bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gestational diabetes affects spatial working memory and lipid metabolism but not oxidative stress biomarkers in second generation offspring rats
Authors: Huerta-Cervantes Maribel , Peña-Montes Donovan J. , Manzo-Avalos Salvador , Salgado-Garciglia Rafael , Cortés-Rojo Christian , Montoya-Pérez Rocío , López-Vázquez Miguel Angel , Olvera-Cortés María Esther , Saavedra-Molina Alfredo 
Published: April 2020

Gestational diabetes (GD) is an impairment of glucose metabolism during pregnancy, an increment of blood glucose in the intrauterine milieu results in abnormal fetal development and increased risk of developing metabolic disorders as diabetes mellitus 2, neurodegenerative diseases and behavior abnormalities in the offspring. In addition, increased oxidative stress in specific brain regions at early ages has been reported in the offspring of experimental models of GD. However, the consequences in the F2 offspring behavior and oxidative stress in cerebral cortex remains unclear. The aim of this study was to evaluate the effect of gestational diabetes in the spatial working memory and oxidative stress of adult male and female F2 GD offspring. Twelve Wistar female rats were placed mate with a control male. Gestation pregnant rats (day 10) were divided randomly in two groups: Gestation control rats (GC) and gestational diabetic rats (GD). GD rats were injected with a single intraperitoneal injection of a low dose of streptozotocin (STZ), only rats exhibiting blood glucose levels 140–300 mg/dL were considered for the study. Four months female rats (F1) were mated with control male rats to obtain F2 offspring. Six‐months old rats (adult) male and female F2 offspring from GC and GD were employed in this work. We evaluated anxiety in the elevated plus maze and open field and spatial working memory in the eight‐arm radial maze. Oxidative stress biomarkers (reactive oxygen species, lipid‐peroxidation, glutathione status) were evaluated in cerebral cortex. We found an impairment in lipid metabolism and a deficiency in spatial working memory of F2 female GD offspring. On the other hand, we did not find changes in oxidative stress biomarkers. In conclusion, these results suggest a transgenerational effect of gestational diabetes in lipid metabolism and cognitive behavior without the involvement of oxidative stress.Support or Funding InformationThe authors appreciate the partial economic support from CIC‐UMSNH (2.16 to ASM).
Kinetic Effects of Inside‐Positive Membrane Potential on the Lactose Efflux of LacY
Authors: Jiang Xiaoxu , Ermolova Natalia , Lim John , Choi Seo Woo , Kaback H. Ronald 
Published: April 2020

LacY, the lactose permease of E. coli., utilizes the free energy released from the downhill movement of H+ in response of an electrochemical H+ gradient (Δμ̃H+, interior negative and/or alkaline) to catalyze the accumulation of lactose against a concentration gradient. In the absence of Δμ̃H+, the downhill transport of lactose in response of a concentration gradient drives uphill translocation of H+ to generate a H+ gradient, and the polarity of the H+ gradient depends on the direction of lactose gradient. In other words, LacY is a lactose/H+ symporter, in which the lactose transport and H+ translocation are obligatorily coupled. LacY functions as a monomer with a total of 12 transmembrane helices arranged into two 6‐helix bundles tethered by a long loop between helix VI and VII. The sugar‐ and H+‐binding sites are located at approximate the center of the permease and can be accessed from either side of the membrane via an alternating access conformational change driven by the binding and dissociation of sugar. In LacY, Glu325 plays an essential role in H+ translocation since replacing Glu325 with neutral amino residues abolishes transport activities involving H+ translocation, but the mutants still bind sugar with normal affinity and catalyze equilibrium exchange. Previous studies have shown that the presence of Δμ̃H+ (interior negative) significantly accelerates lactose influx by decreasing the Km 50‐100‐fold with no change in Kd. The goal of this study is to investigate kinetic effects of Δμ̃H+ on lactose efflux in right‐side‐out membrane vesicles. We measured the initial rate of efflux at different pHs in the absence or presence of an imposed membrane potential (ΔΨ, generated by K+ influx in response of a concentration gradient in the presence of valinomycin, interior positive). The results revealed that the lactose efflux of WT LacY exhibits an apparent pK value near 7.2, which is not affected by the imposition of ΔΨ (interior positive). The initial rate of lactose efflux increases in the presence of imposed ΔΨ (interior positive) at all tested pHs, although enhancement is about two orders of magnitude less than that observed in lactose influx. Moreover, the effect of ΔΨ (interior positive) on the Km of lactose efflux is almost negligible compared to the dramatic change of the Km of lactose influx caused by ΔΨ (interior negative). In contrast to WT LacY, the E325A mutant does not carry out lactose efflux in the absence or presence of imposed ΔΨ (interior positive) at all tested pHs.Support or Funding InformationNSF Eager Grant MCB‐1547801 to H.R.K., NIH Grant 1R01 GM120043 to H.R.K., and a grant from Ruth and Bucky Stein to H.R.K..
Utilizing Knockdowns, Overexpression and Tagging to Study the SUMO Proteases Ulp1 and Ulp2 in Tetrahymena thermophila
Authors: Lillian Jenna , Tessema Liya , Gessner Rachel , Wilson Cierra , Kandl Kim 
Published: April 2020

SUMO, small ubiquitin like modifier, plays a role in post‐translational modification of proteins, altering protein‐protein interactions and regulating cellular processes. Much of what is known about the SUMO pathway and SUMOylation comes from studies conducted in yeast and vertebrates. As a unicellular ciliated eukaryote, Tetrahymena thermophila exhibit complexity and diversity of cellular processes that are not present in yeast, and they possess the advantages of other single celled model organisms, such as short cell division times and ease of growth and genetic manipulation. We aimed to develop a better understanding of the function of the two SUMO proteases, Ulp1 and Ulp2, by knocking out the genes, ULP1 and ULP2, that encode these proteins. We designed knockout plasmid constructs by cloning the 5′ and 3′ flanking sequences of each gene into a vector with a neo resistance marker. The plasmid construct was then introduced into Tetrahymena, and cells were passaged into increasingly higher concentrations of paromomycin to promote phenotypic assortment. Quantitative RT‐PCR suggests that we were able to knockdown ULP2 but not ULP1, despite high paromomycin drug resistance, suggesting that ULP1 is an essential gene in Tetrahymena. We have also created plasmid constructs using a Gateway cloning method that enable us to both overexpress and YFP tag ULP1 and ULP2. Preliminary results show that Ulp1 localizes to the nuclear envelope, and Ulp2 localizes to the nucleus. Future work will focus on characterizing the overexpression phenotypes of ULP1 and ULP2 and the knockdown phenotype of ULP2 in Tetrahymena.Support or Funding InformationThis research was supported by the St. Olaf College Office of Collaborative Undergraduate Research, St. Olaf TRIO McNair Scholars Program (L.T.) and the Beta Beta Beta National Biological Honor Society (J.L.).
Nox4‐related miR‐92a Mimic Improves Alcohol‐Induced Derangements in Alveolar Macrophage Immunometabolism
Authors: Morris Niya L. , Yeligar Samantha M. 
Published: April 2020

ObjectiveIndividuals with a history of alcohol use disorders (AUD) are at increased risk of developing respiratory infections due in part to alveolar macrophage (AM) phagocytic dysfunction. Cellular metabolism and bioenergetics are known to play key roles in immune cell functions. Chronic alcohol exposure increases NADPH oxidase (Nox)4, which increases AM oxidative stress, and may contribute to derangements in AM immunometabolism. Nox4 expression may be post‐transcriptionally regulated by microRNA (miR)‐92a, leading to Nox4 downregulation. Therefore, we hypothesized that chronic alcohol exposure decreases mir‐92a to upregulate Nox4, which increases mitochondrial oxidative stress and dysfunction in immunometabolism, resulting in increased susceptibility to respiratory infections.MethodsMH‐S, a mouse alveolar macrophage cell line, were treated ± 0.08% ethanol (EtOH) for 3 d ± miR‐92a mimic (50 μM, last 1 d). Expression of miR‐92a and Nox4 were measured by qRT‐PCR. Cellular reactive oxygen species (ROS) generation and H2O2 were measured using 2′,7′‐dichlorofluorescein‐diacetate fluorescence and Amplex Red assay, respectively. Co‐localization of Nox4 and mitochondria were assessed by confocal microscopy. Mitochondrial respiration and bioenergetics were measured using an extracellular flux bioanalyzer. In vitro phagocytic capacity was measured using pHrodo Staphylococcus aureus BioParticles conjugate.ResultsIn vitro EtOH: 1) decreased miR‐92a and increased Nox4 expression, 2) increased ROS and H2O2, 3) increased Nox4 co‐localization with mitochondria, 4) decreased mitochondrial respiration and bioenergetics, and 5) decreased AM phagocytic ability. Treatment with miR‐92a mimic reversed these EtOH‐induced impairments.ConclusionsChronic alcohol consumption increases alveolar macrophage oxidative stress, while reducing mitochondrial respiration and phagocytic capacity. Treatment with miR‐92a mimic reduced EtOH‐induced oxidative stress and improved AM immunometabolism and phagocytic function. Our studies reveal that miR‐92a may be a novel therapeutic target in the treatment of pulmonary infections in individuals with a history of AUD.Support or Funding InformationThis work was supported by the National Institute of Alcohol Abuse and Alcoholism R01‐AA026086 (SMY) and by the Emory Acute Lung Injury Training Program T32‐HL116271‐05 (David M. Guidot).
Receptor Mechanisms in Nicotinic Enhancement of Opioid Antinociception
Authors: de Moura Fernando Barreto , Bergman Jack 
Published: April 2020

The use of prescription opioids for pain management can be limited by their adverse behavioral effects, which usually are not measured in the initial preclinical assessment of established or novel candidate analgesics. Recently, we have developed a protocol for concurrently assessing the antinociceptive effects of drugs (an indicator of analgesic promise) and their ability to decrease rates of operant behavior (an indicator of unwanted behavioral effects). Under these procedures, nicotine has been shown to augment the antinociceptive, but not the rate‐decreasing, effects of opioid analgesics with an inverse relationship to efficacy at the m‐opioid receptor‐ e.g., nicotine‐induced enhancement of nalbuphine>oxycodone>fentanyl. The present study was designed to further elucidate nicotinic‐opioid interactions by exploring the role of nicotinic receptor efficacy in the nicotinic augmentation of opioid antinociception. Male squirrel monkeys (n=4) lever‐pressed under a fixed ratio (FR) 10 schedule of 20% (v/v) condensed milk reinforcement. A 30‐s timeout (TO) period followed either the completion of the FR 10 or the elapse of 20‐s (limited hold (LH) 20). During the 30‐s TO, the distal portion of the monkey’s tail was immersed in 35, 50, 52, or 55°C water, and the latency to remove the tail was recorded. Dose response functions for tail‐withdrawal latency and decreases in response rate were generated for the high efficacy m‐opioid agonist fentanyl and the low efficacy m‐agonist nalbuphine alone and in the presence of nicotinic agonists reported to have high or intermediate efficacy‐‐epibatidine (0.00056 mg/kg) or varenicline (0.032 mg/kg), respectively. Fentanyl dose‐dependently increased tail‐withdrawal latencies at all water temperatures and, concomitantly, decreased rates of responding. Nalbuphine did not significantly decrease response rate, and increased tail‐withdrawal latencies at 50 and 52°C, but not at 55°C. Neither epibatidine nor varenicline significantly modified the rate decreasing effects of any of the opioids or the antinociceptive effects of fentanyl. However, the antinociceptive potency of nalbuphine was markedly enhanced by epibatidine (up to ~70‐fold) and only slightly increased by varenicline. The present results add to previous findings with nicotine, showing that nicotinic enhancement of opioid antinociception is inversely related to m‐opioid efficacy. They also extend those findings to suggest that such adjuvant effects are more robustly produced by nicotinic full agonists (nicotine, epibatidine) than a partial agonist (varenicline). In conjunction with previous reports, these findings support the idea that nicotinic agonists may be clinically useful as adjuvants for opioid analgesia.Support or Funding InformationDA035857 and Harvard Medical School Livingston Fellowship
Smooth Muscle Tone Contributes to Bladder Wall Stiffness during Filling
Authors: Saxena Pragya , Tykocki Nathan , Roccabianca Sara 
Published: April 2020

Sensations of bladder fullness are conveyed to the central nervous system by sensory nerves as a response to distention of the urinary bladder. This distention is accompanied by non‐voiding transient pressure events and contractions called “micromotions” that are responsible for the majority of sensory outflow during bladder filling. Changes in smooth muscle contractility along with alterations in wall stiffness and sensory feedback are observed in most UB pathologies. Thus, the mechanical properties of bladder wall may play a crucial role in communicating a sense of bladder fullness. We hypothesize that the contractile apparatus of the urinary bladder is actively involved in maintaining bladder wall stiffness during filling.Whole mouse bladders were dissected and mounted in a custom designed pentaplanar reflected image macroscopy (PRIM) system, for simultaneous measurement of bladder wall micromotions, bladder volume, intravesical pressure, bladder wall stress, and bladder wall strain during ex vivo bladder filling. Bladders were filled at a constant rate (30 μl/min) until intravesical pressure reached 25 mmHg. During filling, bladder wall micromotions led to transient pressure events (0–2 mmHg in amplitude). These micromotions began on the lateral sides of the bladder wall near the trigone and expanded upwards as the bladder filled. Baseline bladder pressure, however, remained relatively low over a large infused volume (~350 μL). Next, we derived the stress versus strain relationship of the bladder wall from the pressure‐volume recordings during filling. At baseline pressures >15 mmHg, intravesical pressure rapidly increased and transient pressure events could no longer be distinguished implying that wall compliance is maintained until larger infused volumes, after which bladder wall stress with respect to strain increases rapidly. To determine if smooth muscle tone altered bladder wall stress during filling, bladder was exposed to myosin inhibitor blebbistatin (10 μM) or PI3 kinase inhibitor wortmannin (10 μM). Both blebbistatin and wortmannin effectively diminished the micromotions and shifted the stress vs strain curve to the right, indicating that the smooth muscle contractile apparatus and smooth muscle tone contribute to bladder wall stiffness, but predominantly only at higher filling volumes. These findings suggest that smooth muscle tone directly increases bladder wall stiffness and that changes in this tone could alter sensory feedback during bladder filling.Support or Funding InformationSupported by NIH K01DK103840 (NRT) and R01DK119615 (NRT).
Long noncoding RNA LINC00844‐mediated molecular network regulates expression of drug metabolizing enzymes and nuclear receptors in HepaRG cells
Authors: Ning Baitang , Li Dongying , Wu Leihong , Knox Bridgett , Chen Si , Tolleson William , Liu Fang , Guo Lei , Tong Weida 
Published: April 2020

Noncoding RNAs, such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), regulate gene expression in many physiological and pathological processes, including drug metabolism. Drug metabolizing enzymes (DMES) are critical components in drug‐induced liver toxicity. In this study, we used human hepatic HepaRG cells treated with 5 or 10 mM acetaminophen (APAP) as our model system and identified LINC00844 as a toxicity‐associated lncRNA. We analyzed the expression profiles and the correlations between the expression of long noncoding RNAs and key enzymes for acetaminophen metabolism in humans. Our results showed that lncRNA LINC00844 is enriched in the liver and its expression correlates positively with that of CYP3A4, CYP2E1, SULT2A1, NR1I2, and HNF4A mRNA transcripts. We demonstrated that LINC00844 regulates the expression of these five genes in HepaRG cells using gain‐ and loss‐of‐function assays. Further, we discovered that LINC00844 was predominantly localized in the cytoplasm and acted as a hsa‐miR‐486‐5p sponge, via direct binding, to protect SULT2A1 from miRNA‐mediated gene silencing. Our data also showed that hsa‐miR‐486‐5p upregulated the expression of CYP3A4, SULT2A1, NR1I2, and HNF4A by positively regulating LINC00844. We depict a LINC00844‐mediated regulatory network that involves miRNA and NRs and influences the expression of DMEs.Support or Funding InformationFDA E0753201
Eukaryotic Initiation Factor 3 Interactions with 5′ and 3′ RNA structures of the Barley Yellow Dwarf Virus Facilitate a Non‐Canonical Translation Mechanism
Authors: Powell Paul , Bhardwaj Usha , Goss Dixie 
Published: April 2020

Barley Yellow Dwarf Virus (BYDV) is a positive strand RNA plant virus that translates without using a 5′ 7‐methylguanosine cap or a 3′ poly‐adenosine tail that are required for the canonical translation mechanism used by most mRNA. BYDV’s non‐canonical translation relies on RNA structures in the 5′ and 3′ untranslated regions (UTRs) that recruit eukaryotic initiation factors (eIFs) and ribosomes. Using a combination of biophysical, biochemical and chemical probing techniques, we have measured direct binding of eukaryotic initiation factors (eIFs) to BYVD 5′ and 3′ UTRs and probed binding sites. Our data show that one of the initiation factors, eIF3, can bind tightly, specifically, and simultaneously to structures in both 5′ and 3′ UTRs. This suggests that eIF3’s mechanistic role includes stabilization of the long‐distance RNA‐RNA interaction and the transfer of 3′‐bound factors/complexes to the 5′ UTR. Further work with RNA chemical probing has identified regions of the BYDV UTRs that interact with eIF3. This data highlights commonalities between BYDV structures and structures found in other viruses (e.g. Hepatitis C virus) and cellular mRNAs (e.g. c_JUN mRNA).Support or Funding InformationNSF MCB‐1902054 to Dixie J. Goss, NSF IGERT‐0965983 to Hunter College
Molecular Basis of Takotsubo Syndrome
Authors: Chandler Selena , Joshi Hirva , Hoff Ellianna , Patel Karishma , Ohanyan Vahagn , Kiedrowski Mathew , Chilian William M. , Dong Feng 
Published: April 2020

IntroductionTakotsubo syndrome (TTS), also known as the “Broken Heart Syndrome” or “Apical Ballooning Syndrome is defined by its characteristic anomaly: when the heart contracts during systole, the apex of the heart dilates as the base of the heart contracts. Severe TTS can lead to cardiogenic shock and death in 3–4% of patients. There is no standard medical therapy for TTS because the mechanism underlying the development of the syndrome is unknown. Our goal is to determine the molecular mechanisms of TTS as a first step towards better treatment plans and outcomes.MethodsOur model of TTS is a Kv1.5 null mouse, with compromised coronary metabolic dilation, subjected to transaortic constriction (TAC). Two weeks after TAC, when Kv1.5 null mouse showed profound apical ballooning during ventricular contraction (echocardiography), myocardial blood flow (MBF) was measured by contrast echocardiography in the base and apex of the left ventricle of mice under control conditions and during acute administration of norepinephrine to increase cardiac work. Hearts were collected and gene expression (RNA deep sequencing) in both the apex and the base were performed and followed by bioinformatic analysis. Wild type (WT) and unstressed Kv1.5 null mice were used as controls. To increase the scientific rigor, we purposefully analyzed RNA expression from different animals with Real‐Time‐PCR, to confirm the sequencing data.ResultsA total of 3875 genes were identified by differentially expressed between TTS vs unstressed Kv1.5 null hearts. Gene Set Enrichment Analysis shows many families of genes downregulated (metabolism) and upregulated (inflammation, hypoxia, apoptosis) in the apical (ballooning) area of the heart in TTS (compared to the base of the heart). RT‐qPCR revealed significant upregulation of the genes for Postn, Lox, and C920009B18Rik (p<0.01) and down‐regulation of the genes Ucp3, Acaa2, Pfkb1, Mir133a‐2 (p<0.05). These significant changes in expression were also shown in the apex of TTS hearts as compared to the apex of control Kv1.5 null hearts (p<0.01). We also found that in the mice with TTS, MBF was lower in the apex than in the base.ConclusionOur results suggest that the apex of the heart in TTS is receiving insufficient perfusion compared to the base, which may be one of the root problems in TTS. There is significant upregulation of structural genes in the apex of TTS hearts versus their bases and the apexes of normal hearts, whereas there is a significant downregulation of genes playing roles in metabolism. This confirms the importance of these specific genes in the development of the anomaly shown in TTS.