bluuebunny/crossref_metadata_2025 huggingface dataset:
Porcine bone marrow‐derived mesenchymal stem cells as cell source for smooth muscle cells in small diameter vascular tissue engineered grafts
Authors: Kolk Felix , Akker Nynke M.S. , van den Molin Daniel G.M. , Post Mark J.
Published: June 2021

Preconditioning of a synthetic graft with autologous cells is considered the best option to obtain functional vascular grafts. These cells are preferably isolated from an easy accessible patient‐autologous source, such as bone marrow or blood. We investigated if bone marrow‐derived mesenchymal stem cells (MSCs) can be isolated, cultured and instructed to gain a vascular smooth muscle cell (SMC) phenotype.

MSCs were isolated from porcine bone marrow by gradient centrifugation and cultured in MSC or SMC‐defined culture media. Cells were analysed for SMC phenotype by determination of mRNA and protein expression of alpha‐SMA, calponin, desmin and collagen.

Porcine MSCs can be reproducibly isolated and cultured. No eminent differences in growth dynamics (doubling time, morphology) were observed between animals. Cells grew in multiple layers and showed mesenchymal morphology with an elongated cell shape. Induction of expression of all SMC markers, both at mRNA and protein level, was observed and was highest in cells cultured in MSC‐defined medium.

We conclude that porcine MSCs can be reproducibly isolated and differentiated into cells with SMC‐phenotype with sufficient efficacy. We show that the current protocol allows the MSCs to be readily used for application in small diameter vascular grafts.
Six Weeks of a Low Protein Diet Impairs Calcium Homeostasis
Authors: Sullivan Rebecca Raphael , Rosewater Irina , Caseria Donna , O'Brien Kimberly , Kerstetter Jane , Insogna Karl
Published: June 2021

Restricting dietary protein to 0.7 g/kg for one week has been reported to impair intestinal calcium absorption, in adults. The objective of this study was to determine if longer‐term ingestion of a low protein diet adversely affects mineral homeostasis. Eleven healthy, young women participated in a 6.5‐week diet intervention trial during which they ingested a low protein diet controlled in nutrients known to affect calcium metabolism. The protocol consisted of a 3‐week adjustment diet followed by 6.5 weeks of a low protein diet (0.7 g/kg/d, 800 mg Ca/d, 2.3 g Na/d). All foods were weighed in a metabolic kitchen and subjects were required to consume all foods provided. Dual stable calcium isotopes were used to quantify intestinal absorption and skeletal turnover. Calcium absorption declined significantly (p<0.05) at week 6.5 compared to baseline (20.5±1.5% vs 22.7±4.6%, paired t‐test). A slight decline in urinary calcium paralleled absorption (157±55.7 mg/d vs 118.9±60.9, paired t‐test, p=0.07). Preliminary kinetic analyses indicate more negative bone balance and increased bone resorption when comparing week 1 to week 6.5 of the low protein diet (p=0.03). PTH and 1,25(OH)2vitamin D rose initially and then returned towards baseline (p<0.05 for both time trends), perhaps declining as resorption rates increased. These data are consistent with the conclusion that increased bone loss occurs during a low protein diet.
The associations between sleep quality and eating patterns in young adults
Authors: Alano Ryan , McEligot Archana , Johnson Sara , McMahan Shari , Mullapudi Lakshmi , Sassarini Fiorella
Published: June 2021

The objective of the present study was to examine the associations between sleep quality and eating patterns, including missing meals, snacking after dinner, and considering nutrition when choosing foods, in a college‐aged young adults. Participants consisted of both men and women at least 18 years of age, from a university campus in Southern California (n = 346). Sleep and eating pattern data were collected via a 17‐item sleep assessment questionnaire and a 5‐item sociocultural questionnaire, both of which were self‐administered. Sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI). Univariate analyses, including chi‐square tests and a Spearman correlation coefficient, were used to assess the relationship between sleep quality and PSQI with eating behaviors. We found that missing a meal in the day was associated with sleep quality (χ2 = 15.04, p < 0.05) and that snacking after dinner was associated with trouble falling asleep (χ2 = 8.47, p < 0.05). Furthermore, we found that considering nutrition when incorporating food into diet was inversely correlated with PSQI (r = −0.14, p < 0.05). These results suggest that sleep quality is significantly associated to eating habits, a finding which has implications for nutrition education related to improving sleep. Specifically, promoting the importance of regular eating habits, not eating prior to bed, and improving nutrition education may influence better quality sleep for young adults. Supported by CDC grant #1H75DP001814‐01.
Inhibition of TNF‐α‐induced Adhesion Molecule Expression by Polar Lichen Extracts in mouse Vascular Smooth Muscle Cells via Downregulation of the JNK/p38 and NF‐κB Signaling Pathway
Authors: Byeon Hye‐eun , Yim Joung‐Han , Lee Hong‐Kum , Pyo Suhkneung
Published: June 2021

Adhesion of circulating leucocytes to the endothelial cell and their migration into arterial intima contribute to the inflammatory reaction as well as the formation of atherosclerotic lesions. Cellular adhesion molecules, which are rapidly induced by pro‐ atherosclerotic conditions, are responsible in such processes. Lichen‐derived compounds have been known to have a variety of biological activities, but the effect of polar lichen extracts (PLEs) on adhesion molecule expression is not known yet. In the present study, we examined whether PLEs are able to inhibit the expression of adhesion molecules induced by TNF‐α in mouse vascular smooth muscle cells (VSMCs). ELISA and immunoblotting analysis showed that PLE 1 suppressed the expression of VCAM‐1 and ICAM‐1 in surface and total cellular protein levels, while PLE 2 only attenuated VCAM‐1 expression. PLEs also abrogated NF‐κB activation and phosphorlyation of JNK and p38 induced by TNF‐α. Adhesion assay demonstrated that adhesion of monocytes to VSMCs was decreased by PLEs. Overall, these results suggest that PLEs downregulates TNF‐α‐induced adhesion molecule expression through the inhibition of JNK/p38 and NF‐κB signaling pathway and may be considered as a therapeutic strategy to reduce the risk of atherosclerosis.
Interleukin 21 can induce granzyme B‐secreting cytotoxic B lymphocytes
Authors: Hagn Magdalena , Sontheimer Kai , Dahlke Karen , Beyer Thamara , Lunov Oleg , Fabricius Dorit , Tron Kyrylo , Nienhaus G. Ulrich , Simmet Thomas , Jahrsdörfer Bernd
Published: June 2021

Interleukin 21 (IL‐21) is a promising cytokine for the treatment of tumors and infections. Recently, IL‐21 was identified as an inducer of plasma cells when combined with CD40 ligand (CD40L). Here we show that in the absence of CD40L B cells (BC) rather differentiate into granzyme B (GzmB)‐secreting cytotoxic cells. GzmB induction in BC requires IL‐21 and BC receptor cross‐linking, and involves phosphorylation of SYK, JAK1/3 and STAT1/3. CD40L effectively suppresses GzmB in BC, suggesting that GzmB‐secreting BC play a role early during inflammation, before CD40L‐expressing T cells are present. This is underlined by our observation that T cell receptor (TCR)‐stimulated, but not fully activated CD4+ T cells express IL‐21 and induce GzmB in co‐cultured BC in an IL‐21 receptor‐dependent manner. BC‐derived GzmB is enzymatically active and induces apoptosis in various tumor cell lines. Our data reveal a novel role of IL‐21‐activated BC, which involves GzmB secretion and cytotoxicity. Our findings may have implications for the understanding of tumor immunosurveillance and early anti‐viral immune responses, and may open novel approaches for the immunotherapy of neoplastic and viral diseases.
Viral Antigens Trigger Granzyme B Secretion by Human CD5+ B1 Cells in the Presence of Interleukin 21
Authors: Sontheimer Kai , Hagn Magdalena , Ebel Verena , Beyer Thamara , Simmet Thomas , Jahrsdörfer Bernd
Published: June 2021

CD5+ B1 cells are thought to play a key role in innate immune responses by secreting natural immunoglobulins. Here we demonstrate that human B1 cells (B1C) are able to express the pro‐apoptotic serine protease granzyme B (GzmB) in response to viruses including tick‐borne encephalitis, rabies and hepatitis B virus. We reveal that this response depends on B1C receptor stimulation, TLR9 engagement and interleukin 21 (IL‐21), a cytokine primarily found in the acute phase of viral infections. Up to 35% of freshly isolated B1C directly respond with GzmB expression to stimulation with inactivated viruses in the presence of IL‐21. Of note, GzmB is secreted by B1C in an enzymatically active state, reaching levels comparable to those secreted by cytotoxic cells and its induction requires activation of similar signaling pathways as in CTL and NK cells. Our findings suggest GzmB secretion by B1C represents a novel innate immune response mechanism. GzmB‐secreting B1C may play a role in the early anti‐viral immune defense, and may contribute to elevated serum GzmB levels found in viral diseases. Further studies will elucidate whether B1C cells exhibit cytotoxicity towards virus‐infected or tumor cells.
Changes in autonomic control of the cardiovascular system in the Wistar audiogenic rat (WAR) strain, an experimental model of epilepsy
Authors: Fazan Rubens , Oliveira Mauro , Oliveira Jose Antonio Cortes , Salgado Helio Cesar , Garcia‐Cairasco Norberto
Published: June 2021

This study aimed to evaluate autonomic cardiovascular modulation and baroreflex control of heart rate (HR) in Wistar audiogenic rats (WAR), an epileptic rat strain. We studied spontaneous baroreflex (sequence method) as well as reflex changes in HR evoked by phenylephrine or nitroprusside‐induced changes in arterial pressure (AP). Blockers of autonomic receptors (atropine and propranolol) were used to measure cardiac autonomic tone. Pulse interval (PI) and AP variability analyses were performed by FFT spectral analysis to evaluate cardiovascular sympathovagal modulation. In WAR, AP and HR were higher (109±2 mmHg and 366±9 bpm) as compared with Wistar rats (101±2 mmHg and 326±10 bpm). Power in the low frequency (0.2–0.8 Hz) band, a marker of sympathetic activity, was higher in the AP and PI spectra of WAR than in those of Wistar rats. High frequency (0.8–3 Hz) power of PI spectra, which is linked to cardiac vagal modulation, was lower in WAR. Both strains showed similar vagal tone (91±13 vs. 94±11 bpm) but sympathetic tone was higher in WAR (30±4 vs. 14±4 bpm). No differences were found in the gains of evoked (1.32±0.1 vs. 1.35±0.2 ms/mmHg) or spontaneous (1.34±0.2 vs. 2.04±0.2 ms/mmHg) baroreflex sensitivity. Higher AP and HR are well known to be associated with sympathetic predominance and with threatening cardiovascular events, which are typical causes of sudden unexpected death in epilepsy. Support: FAPESP, CNPq.
Tunneling membrane nanotubes generate local calcium signals and may actively propagate calcium signals between cells
Authors: Smith Ian F , Shuai Jianwei , Parker Ian
Published: June 2021

Cells have long been known to employ gap junctions and synapses to communicate with their neighbors. A new mechanism has recently been proposed following the discovery of tunneling membrane nanotubes (TNTs) between cells [1]. TNTs are dynamic membrane protrusions with lengths up to several tens of microns and diameters of 50–800nm, which permit the exchange of membrane components and cytoplasmic molecules between neighboring cells. Ca2+ diffusion along TNTs has been proposed as a means of intercellular communication [2], yet our modeling simulations show that passive diffusion alone is insufficient to account for efficient transmission of Ca2+ between cells. Instead, we observe local spontaneous and inositol trisphosphate (IP3)‐evoked mediated Ca2+ signals within the length of TNTs formed between cultured SHSY‐5Y neuroblastoma cells. Moreover, immunostaining demonstrates the presence of both ER and IP3 receptors along the TNT. We propose that IP3Rs are involved in actively propagating intercellular Ca2+ signals along TNTs, acting as amplification sites to overcome limitations of passive diffusion in a chemical analog of electrical transmission of action potentials along axons.

Supported by grants NIH GM 40871 and GM65830.
Do vertebrate rhodopsins contain an allosteric binding site(s) for retinoids?
Authors: Isayama Tomoki , Miyazono Sadaharu , Okada Tetsuji , Kono Masahiro , Crouch Rosalie , Zimmerman Anita , Makino Clint
Published: June 2021

We found by microspectrophotometry that β‐ionone partitioned into the outer segment membranes of salamander green‐sensitive (GS) and blue‐sensitive (BS) rods with greater uptake into BS rods. Electrophysiology showed that BS rod rhodopsin, but not that of GS rods, was activated and bleached by β‐ionone. But biochemical assays using rhodopsins formed from recombinant salamander GS rod and BS rod opsins and 11‐cis retinal (A1) showed little activation by β‐ionone. Since salamander photoreceptors normally contain both A1 and 3‐dehydro 11‐cis retinal (A2)‐based visual pigments, we hypothesized that only the latter activated pharmacologically. We recorded BS rods whose native chromophore had been partially replaced by A1. Lowering the amount of A2 rhodopsin decreased pharmacological activation. Because β‐ionone does not occur naturally in the retina, we also examined the effects of vitamin A on rod activity. In contrast to β‐ionone, vitamin A activated rhodopsin in both GS and BS rods, and also sensitized rods to UV light, presumably by resonance energy transfer. Thus the different effects of retinoids on rods argue for the existence of allosteric binding sites on rhodopsins.

Research Support: NEI EY011358 and Lions of Massachusetts
Differentiating racial differences in oxidative stress levels: in vitro and in vivo
Authors: Feairheller Deborah L , Park Joon‐Young , Sturgeon Kathleen M , Kim Boa , Williamson Sheara T , Diaz Keith M , Brown Michael D
Published: June 2021

Black (B) ethnicity has become a strong independent risk factor for cardiovascular disease, endothelial dysfunction, and enhanced oxidative stress. Identifying the racial differences in specific origins of oxidative stress is paramount to guiding future mechanistic research. We examined racial differences in oxidative stress markers, in healthy B (N=9) and White (W) (N=9) young adults (21± .4 yrs) as well as in B (N=4) and W (N=4) Human Umbilical Vein Endothelial Cells (HUVECs). A heightened oxidative stress level in B was confirmed. In unstimulated culture, B HUVECs had significantly lower superoxide dismutase activity (SOD) indicating active O2− scavenging. We also found enhanced nitric oxide (NO) levels in B HUVECs suggesting overproduction of NO in response to increased O2−. Finally, B HUVECs had higher levels of total antioxidant capacity (TAC), a measure of all antioxidants including those that quench ONOO−. Likewise in humans, we found higher oxidative stress in B adults through significantly higher plasma levels of protein carbonyls, TAC, and SOD. B adults also had lower plasma NO levels relative to W, which is consistent with previous research demonstrating higher ADMA and lower NO levels in young B males. In conclusion, based on these preliminary findings, our future mechanistic research will focus on expression of SOD and NOS isoforms to identify where the racial differences exist in activation of these oxidative stress markers. NIH/NHLBI RO1 HL085497 & NIH/NIA KO1 AG019640
Differential calcineurin/NFATc1‐c3 signaling in the regulation of the skeletal muscle fiber phenotype
Authors: Kulig Ewa M , St‐Louis Mathieu , Eibl Joe K , Pavlath Grace , Michel Robin N
Published: June 2021

Involvement of Cn/NFAT signaling in the regulation of the skeletal muscle fiber phenotype is recognized, however, the role of the various Cn‐regulated NFAT isoforms in this process remains rudimentary. We thus assessed the expression, localization and function of NFAT isoforms in muscle fibers of wild type or transgenic NFATc2 or NFATc3 deficient mice under normal weightbearing or functional compensatory work overload conditions. Immunohistochemical analyses indicated that NFAT isoforms have distinct roles in the regulation of muscle remodeling associated with compensatory growth. Semi‐quantitative RT‐PCR did not reveal compensatory up‐regulation of surviving NFAT isoform mRNA levels in transgenic mice. On the other hand, western blotting and immunofluorescence data showed NFAT protein localization to be differentially regulated during compensatory growth. Moreover, gene array analyses showed distinct gene expression patterns across conditions. Taken together, these results show that members of the NFAT family of transcription factors have distinct roles in the expression of the muscle fiber phenotype and a limited capacity to substitute for one another during conditions of rapid fiber growth. Supported by NSERC, CIHR and CRC to RNM. EMK is a CIHR scholar.
Voltage‐dependent potassium currents of novel threadlike structure on the surface of internal organs in rats
Authors: Han Tae Hee , Choi Jae‐Hong , Lim Chae Jeong , Lee Byung Cheon , Soh Kwang Sup , Lee So Yeong , Ryu Pan Dong
Published: June 2021

The electrophysiological properties of novel threadlike structure (NTS) on the surface of internal organs were investigated using whole cell patch clamp technique. NTS had well‐developed branches and were joined to a corpuscle (NTSC). Voltage clamp analysis revealed that the current‐voltage relationship (I–V) was highly non‐linear and can be divided into four types (Type I ~ IV) by slope conductance and their passive membrane properties in NTS cells. The outward current was inhibited, while residual currents were existed in outward and inward currents, by the voltage‐dependent potassium (Kv) channel blocker, tetraethylammonium (TEA), in a concentration‐dependent manner (IC50: 4.30 ± 1.71 and 4.43 ± 1.92 mM at +60 mV and −60 mV, respectively). These results indicated that the current was mainly carried by K+ ions. In addition, the resting membrane potential was depolarized by administration of 30 mM TEA. A systematic screening using RT‐PCR technique showed that genes of Kv1.2, Kv1.3, Kv2.1, Kv3.2, Kv3.3, Kv3.4, Kv4.1, Kv4.2 and Kv4.3 were expressed in NTSC. Taken together, our results indicated that Kv channel has an important role in regulating the membrane potential of NTSC cells.
Activation of Neutral Sphingomyelinase‐2 and Protein Phosphatase 2A by Interleukin‐1B: role in IRAK‐1 degradation and intracellular translocation
Authors: Dobierzewska Aneta , Giltiay Natalia , Rutkute Kristina , Karakashian Alexander , Nikolova‐Karakashian Mariana
Published: June 2021

The objective of this study is to understand the role of Neutral sphingomyelinase‐2 (NSMase‐2) in hepatic response to Interleukin 1β (IL‐1β). The IL‐1β signaling cascade is initiated by rapid phosphorylation of IL‐1β receptor activated kinase‐1 (IRAK‐1) in response to ligand binding. Subsequently, the phoshporylated IRAK‐1 is ubiquitinated and degraded and the rate of its degradation is known to affect the magnitude of IL‐1β response. We show that the activity of protein phosphatase 2A (PP2A) gradually increases in response to IL‐1β stimulation with a maximum effect seen 30 minutes after the stimulation. The PP2A catalytic subunit directly interacts with IRAK‐1, indicating that IRAK‐1 is a novel PP2A substrate. Furthermore, treatment of primary rat hepatocytes or HEK293cells overexpressing the IL‐1β receptor with PP2A inhibitor, okadaic acid, enhances the rate of IL‐1β‐induced IRAK‐1 ubiquitination and degradation.

NSMase‐2 mediates the activation of PP2A by IL‐1β. Overexpression of NSMase‐2 enhances PP2A activity and delays IRAK‐1 degradation. In contrast, NSMase‐2 silencing or inhibition by pharmacological inhibitor causes faster IRAK‐1 degradation. NSMase‐2 also plays a role in IRAK‐1 intracellular translocation in response to IL‐1β. These experiment identify NSMase 2‐and PP2A as novel physiological regulators of IRAK‐1 phosphorylation.

Supported by NIA RO1 AG026711.
A modified dileucine motif mediates the internalization of a G‐protein activated inward potassium channel
Authors: Rangel Claudia Iveth , Díaz‐Bello Beatriz , Padilla Teresa , López Zinaeli , Salvador Carolina , Escobar Laura
Published: June 2021

G‐protein‐activated inward rectifying potassium channels (GIRK) participate in inhibitory synapsis, where neurotransmitters activate Gi/Go protein coupled receptors. Gβγ dimers bind and open GIRK channels, hyperpolaryzing the postsynaptic membrane. GIRK (GIRK1‐4) subunits form heteromultimers of GIRK1 in mammals. GIRK5 channels are expressed in Xenopus laevis oocytes. We previously found that phosphorylation of a tyrosine (Y16) at the NH2‐terminal of GIRK5, keeps the channels internalized (Mora and Escobar, 2005). The sequence E17XXXLI22, reported as an internalization and/or a lysosomal targeting motif, is localized upstream of Y16. Therefore, we aim to explore the role of this dileucine motif in the GIRK5 functional expression in oocytes. We performed alanine scanning mutagenesis and whole cell voltage‐clamp recordings. The single mutants L15A, E17A, S18A, P19A, Q20A, L21A and Q23A did not produce inwardly rectifying currents. In contrast, Y16A, I22A and the quadruple mutant Y16A/E17A/L21A/I22A displayed inward current amplitudes according to the number of mutant residues. In conclusion, our results suggest that the sequence Y16XXXXXI22 at the NH2‐term of GIRK5 determines the internalization of the GIRK5 channels in Xenopus laevis oocytes.
Carotid body chemoreceptors removal (CBX) blocks resting bradycardia and reduces oxytocin immunoreactivity (OTir) in PVN preautonomic subnuclei.
Authors: Cruz Josiane Campos , Cavalleri Marina Tuppy , Michelini Lisete Compagno
Published: June 2021

We showed that sinoaortic denervation prevents training (T)‐induced bradycardia in normotensive rats (Exp Physiol 2009;94:630–640; 947–960) as well prevent T‐induced plasticity in hypothalamic paraventricular (PVN) neurons (unpublished). We sought to investigate whether peripheral chemoreceptors also mediate these responses. Rats submitted to CBX or SHAM surgery were trained or kept sedentary (S) for 3 month. After functional recordings, rats were anesthetized, perfused (PFA 4%) and brains prepared for OT immunohistochemistry (1ary antibody 1:20.000). CBX abolished pressor and bradicardic responses to chemoreflex activation (KCN 40 ƒÝg, iv) in S and T rats, without changing baroreflex function (phenylephrine/sodium nitroprusside). T caused resting bradycardia and marked increase in the number of OTir cells in the ventromedial [29¡Ó3 vs. 12¡Ó1, n=3 (VM)] and posterior [28¡Ó1vs. 21¡Ó1, n=3 (PT)] PVN, but not in the dorsal cap. CBX blocked T‐induced bradycardia, caused decrease in the number of OTir cells (6¡Ó1 in VM, 14¡Ó1 in PT, n=3) and blocked T‐induced increases in OTir cells only in the VM. Conclusions: Chemoreceptor signaling mediates T‐induced bradycardia by improving OTergic control within the VM PVN. It is also important to maintain tonic OTergic control in some preautonomic PVN subnuclei. Financial Support: CNPq, FAPESP.
HotSpot: A Novel Methyltransferase Assay Platform for Drug Screening and Discovery
Authors: Horiuchi Kurumi Y , Eason Mia M , Ma Haiching
Published: June 2021

Epigenetic regulation has been studied extensively in the past few years, and many studies indicate that it is strictly involved in tumor establishment and progression. Namely, Histone methyltransferases (HMTs) and DNA methyltransferases (DNMTs) are a large group of enzymes that specifically methylate histone or DNA using S‐adenosyl‐L‐methionine (AdoMet) as a methyl donor. However, there is no widely accepted HTS assay formats and reference inhibitors are available. We have developed and successfully commercialized an extremely low cost miniaturized radioisotope reaction system, the HotSpotSM platform, for kinase screening and profiling to provide services in the drug discovery community. We have effectively adapted this HotSpotSM platform for methyltransferase assays. In this gold standard radioisotope format, tritium‐labeled AdoMet (3H‐AdoMet) was used as a methyl donor and total methylations on substrate were read‐out, which gave high signal/background ratio resulted in high Z’ values. Here, we demonstrate HMTs and DNMTs profiling, kinetic studies, mode of inhibition studies, and HTS can be performed with this platform, which will become a low cost high productive service product to help the drug discovery activities in the emerging epigenetics field. This work was funded in part by NIH SBIR grants R43CA139621, and NIH grants R01HG003818 to H.M.
Supraphysiological testosterone administration alters renal 25‐hydroxyvitamin D‐3 1α‐hydroxylase protein expression in female rodents
Authors: Lipinska Judyta Anna , Conover Christine F , Yarrow Joshua F , McCoy Sean C , Santillana Cesar A , Borst Stephen E
Published: June 2021

The enzyme 25‐hydroxyvitamin D 1α‐hydroxylase (CYP27B1) converts circulating 25‐hydroxyvitamin D to its active form, 1,25‐dihydroxyvitamin D. The effects of testosterone (T) on CYP27B1 expression within the kidney, an androgen sensitive organ, remain unclear. The purpose of this study was to determine whether gonadectomy (GNX) or T alter renal CYP27B1 protein expression. 3 month old male and female F344 rats (n = 5/group) received SHAM surgery + vehicle (V), GNX + V, or GNX + supraphysiological T‐enanthate (GNX+TE) for 28 days. The kidney was removed at sacrifice and CYP27B1 expression was measured in the renal cortex by Western blot. GNX+TE increased kidney mass by 19% and 28% in males and females compared with Shams and by 29% and 26% compared with GNX + V (p < 0.05). No differences in renal CYP27B1 expression were present between intact males and females. GNX did not alter CYP27B1 protein expression in either sex. No change in CYP27B1 protein expression was observed in GNX+TE males. However, renal cortex CYP27B1 protein expression was reduced by 28% in GNX+TE females compared with SHAMs (p < 0.05). Our results indicate that supraphysiological TE down‐regulates renal cortex CYP27B1 protein expression in female rodents, through a yet to be identified mechanism. Future studies examining the effects of T on the activity and expression of CYP27B1 should account for androgen induced alterations in kidney mass.
Genetic ablation of cyclophilin D, a component of the mitochondrial permeability transition pore, improves insulin sensitivity in high‐fat fed mice
Authors: Akhtar Yasir Naeem , Lira Vitor A , Zhang Mei , Okutsu Mitsuharu , Hoehn Kyle L , Yan Zhen
Published: June 2021

Mitochondrial dysfunction has been implicated in the development of insulin resistance. Inhibition or genetic ablation of cyclophilin D (CypD), a critical protein of the mitochondrial permeability transition pore (PTP), has been shown to protect against mitochondrial injury in skeletal muscle. We hypothesized that CypD knockout (KO) mice would be protected from high‐fat diet‐induced insulin resistance. We subjected 8‐week old male CypD KO (n=8) and wild‐type (WT) littermates (n=4) to 10 weeks of high fat feeding (60%). Before the diet intervention, CypD KO and WT littermates had similar body weight, food intake, insulin tolerance test (ITT) and glucose tolerance test (GTT) profiles. After high‐fat feeding, CypD KO mice had significantly lower glucose levels on GTT (p < 0.005 for the area under the curve, AUC) and lower fasting glucose levels compared with WT mice (117.5±17.2 vs. 166.8±36.1, p < 0.01). ITT showed generally lower glucose levels in the CypD KO mice with a statistical difference at 30 minutes (85.5±32.5 vs. 140.4±55.3, p < 0.05). Therefore, insulin resistance induced by high fat diet is attenuated in mice lacking a critical component of the mitochondrial PTP.
Hepatic lipid saturation indices and insulin resistance in non‐alcoholic fatty liver disease (NAFLD)
Authors: Haus Jacob M , Solomon Thomas PJ , Lu Lan , McCullough Arthur J , Flask Chris A , Kirwan John P
Published: June 2021

NAFLD is a prevalent condition associated with obesity and insulin resistance (IR). Evidence suggests saturated lipids play a key role in the development of NAFLD while polyunsaturated lipids may be protective. Our aim was to examine the relationships between indices of lipid saturation (SI), polyunsaturation (PUI) and total fat content in the liver with measures of peripheral (GDR) and hepatic insulin resistance (HGP). Obese (n=21; BMI: 33.8±0.97 kg/m2) subjects with suspected or diagnosed NAFLD underwent measures of liver lipid saturation via proton MRS, visceral adiposity (VAT) and euglycemic clamp with 2H2‐glucose infusion. Total hepatic triglyceride content (HTGC; 15.3±2.0%) was negatively correlated (R=− 0.39; p<0.05) with GDR, and positively correlated (R=0.55; p<0.05) with VAT. However, SI (95.8±0.3%) did not correlate with HTGC, GDR or HGP, while PUI (0.3±0.03%) was inversely and paradoxically associated with GDR (R=− 0.55; p<0.05). PUI was also found to be correlated with VAT, BMI, and HTGC (R=0.74, 0.54, 0.52; p<0.05). In obese subjects with NAFLD, MRS derived indices of liver lipid saturation appear to be linked to measures of adiposity such as VAT, BMI and total HTGC. Further examination in a more heterogeneous population is required to validate the diagnostic utility of liver lipid saturation indices with NAFLD.
Juvenile Murine Distal Colon Exhibits Increased CFTR, ENAC and Basolateral Transport
Authors: Gawenis Lara , Gawenis James
Published: June 2021

Previous studies suggest increased epithelial ion transport in neonatal colon. We hypothesized that apical and basolateral transport activities are increased in distal colon of juvenile mice and that this tissue is a model for studying transport during intestinal maturation. Short‐circuit current (Isc) responses to cAMP and inhibitors were measured in distal colon from adult (2–4 months) and juvenile (14–16 days) wild‐type mice. In physiologic Ringers (Cl− and HCO3− containing), the Isc response to amiloride and the peak cAMP‐induced increase in Isc were significantly greater in juvenile as compared to adult tissues. The increase in peak Isc was abolished by inhibition of carbonic anhydrases or EIPA (to inhibit coupled NHE1 Na+/H+ exchanger/AE2 Cl−/HCO3− exchanger). Removal of HCO3− from the perfusion Ringers (Cl−‐only) also abolished the difference between juvenile and adult distal colon; however, the ÄIsc response to cAMP was increased or unchanged due to increased NKCC1 activity. Together these findings support the hypothesis that apical channel activity facilitated by the basolateral NHE1/AE2/CA transport metabolon enhances Cl− secretion in juvenile distal colon. Current studies are focused on evaluating the altered activity to determine if these changes reflect changes in expression or intestinal morphology in the post‐weaning period (as the intestine matures). Supported by AGA‐FDHN and CFFT.
Expression of BK (large conductance calcium‐activated K) channel subunits and oxygen sensitivity of glomus cells in mice
Authors: Shirahata Machiko , Kostuk E. , Otsubo T. , Balbir A. , Fujii K. , Pichard L.
Published: June 2021

The carotid body (CB) is a primary oxygen sensor, and oxygen sensitivity of K channels in glomus cells (GCs) may be responsible for hypoxic sensing. Hypoxic sensitivity of the CB appears to be genetically determined. In mice, the DBA/2J strain responds vigorously to hypoxia, but the A/J strain responds weakly. We have hypothesized that differential expression of oxygen‐sensitive K channels, especially BK channels, causes different hypoxic sensitivity of the two strains of mice. Gene expression of BK channel subunits in the CB was examined with RT‐PCR analysis. Actin was used as a reference gene after validating its use among total five housekeeping genes. We found that α and β2 subunits were more expressed in DBA/2J mice than A/J mice. No differences were found in the expression of the β4 subunit between the two strains. The role of BK channels in hypoxic sensitivity was examined in patch clamp experiments using undissociated CBs. Voltage‐dependent outward K current in GCs was inhibited by 4‐aminopyridine in both mice. Iberiotoxin significantly inhibited K current in DBA/2J mice, but not in A/J mice. With decreasing PO2 to ~70 mmHg, K current declined in GCs of DBA/2J, but not A/J mice. With the presence of iberiotoxin, mild hypoxia did not inhibit K current in either strain of mice. In accordance with these results, mild hypoxia increased intracellular Ca2+ in GCs of DBA/2J mice, but not in A/J mice. The data suggest that BKαβ2 channels are extremely sensitive to PO2 changes, and thus, GCs in DBA/2J mice are sensitive to mild hypoxia. It is possible that BKβ4 subunits interferes functional expression of BK channels. In A/J mice, in addition to lower expression of pore‐forming α subunits, higher expression of β4 subunits relative to other subunits may cause lower hypoxic sensitivity of the GC. Support: HL81345, AHA09GRNT2080158
Oxygen Generating Biomaterials for Ischemic Tissue Salvage and Function
Authors: Corona Benjamin T , Ward Catherine L , Yoo James J , Harrison Benjamin S , Christ George J
Published: June 2021

Skeletal muscle viability is lost when there is a prolonged interruption of blood supply. The ensuing lack of oxygen, or hypoxia, results in ischemia and necrosis, which greatly deteriorates skeletal muscle function. The application of a material that generates oxygen could maintain skeletal muscle viability under such hypoxic conditions. A solution of particulate oxygen generators (POGs) was created and injected into an isolated rat extensor digitorum longus (EDL) muscle, a tissue that will experience oxygen diffusion limitations due to its volume. The muscle was placed in an in vitro organ bath, wherein the core of the EDL muscle is oxygen‐deprived, and assessed at 37°C for functional capacity during a 1.5‐hour contractile test. The injection of POGs subsided the decline of active force production and rise in baseline tension during the contractile test, compared to EDL muscle injected with vehicle‐only. These findings suggest that POGs attenuated hypoxia in isolated EDL muscle.
A genetic approach to optically investigate P2X2 receptor activation in vivo using an activity‐dependent FRET based reporter tag
Authors: Kracun Sebastian , Khakh Baljit S.
Published: June 2021

P2X receptors are ATP‐gated cation selective ion‐channels found throughout the body, including the CNS where ATP signaling is proposed to contribute to normal brain physiology, whereas aberrant P2X receptor expression may result in phenotypes relating to nervous system dysfunction, including epilepsy. However, a precise mechanistic understanding of P2X receptors in the brain does not exist and their role in epileptiform activity remains unclear. Our goal was to generate transgenic P2X2 reporter mice and, thus investigate in vivo P2X2 receptor mediated ATP signaling and its involvement in the hyperexcitability of neural networks. We have developed a non‐invasive approach to optically track the location and activation of P2X2 receptors, using a calcium sensitive FRET based reporter, with sensitivity equal to whole‐cell patch clamp recording (P2X2‐YC; Nature Methods, 2008). Here, we describe the generation of lentiviruses expressing P2X2‐YC and the DNA construct design and bacterial artificial chromosome (BAC) based transgenic method to generate mice expressing P2X2‐YC under the influence of the CaMKIIα gene promoter. We have successfully generated six ‘founder’ mouse lines verified by genomic PCR and have tested lentiviruses in vitro. The mice and viruses will be further characterised using immunohistochemistry, fluorescence imaging and electrophysiology to assess P2X2‐YC gene expression.

This research was supported by the NIH.
Comparative Analysis of the Biological Activities of Different NS1 Variants from H5N1 Avian Influenza Virus
Authors: Zhang Chi
Published: June 2021

The nonstructural protein NS1 contributes significantly to the pathogenicity of H5N1 avian influenza virus. It has been reported that five amino acids missing at 80–84 sites or mutation from aspartic acid to glutamic acid at position 92 (D92E) in NS1 play a key role in the enhanced virulence of H5N1 influenza virus. In order to elucidate whether the elevated virulence of virus is associated with the changes of cytokines induction/resistance and cellular apoptotic response caused by these NS1 mutations, we constructed three mutant NS1 proteins: NS51 with 5aa deletion at position 80–84; NS51(I) with 5aa insertion at position 80–84, and NS51(IM) with 5aa insertion and D92E replacement. We observed that 5aa deletion enhanced resistance to TNF¦Á response and D92E mutation resulted in the lost of NS1 inhibition in IFN induction. Our results also revealed that all three variants interfered similarly the transcriptional activity of p53. The above findings help to understand the role of NS1 in H5N1 avian influenza virus.
Functional Consequences of Nucleotide Binding to the Proteasomal ATPases
Authors: Smith David M , Fraga Hugo , Reis Christian , Goldberg Alfred L
Published: June 2021

Protein degradation by the eukaryotic 26S proteasome or the homologous archaeal PAN‐20S proteasome complex is a multistep process that requires ATP hydrolysis by the proteasome‐associated AAA ATPase complex. However, the mechanisms by which these hexameric ATPases utilize ATP to promote protein breakdown and activate proteasomal function are poorly understood. Although PAN contains six identical ATPase subunits, we found that it exhibits three types of binding sites: 2 high affinity conformations (Kd=0.2uM), 2 with lower affinity (Kd=60uM), and 2 with conformations that fail to bind ATP. In fact, PAN never bound more than four of any type of nucleotide (ADP, ATP, or the nonhydroylyzable analog ATPγS), even at high concentrations. ATP binding to the high and lower affinity sites has distinct functional consequences on the proteasome. With two ATPγS molecules bound, PAN maximally stimulates opening of the gated channel for substrate entry into the 20S proteasome and has a high affinity for the 20S as well as for protein substrate. However, the binding of 4 ATPγS reduces PAN's ability to stimulate gate opening as well as its affinity for the 20S and substrates. These functional consequences of nucleotide binding are conserved in the mammalian 26S proteasome, since gate opening exhibited nearly identical multiphasic dependence on ATPγS concentration as found for the PAN‐20S complex. Because ATP binding drives the association of the C‐termini of the ATPase with the 20S and only two ATPase subunits bind ATP for maximal function it's likely that only two ATPases’ C‐termini dock into the 20S at any time and in a predictable pattern. This observation suggests how the hexameric ATPase ring associates with the heptameric 20S proteasome to regulate substrate degradation.
The molecular mechanism of the Hsp70‐lipid interaction (1002.6)
Authors: McCallister Chelsea , Nikolaidis Nikolas
Published: June 2021

Hsp70s are molecular chaperones essential for cellular homeostasis and survival. Several Hsp70s associate with cellular membranes, where they bind to specific lipids. The association of Hsp70s with lipids anchors them to the plasma membrane, where they serve several functions, stabilizes the lysosomal membrane, and is required for microautophagy. Despite these essential functions the Hsp70‐lipid interactions remain largely uncharacterized. To understand the molecular mechanism of these interactions we characterized the binding of HSPA1A, a cytosolic Hsp70, to lipids. We determined the binding affinities of recombinant HSPA1A to several negatively charged phospholipids and established that allosteric communication between the nucleotide‐binding and substrate‐binding protein domains is important for lipid‐binding. We also determined that a conformational change caused by nucleotide‐binding reduces lipid‐binding for a particular subset of lipids, while binding of protein substrates has no effect on the lipid‐binding. Additionally, we determined that the HSPA1A‐lipid interaction is not purely electrostatic, but it also depends on other forces. Furthermore, we found that HSPA1A embeds in membranes only when bound to specific lipids and that membrane embedding is independent of the affinity of the interaction. Finally, we observed that while lipid‐binding does not affect the ability of HSPA1A to hydrolyze ATP, it minimizes the refolding activity of the protein suggesting that lipid‐binding could represent a new regulatory mechanism of chaperone function.

Grant Funding Source: Supported by funds from CSUPERB and CSUF to NN, and HHMI to CM and NN
Insulin‐stimulated neuronal nitric oxide synthase phosphorylation is AKT2‐dependent in C2C12 skeletal muscle cells (1013.13)
Authors: Hinchee‐Rodriguez Kathryn , Adamo Martin , Masters Bettie Sue , Roman Linda
Published: June 2021

Type 2 Diabetes (T2DM) is a major cause of death in the United States, and has become a global pandemic. T2DM results from reduced insulin sensitivity coupled with a relative failure of insulin secretion. Reduced insulin sensitivity is associated with reduced nitric oxide synthase (NOS) activity and impaired glucose uptake in T2DM skeletal muscle. Upon insulin stimulation, nitric oxide synthesis increases in normal adult skeletal muscle, whereas no such increase is observed in T2DM adults, which is significant given that nitric oxide stimulates glucose uptake in skeletal muscle. We have shown that neuronal NOS (nNOS) is phosphorylated at Ser1450, resulting in its activation, in response to insulin in skeletal muscle, but it remains unknown which kinase(s) in the insulin signaling cascade is responsible. Immunoblot analyses indicate that in C2C12 myotubes, insulin‐stimulated nNOS phosphorylation is dependent on one or more AKT isoforms, as AKTi, which prevents AKT1/2 activation, inhibited nNOS phosphorylation. Moreover, the AKT2‐specific inhibitor, AKT2i, also attenuated nNOS phosphorylation, implicating this isoform specifically. These results are consistent with the essential role of AKT2 in mediating glucose uptake in skeletal muscle and show, for the first time, that C2C12 myotube nNOS phosphorylation, and its associated increase in activity, are dependent on insulin signaling‐activated AKT2.

Grant Funding Source: Supported by NIH Grant No. GM052419 to BSM/LJR
Pregnancy in Mbale, Uganda: perceptions of food security and influences on women's breastfeeding intentions (1014.7)
Authors: Ickes Scott , Pugh Laura
Published: June 2021

Pregnancy is a nutritionally vulnerable time for both the mother and the child; nutritional demands increase, as do the consequences for failing to meet these needs. Traditional beliefs, social support, psychological health, food security, other responsibilities, income, and autonomy in decisions all play roles in what women eat during pregnancy. The purpose of this study was to understand women’s perceptions of food security during pregnancy, and to assess the concordance of desired versus actual dietary changes during pregnancy among low‐income pregnant women in Mbale, District, Uganda. We conducted semi‐structured, in‐depth interviews (n=15) and one focus group (n=8) among pregnant women living in slum and rural settings. The majority of women report eating less during pregnancy. Often they describe eating fewer meals in a day, in addition to cutting out staple foods due to nausea. All women changed their eating habits during pregnancy, both cutting out foods that made them feel sick and introducing foods they considered to be necessary or healthier. Only two women felt they were in control of what and how much they ate; all others said that resource constraints prevent eating how much and what kinds of food they’d prefer. Half of the woman also worried that their household did not always get enough total food. Few women receive support for either food or responsibilities during their pregnancies. When asked, most women intended to exclusively breastfeed, but frequently misconstrued the definition of exclusive. Pregnancy should be a renewed focus among nutrition programs in Uganda, to dispel misconceptions around correct eating habits and help women form strong and achievable breastfeeding intentions. It is vital that even food insecure women gain access to adequate calories and nutrients in order to prevent low birth weight and stunted babies, and to give them enough energy reserves to feel confident in sufficient high quality breast milk.

Grant Funding Source: Supported by a College of William and Mary Monroe Scholarship
Association between dairy intake and brain glutathione levels in older adults (1018.4)
Authors: Choi In‐Young , Lee Phil , Denney Douglas , Spaeth Kendra , Nash Olivia , Roth Alexandra , Lierman Jo , Sullivan Debra
Published: June 2021

Glutathione (GSH) plays a key role in protecting cells against oxidative stress. A reduction in GSH levels is noted in brain tissue undergoing oxidative stress in health and disease. We investigated the effect of dietary factors, specifically dairy intake, on oxidative stress, using a unique, noninvasive measure of brain GSH levels in older adults. Healthy subjects (n=57; 68 ± 6.1 yrs; BMI = 25 ± 4) whose daily dairy consumption was low (n=20; < 1 serving/day), moderate (n=22; >1,<3) or at recommended levels (15, >3) were recruited. Brain GSH was measured using a specially designed 1H MRSI technique for GSH at 3 T. Dietary intake was assessed with 7‐day food records, entered into NDS‐R. Total dairy servings were correlated with GSH levels in the frontal (Spearman Rs = .37, p = .004) and parietal regions (Rs = .46, p < .001). Correlations were also found between frontal GSH and milk (Rs = .36, p =.006) and between parietal GSH and milk (Rs = .38, p = .004), cheese (Rs = .40, p = .002) and calcium (Rs = .31, p = .022), but were lower than those for total dairy servings. The dairy intake groups did not differ in age, BMI, or fat consumption. Recommended dairy consumers had higher energy, carbohydrate, and protein intake, but these differences did not account for the relationship between dairy servings and GSH. The results suggest that increased dairy intake may be beneficial to brain health in aging via increasing cerebral antioxidant capacity.

Grant Funding Source: Supported by Dairy Research Institute
Of Soda and Sick Days: a field study of firefighter diets (1019.12)
Authors: Kniffin Kevin , Wansink Brian
Published: June 2021

We present the results of a survey study of on‐ and off‐duty eating behaviors among professional firefighters in a large city in the United States. Of particular interest, we find a significant positive correlation (r = .242) between the amount of soda that a person consumes and the number of sick days they reported for the previous year. Notably, we do not find relationships between the amount of soda consumption and the presence of worksite vending machines. Similarly, we do not find a relationship between the amount of soda consumption and a person's overall health. Our findings invite closer investigation into the potential influence of soda consumption for short‐term illnesses that disrupt a person's ability to work.