 shifts this equilibrium towards a wrapped state which represses TF binding without fully occluding it. Details of this TF binding mechanism remain unknown, including the structural dynamics of H1 domains during TF binding and which domains affect wrapping and TF binding. Using in vitro fluorescence‐based assays, we have determined the structural dynamics of H1 during TF binding to a nucleosome. H1 binds nucleosomes via electrostatic interactions with DNA and is composed of 3 domains: The winged helix domain that binds the nucleosome dyad and linker DNA, and the intrinsically disordered C terminal and N terminal domains that bind linker DNA. Previous work implied that H1 remains bound to a nucleosome during TF binding, but the details of H1 domain locations were unexplored. We have attached fluorophores and fluorophore quenchers to all 3 H1 domains and either the DNA or histone octamer to measure FRET and fluorophore quenching. This allows us to monitor the movement of the H1 domains relative to the DNA and histone octamer during TF binding. Our results indicate that H1 remains bound to the nucleosome dyad during a TF binding event, and the H1 C terminal domain dissociates from the linker DNA to remain at the dyad. These data further understanding of the mechanistic details of H1 suppression of TF binding to nucleosomes. Future experiments will focus on which H1 domains affect DNA wrapping and TF binding to a nucleosome to further understanding of how H1 regulates TF binding and the implications for transcription.Support or Funding InformationNIH R01‐GM121966NIH T32‐GM086252OSU University Fellowship
Sigma‐1 Receptor Agonists Are Renoprotective in Experimental Kidney Transplantation
Authors: Hosszu Adam , Lakat Tamas , Balogh Dora Bianka , Lenart Lilla , Rimaszombati Fruzsina , Saeed Adar , Toth Akos , Hodrea Judit , Wagner Laszlo Jozsef , Szabo Attila Jozsef , Fekete Andrea 
Published: April 2020

Introduction and aimsKidney transplantation (Tx) is associated with better quality of life and reduced costs compared to dialysis, but the shortage in donor organs is a limiting factor. Graft survival is highly dependent on the extent of ischemia/reperfusion injury (IRI) during Tx. We recently described the renoprotective effects of Sigma‐1 receptor (S1R) agonist treatment in renal IRI. Thus, our aim was to develop a preservation solution which minimizes ischemic graft damage in order to improve Tx outcomes and to increase the number of organs suitable for Tx.MethodsKidneys of male Wistar rats were perfused and placed in ice cold (i) Custodiol preservation solution; Custodiol containing S1R agonists (ii) fluvoxamine or (iii) SA‐4503 for 2 hours, then autotransplanted and sacrificed 24 hours after reperfusion. Sham‐operated rats served as controls. In a second experiment kidneys of rats as well as wild‐type and S1R knockout mice were perfused and placed in ice cold Custodiol or Custodiol containing various selective S1R agonists for 2/3/8/24 hours and tissue samples were collected.ResultsS1R agonists mitigated renal functional impairment and tubular dilatation following Tx. Expression of early and sensitive tubular injury markers Kim1 and Ngal were markedly less elevated in S1R agonist‐treated kidneys. S1R agonists alleviated renal apoptosis as shown on TUNEL‐stained kidney sections, decreased apoptotic Bax expression, while increased anti‐apoptotic Bcl2 expression. Reduced number of CD45+ leukocytes and reduced inflammatory cytokine (Mcp1, Il1a, Il6, Tnf) expressions confirmed the anti‐inflammatory effect of S1R agonists. All S1R agonists mitigated cold ischemic structural kidney damage at all time points.ConclusionThe addition of S1R agonists to the preservation solution during Tx improves graft function and alleviates structural damage, thus improving long‐term outcomes. S1R agonists reduce graft injury during cold storage, therefore the number of transplantable donor organs can be increased.Support or Funding InformationGrants: VKE‐2017‐00006; OTKA PD‐131637; FK‐124491; K‐116928; NN‐114607; FIKP; STIA_18_KF
Characterization of a Human ALDH2 Mutant that Causes Alcohol Flushing in Non‐east Asians
Authors: Rwere Freeborn , White Joseph R. , Chen Che-Hong , Mochly-Rosen Daria , Gross Eric R. 
Published: April 2020

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a ~55 kDa protein responsible for the metabolism of the alcohol derived intermediate, acetaldehyde, in addition to a variety of toxic aldehydes including 4‐hydroxynonenal (4HNE) and acrolein. A single nucleotide polymorphism (SNP) of the ALDH2 gene (rs671) is highly prevalent among East Asians with ~540 million people in the world with the ALDH2 genetic variant (ALDH2*2 or E504K) that causes facial flushing and tachycardia after alcohol consumption due inefficient acetaldehyde metabolism. However, anecdotal reports exist of non‐East Asians who flush after alcohol consumption. Therefore, we hypothesize that other dysfunctional human genetic variants exist, besides ALDH2*2, that cause inefficient acetaldehyde metabolism.To address our hypothesis, after receiving IRB approval, we recruited human volunteers with self‐reported alcohol‐induced facial flushing. Volunteers were genotyped in addition to being subjected to an alcohol challenge (0.25g/kg) where levels of acetaldehyde in the breath were quantified by gas mass spectrometry for the 2 hours after an alcohol challenge. In addition, to support these findings, we carried out site‐directed mutagenesis, expressed, and purified an ALDH2 mutant we identified in humans using a nickel affinity column for histidine tagged protein. We measured enzymatic activity of the ADLH2 mutant using wild type ALDH2 and ALDH2*2 as comparators. All data are presented as mean ± SEM. Statistical analysis was performed by ANOVA and Tukey’s post hoc with significance set at P<0.05.We discovered a novel non‐East Asian ALDH2 mutation (R114W) that also causes alcohol‐induced facial flushing and acetaldehyde accumulation. Acetaldehyde levels measured in human breath showed that a human heterozygous for ALDH2 R114W had ~30% more acetaldehyde accumulation compared to homozygote wild‐type ALDH2 human subjects. The heart rate of ALDH2 R114W was not affected by alcohol intake, whereas skin temperature increased after alcohol consumption. To confirm these findings in humans, enzymatic activity of the purified proteins showed that E504K and R114W had 49±3 and 603±11 nmol/min/mg protein, respectively compared to 1209±15 nmol/min/mg protein for wild‐type ALDH2 (n=3, p<0.05). When mixed in the 1:1 ratio with wild‐type enzyme to mimic the heterozygous condition, the activity of E504K and R114W increased to 683±6 and 838±16 nmol/min/mg of pure protein, respectively, but still significantly below the activity of the wild‐type ALDH2. The results presented here demonstrate that a newly found ALDH2 variant, R114W, is also responsible for alcohol‐induced acetaldehyde accumulation and facial flushing. In conclusion, individuals carrying this mutation are more potentially also more susceptible to esophageal cancer similar to those heterozygous for the ALDH2*2 genetic variant.Support or Funding InformationNIGMS HL119522 (ERG) and a Stanford ChEM‐H seed grant (ERG, DMR).