bluuebunny/crossref_metadata_2025 huggingface dataset:
Deducing the Origin of Hsp70 Binding Selectivity by Structural Study
Authors: Jansen Rachel M. , Clerico Eugenia M. , Ozden Can , Gierasch Lila M.
Published: April 2020

Hsp70s are a family of molecular chaperone proteins that aid in the folding of other proteins under physiological and stress conditions. Chaperones prevent protein misfolding and aggregation, characteristic of many neurodegenerative diseases. Hsp70s perform their chaperoning functions through interactions between their substrate binding domain (SBD) and short hydrophobic sequences of their non‐native protein clients that are exposed to the solvent. The details of the interaction between the chaperone and diverse sequences remain poorly understood. Much of the work on the interaction between Hsp70s and client proteins has been done using DnaK, the E.coli Hsp70 and short peptides as surrogates for the client protein. In 1996, the 3D structure of the SBD of DnaK bound to one model peptide was determined using x‐ray crystallography (Zhu et al., 1996), and more recently, the forward and reverse binding between the SBD and a few small peptides has been observed (Zahn el al., 2013). Therefore, it is known that the SBD can accommodate numerous sequences but is nonetheless selective in which sequences it binds. This project aims to understand this “promiscuous selectivity” through analyzing the atomic resolution structures of SBD bound to various peptides. To begin dissecting the selective promiscuity of SBD binding to various sequences we are using proPhoA, an endogenous substrate of DnaK, that has been shown to have five DnaK binding sites. We have determined structures of the SBD of DnaK bound to three of the five binding sites in proPhoA using x‐ray crystallography. These structures reveal a variety of binding modes despite a uniformity of the backbone conformation for the bound peptides. Using this detailed atomic resolution information, we are dissecting how the peptides bind in the SBD and if the SBD remodels to accommodate these unique but specific sequences. This information is shedding light on the basis of Hsp70/substrate recognition.

Support or Funding Information

1. NIH R35 GM118161 MIRA Grant "Protein folding in the Cell: Challenges and Coping Mechanisms" 6/1/16 – 5/31/2021

2. University of Massachusetts ‐ Amherst ‐ Biochemistry and Molecular Biology Department
Understanding the Mechanism of Fibroblast Growth Factor 21 Action in the Paraventricular Nucleus of the Hypothalamus: A Promising Candidate in the Treatment of Obesity and Metabolic Diseases
Authors: West Chloe Lily , Wanders Desiree , Gettys Thomas W , Land Hannah , Stern Javier
Published: April 2020

Fibroblast Growth Factor 21 (FGF21), when administered pharmacologically reduces body weight, increases insulin sensitivity and energy expenditure, and induces browning of white adipose tissue (WAT). Research has shown that FGF21 signaling in the brain is required for the induction of these metabolic benefits, with the paraventricular nucleus of the hypothalamus (PVN) as a potential primary site of action. However, the neuronal circuits and precise mechanisms mediating FGF21’s metabolic effects remain incompletely understood. Further, the role of the sympathetic nervous system (SNS) in this process remains unclear. Here, we aimed to address these questions using dietary methionine restriction (MR) as a tool to stimulate endogenous FGF21 production in WT and FGF21 KO mice fed a control or a high‐fat diet. Dietary MR rapidly and persistently increases serum FGF21 concentration by up to 8‐fold (p<0.0001). We found that in WT mice, MR increased energy expenditure and thermogenic markers in iWAT and BAT, an indication of increased SNS output to these tissues in WT, but not FGF21 KO mice. Additionally, we observed increased ERK phosphorylation in the PVN of the WT but not FGF21 KO mice, supporting PVN neurons as a likely target mediating FGF21 actions. To further investigate the role of FGF21 in the PVN, we used whole‐cell patch clamp recordings in retrogradely‐labelled presympathetic PVN neurons projecting to the rostral ventrolateral medulla (RVLM), and measured the effects of the FGF21 analog LY2405319 (500nM) on the firing activity of PVN‐RVLM neurons. Recordings in current‐clamp mode showed that FGF21 either increased (33% of recorded neurons) or decreased (67% of recorded neurons) the firing activity of these neurons suggesting that FGF21 effects may depend on their projecting targets. Recordings in voltage‐clamp mode also showed that FGF21 significantly decreased the degree of activity of GABAergic inhibitory inputs into these neurons. Thus, LY2405319 inhibited the frequency of GABA inhibitory postsynaptic current (IPSC) (42.02% decrease, p< 0.05), suggesting that FGF21 effects on presympathetic neurons in the PVN could be mediated by direct and/or indirect actions, including modulation of local network activity, such as local GABAergic interneurons. Taken together, our data support the hypothesis that FGF21 signals through PVN neurons to increase SNS output to adipose tissue, resulting in increased energy expenditure, reduced body weight, and improved insulin sensitivity.
Role for NADH‐sensitive Kv channels in the myocardial‐vascular signaling axis.
Authors: Nystoriak Matthew A. , Ohanyan Vahagn , Dwenger Marc M. , Chilian William M , Bhatnagar Aruni
Published: April 2020

Voltage‐gated potassium (Kv) channels expressed in the coronary vasculature play a pivotal role in coupling oxygen delivery with myocardial metabolic demand. Despite their importance in maintaining adequate perfusion to support proper cardiac function, the processes by which these channels respond to metabolic cues from the myocardium to elicit smooth muscle hyperpolarization and rapid relaxation are unclear. We tested the hypothesis that the heteromeric auxiliary Kvβ subunit (Kvβ1:Kvβ2) complex facilitates increases in Kv1 channel activity and coronary vasodilation during periods of elevated myocardial oxygen consumption. The Kvβ proteins are functional aldo‐keto reductases, and thus catalyze nicotinamide cofactor‐ (i.e., NAD(P)H) dependent reduction of a range of aldehyde substrates. Intriguingly, these subunits also differentially modify the voltage‐sensitivity of Kv1 channels in a manner that depends upon the redox state of bound cofactor. Imaging mass spectrometry revealed that lactate:pyruvate (used as a surrogate for NADH:NAD+) was significantly increased in intramyocardial arteries in hearts from mice acutely treated with dobutamine to drive elevated cardiac workload versus those from vehicle‐treated mice. Elevation of NADH:NAD+ ratio in coronary arterial myocytes following perfusion of sodium lactate reversibly increased whole cell Kv currents and shifted the voltage‐dependence of activation by ~−15 mV. Moreover, application of 1 mM NADH, but not NAD+, increased single Kv1 channel currents in excised membrane patches from coronary arterial myocytes of wild type mice. In contrast, ablation of Kvβ2 abolished the NADH sensitivity of Kv1 activity, whereas ablation of Kvβ1 had little effect. Consistent with this, we found that vasodilation of coronary arteries to reduced bath oxygen (to ~3%) and the relationship between cardiac work and myocardial blood flow was significantly blunted in Kvβ2−/− arteries/mice when compared with wild type arteries/mice. Together, our results suggest that the Kvβ complex is a functional sensing unit that modulates native coronary Kv1 channel activity upon changes in pyridine nucleotide redox ratio, which may impart a profound influence on the control of myocardial blood flow.

Support or Funding Information

This work is supported by grants from the National Institutes of Health (HL142710, GM103492).
Tertiary sulci, transcriptomics, and functional gradients in human cortex
Authors: Weiner Kevin S.
Published: April 2020

Understanding how the anatomical structure of the brain across spatial scales generates a flexible infrastructure of functional representations subserving the complex aspects of human cognition is a major goal of neuroanatomists, cognitive neuroanatomists, and cognitive neuroscientists. Traditionally, it is well‐accepted that deep, primary indentations in the cerebral cortex, or sulci, serve as landmarks of functional and architectonic areas in primary sensory cortex, but not in association cortex. Nevertheless, findings from the last decade show that shallow, tertiary sulci that emerge late in gestation and continue to develop after birth serve as landmarks identifying microarchitectonic and functional transitions in human visual association cortex (Weiner, 2019, The Anatomical Record), which is consistent with classic anatomical theories (Sanides, 1964, Neuropsychologia). What factors might explain this unexpected anatomical‐functional coupling across spatial scales in visual association cortex? In this talk, I will first briefly summarize a series of studies from my lab revealing the contribution of gene expression (e.g. transcriptomics) in both the arealization of cortical hierarchies, as well as the topological layout of functional maps within areas. I will then discuss additional studies from my lab showing that the anatomical‐functional correspondences identified in human visual association cortex also a) extend to lateral prefrontal and posterior cingulate cortices and b) have behavioral implications. Finally, I will show how the combination of these findings have immediate translational, developmental, and evolutionary applications for understanding how the hominoid brain efficiently leverages shallow, rather than deep, sulci to house cognitively, microarchitectonically, and functionally complex transitions in association cortices ‐ a cortical organization that enables a fast read‐out of neural information that contributes to efficient human behavior.
TEV‐FRET: High‐Throughput Assay for Determining Protease Activity
Authors: Johnson Maddi , Page Richard C.
Published: April 2020

The production of proteins for structural studies has been greatly aided by the use of fusion tags to increase expression yields or facilitate preparative‐scale purification with high yields and high purity. Isolating the desired protein construct from the fusion protein is commonly performed using tobacco etch virus (TEV) protease. Here we develop a high‐throughput assay for testing fusion protein cleavage by TEV protease. Initial results with a TEV‐targeted peptide containing a FRET pair allow for rapid assessment of cleavage efficiency and activity using FRET. Future versions of the assay will capitalize on ELISA with specific N‐ and C‐terminal targets for identifying cleavage efficiency and activity. These results are outcomes of a project executed by members of the Miami University ASBMB Undergraduate Student Chapter.

Support or Funding Information

NIGMS R35 GM128595
Mode of Protein Targeting to the Proteasome Determines Substrate Fate
Authors: Braganca Christopher Eric , Kraut Daniel Adam
Published: April 2020

The Ubiquitin Proteasome System (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Proteins targeted for degradation are first tagged with a polyubiquitin chain through a series of enzyme‐catalyzed reactions before degradation. After polyubiquitination, the 26S proteasome hydrolyzes ATP to processively unfold substrates before they are degraded in the 20S core particle. Not all substrates are degraded well by the proteasome, and many factors underlying the proteasome’s unfolding ability remain unknown. We set out to investigate the role of targeting by comparing a linear Ub4 proteasome‐binding tag with the UBL domain from yeast Rad23, which is also commonly used in degradation experiments. We found that the UBL domain allows for degradation of stable sGFP‐containing substrates, while the Ub4 tag does not. Replacing sGFP with a less stable variant allows degradation in either case. Our results suggest that different proteasome‐binding tags, which are presumably recognized by different ubiquitin receptors, can lead to different proteasome unfolding abilities.

Support or Funding Information

CEB is a Beckman Scholar. This material is based upon work supported by the National Science Foundation under Grant No. 1515229 to DAK.
Improving Protein Translation in an Attenuated Vaccinia Virus System using Translation Enhancing Elements
Authors: Juba Amber N. , Wellensiek Brian P.
Published: April 2020

Developing effective and safe vaccines without the necessity for booster vaccinations is essential to providing lasting immunity for global populations. The vaccinia virus, which was successfully used as a vaccine in the elimination of small pox, is an attractive agent for vaccine development given the ease with which it can be modified. One major drawback to vaccinia virus use however, is the potential for harmful systemic vaccinia reactions. To improve safety, the virus used as an immunogen can be attenuated. Yet, while attenuation can improve safety concerns, it also reduces the efficacy of the vaccine. This reduction is largely attributed to a decreased amount of protein delivered to the immune system. In an effort to improve vaccines exhibiting less immunogenicity, we aim to increase protein translation in an attenuated virus system by using translation enhancing elements (TEEs) previously discovered from the human genome. One in particular, termed 5d5, acts as a vaccinia virus promoter and a TEE. Here we explore combining other TEEs demonstrating high levels of activity with 5d5 to determine whether we can increase protein expression in an attenuated virus system. The effect of each combination on protein expression is measured by transfection/infection assays using luciferase based plasmids. We use wild‐type and attenuated vaccinia virus strains (Copenhagen and NYVAC, respectively) in our model system to compare luciferase expression levels between the two for each of the constructs. Although we did not find any significant increases in translation when combining TEEs with 5d5, we did show for the first time at least a 2‐fold increase in translation when using 5d5 in the NYVAC system when compared to expression when unmodified virus is used.

Support or Funding Information

Midwestern University intramural funding
Foliar Fungicides Reduce Short Term Non‐Target Soil Microbial Activity and Community Structure
Authors: Kosel Kelby , Collins Harold
Published: April 2020

Pesticides are essential in modern agricultural practices and are used frequently for the protection of crops. Fungicides are a group of pesticides that inhibit the establishment of fungal pathogens. Our systematic knowledge of the different affects fungicides have on non‐target organisms, unfortunately, is inadequate. It is difficult to determine the range of organisms affected by fungicides because of the variability and sensitivity of both eukaryotes and prokaryotes. A laboratory study was initiated to assess non‐target effects of fungicides on soil microbes. This study examined the effect of the common fungicides Tilt and Trivapro at different concentrations and incubation periods for their non‐target effects on soil microbial communities and selected activities. Three rates, 0X, 1X (0.0058 μL/cm2) and 2X (0.0116 μL/cm2) of each fungicide were applied to 100 g of Bastrop soil, common to the Blackland Prairie of Central Texas. Significantly lower CO2 was respired from amended soils compared to the control. Additionally, nitrate mineralization concentration decreased over the 30‐day trial period compared to the control. Low carbon mineralization and nitrogen mineralization rates are both attributed to a decrease in soil microbial activity. Plate counts indicate fungi (PDA) and bacteria (TSA) showed variability at two sampling dates indicating that treatment effects could not be determined solely on population counts. Our results suggest these fungicides at 1X and 2X rates have significant effects on non‐target organismal activity and community structure. Future studies will examine field applications and reassess results.

Support or Funding Information

USDA Agricultural Research Service CRIS #3098‐13610‐008‐00D
ROLE OF SPINAL AT1, IONOTROPIC GLUTAMATERGIC AND GABAERGIC RECEPTORS ON CONTROL OF RENAL SYMPATHETIC NERVE ACTIVITY IN 2K‐1C RATS
Authors: de Oliveira Milanez Maycon Igor , Nishi Erika Emy , Rocha Antônio Augusto , Bergamaschi Cássia Toledo , Campos Ruy Ribeiro
Published: April 2020

Previous studies performed in our laboratory showed a higher glutamate and angiotensin receptors activation, as well as an alteration in GABAergic pathways in central nuclei involved in the cardiovascular control that may lead to sympathoexcitation in renovascular hypertensive rats (2K‐1C). However, the role of the spinal cord neurons on the renal sympathoexcitation remains unclear in this model. Thus, we aimed to assess the influence of spinal glutamatergic and GABAergic (ionotropic receptors), as well as angiotensin II type 1 receptors (AT1) receptors on the renal sympathetic nerve activity (rSNA) in the 2K‐1C rats. Male Wistar rats (250–300 gr) were used to induce renovascular hypertension by clipping the renal artery with a silver clip (0.2 mm). After six weeks, a polyethylene catheter (PE‐10) was inserted into the subarachnoid space and advanced to the T10–11 vertebrae level in urethane‐anaesthetized rats (1.2 g/Kg, iv). All experiments were approved by the Institutional Ethical Committee of UNIFESP. The effects of intrathecal (i.t.) injection of kynurenic acid (KYN) (160 nmol in 2 μl), losartan (Los) (2nmol in 2 μl) or bicuculline (Bic) (8 mM in 2 μl) on blood pressure (BP) and rSNA were investigated. KYN i.t. injection induced a larger and significant fall on rSNA in the 2K‐1C compared to control rats. Intrathecally Los evoked a larger and significant fall in rSNA in the 2K‐1C compared to control group. KYN decreased BP similarly in both, control and 2K‐1C groups, however, Los significantly decreased BP in the 2K‐1C group only. Intrathecally Bic triggered a more intense BP and rSNA reponses in 2K‐1C compared to control rats. Gene expression analysis by qRT‐PCR showed a spinal (T11–12) AT1 mRNA upregulation in the 2K‐1C rats (1.71‐fold change), while no changes were observed in AT2, ionotropic glutamate and GABA receptors subunits. Therefore, our data show that ionotropic spinal cord excitatory amino acid, GABAergic and AT1 receptors play a significant role in the control of rSNA in the 2K‐1C model that may contribute to the maintenance of hypertension. The participation of spinal AT1 receptors seems to be more important in the establishment of sympathoexcitation in this model. The origin of those projections remains unclear.

Support or Funding Information

CNPq, FAPESP (2018/01898‐4) and CAPES
Streamlining FEF25‐75 and Peak Flow Calculations for Use with Physiological Data Acquisition Software
Authors: Tomasson Erik P. , Welper Hannah M.E. , Bates Melissa L.
Published: April 2020

Our lab has developed novel techniques to quantify the lung‐structure function relationship, but is also interested in how lung structure impacts ventilation during exercise. But, a limitation to evaluating pulmonary function is that the most commonly used data analysis packages in exercise physiology do not include functions to calculate mid‐expiratory flow (FEF25‐75). To date, we have been calculating FEF25‐75 values manually or switching to equipment that includes a spirometry analysis program. We set out to write a program to automate these calculations so that they could be made in the context of exercise physiology studies. The data is first downsampled to yield a flow value every hundred milliseconds. Then, the program makes a running sum of all the flow values in each breath to determine the expiratory volumes of every data point. Flow values within 25% and 75% of the total volume are then averaged to calculate the breath’s FEF25‐75. Every flow value is then passed through python’s built‐in maximum function to determine peak flow. The FEF25‐75 and peak flow values for each of the breaths are then averaged to return a single FEF25‐75 and peak flow for each breath set. We found that nine breaths are necessary to ensure representative accuracy >95%. Upon further testing, with sets of nine breaths, the program calculated FEF25‐75 values and peak flow values consistently to within 3.13% and 3.64% of the manually calculated values respectively. The average deviation from the manually calculated values is 1.23% for FEF25‐75 and 0.92% for peak flow. The increased efficiency that this program brings to small airway analysis can streamline many projects and serve as a platform for more useful research into lung function.

Support or Funding Information

This work was supported by the University of Iowa Old Gold Fellowship Program (Bates)
Minimum Inhibitory Concentration Assays for Multidrug Resistant Gram‐Negative Bacterial Pathogens
Authors: Edgar Jennifer M. , Zhao Hang , Brakke Sabrina A. , Wise John G.
Published: April 2020

Antibiotic resistance by bacterial pathogens is a persistent problem in medicine. One method of resistance is through RND efflux transporters, which can move antibiotics out of the cells and the periplasm, thus preventing cell death. Many of these transporters can move several very different antibiotics out of the cells, thus conferring multidrug resistance to the pathogens. This project focuses on one such RND transporter found in Neisseria gonorrhoeae known as mtrCDE. Our goal is to identify potential inhibitors of mtrCDE by observing their effects on the minimum inhibitory concentrations (MICs) of antibiotics. Ultimately, we aim to develop inhibitors of mtrCDE with the goal of restoring antibiotic sensitivity to antibiotic‐resistant N. gonorrhoeae. Potential inhibitors have been identified through computational means. In the system described here, the genes for mtrCDE have been transformed into an E. coli strain that has had the homologous E. coli RND transporter deleted for facilitating testing. We have attempted to optimize MIC assays to determine the minimum concentration of antibiotic required to inhibit the growth of cells expressing the mtrCDE genes. To date, the largest effects in these assays have been observed with the detergent Triton X‐100. In addition, the plasmid encoding mtrCDE included a gene conferring chloramphenicol resistance for selection. The media for initial growth of cells contained chloramphenicol to maintain this selection. Initial studies showed that inclusion of chloramphenicol with other antibiotics overwhelms even resistant bacteria. The effects of inclusion of dimethyl sulfoxide (DMSO), a commonly used carrier vehicle for experimental compounds, was tested in the assays since our compound libraries use this solvent. Experiments suggested that inclusion of low concentrations of DMSO does not affect the MIC values observed, but that the cells do reflect a dose response to varying concentrations of DMSO that needs to be carefully controlled. Continuing optimization of these assays with various antibiotics is being performed to determine MIC values, compare them with reported MIC values, and identify antibiotics that are effluxed by mtrCDE that have no published MIC values for N. gonorrhoeae or E. coli. We report here these and other results of our MIC assay optimization for multidrug resistant, gram‐negative bacterial pathogens.

Support or Funding Information

Department of Biological Sciences, the Center for Drug Discovery, Design and Delivery (CD4), the Center for Scientific Computation, Engaged Learning, and the Hamilton Scholars Program, Southern Methodist University, Dallas, TX
From Cells‐to‐Organism: Impact of Dyslipidemia on Inwardly Rectifying K+ Channels and Cerebral Vascular Function
Authors: Kowalewska Paulina M. , Sancho Maria , Fabris Sergio , Huff Murray W. , Gros Robert , Welsh Donald G.
Published: April 2020

Recent studies have noted that dyslipidemia can diminish dilatory function of resistance arteries by altering the activity of ion channels that set membrane potential. Focusing on the cerebral circulation, this study determined whether inwardly rectifying K+ (KIR) channels are targeted early in dyslipidemia and if alterations impact blood flow control. Experiments began at the cellular level (patch‐clamp electrophysiology) and then were extended to isolated arteries (pressure myography) and whole animals (arterial spin‐labelling magnetic resonance imaging). Initial lipid analysis confirmed the dyslipidemic state of LDLR−/− (normal chow) and C57BL/6 mice fed a high‐cholesterol high‐fat (HFHC) diet for 8 weeks; note aortic plaque formation was absent in these animal models. Patch‐clamp electrophysiology revealed a marked reduction in endothelial but not smooth muscle KIR activity in both dyslipidemic models. Flow activation of KIR was also reduced in endothelial cells from LDLR−/− and HFHC mice. However, endothelial KIR activity was recovered in dyslipidemic mice by depleting the plasma membrane of cholesterol. Consistent with these cellular changes, we observed diminished shear‐induced vasodilation in cerebral arteries isolated from dyslipidemic animals. To our surprise, these cellular/tissue level changes didn’t alter cerebral blood flow at baseline or in response to a blood pressure challenge (phenylephrine injection through a peritoneal catheter, 0.816 mg/kg). The maintenance of blood flow control was observed across a full range of structures including the cortex, cerebral nuclei, hippocampus, thalamus, hypothalamus and midbrain. In closing, our findings highlight that while KIR channel function is targeted early on in dyslipidemia, compensatory mechanisms can maintain brain blood flow to preserve neurological function.

Support or Funding Information

Canadian Institutes of Health Research
Prevention of T Cell Activation in Response to Placental Ischemia Improves Hypertension and Natural Killer Cell Number During Pregnancy
Authors: Campbell Nathan Edward , Deer Evangeline , Amaral Lorena , Reeve Krisin , Fitzgerald Sarah , Herrock Owen , Franks Michael , Usry Nathan , Ibrahim Tarek , Lamarca Babbette
Published: April 2020

Preeclampsia (PE), new onset hypertension during pregnancy, is the leading cause of death and morbidity world‐wide for the mother and fetus during pregnancy. The Reduced Uterine Perfusion Pressure Rat Model of Preeclampsia (RUPP) exhibits many characteristics of PE including hypertension, suppressed regulatory T cells (TRegS) associated with increased CD4+ T cells and B cells secreting agonistic autoantibodies to the AngII receptor (AT1‐AA). We have previously shown that blockade of T‐helper cells improves blood pressure and lowers AT1‐AA secretion. A potential mechanism for the decreased blood pressure is decreased cytolytic natural killer (cNK) cells. Abatacept is a fusion molecule designed to inhibit T cell co‐stimulation in response to antigens and is used to treat autoimmune diseases. We hypothesize that treatment with Abatacept will prevent the activation of T‐helper cells and therefore lower AT1‐AA as a mechanism to lower the activity of NK cells in response to placental ischemia in RUPP rats. Abatacept was given on day 13 via the jugular vein. On day 19, blood and tissues were collected, blood pressure (MAP), pup weight and NK cells were measured by flow cytometry in the blood and placenta. A one‐way ANOVA was used for statistical analysis. On GD19, MAP significantly increased in RUPP 119±2 mmHg (n=7, p<0.05) compared to NP controls 101±2 mmHg (n=7) and was normalized with abatacept (100±2.02 mmHg (n=10, p<0.05). Compared to the NP controls (2.216±0.054, n=7), pup weight significantly decreased in RUPP (1.919±0.113, n=7, p<0.05) and was 1.934± 0.050, with Abatacept (n=10, p<0.05). Circulating and placental total NK cells were 45±9, 59±11 % gate in NP rats (n=7, n=5), 59±4, 72±13 % gate in RUPP rats (n=7, n=4), which significantly decreased to 32±7, 25±9 % gate with Abatacept (n=10, p<0.05; n=10, p<0.05). Our findings indicate that prevention of T cell activation lowers total NK cell number and blood pressure in response to placental ischemia of pregnancy.

Support or Funding Information

2R01HD067541P20GM121334 T32HL105324
Stress Preconditioning Effects on Heart Rate and Blood Pressure under Circumstances of Gastric and Small Intestine Injury Induced by Indomethacin in Conscious Rats
Authors: Filaretova Ludmila , Yarushkina Natalia
Published: April 2020

Nonsteroidal anti‐inflammatory drugs (NSAIDs) are commonly used medications. However NSAIDs exert side effects not only in the stomach and the small intestine but also in cardiovascular system. A disorder of the circadian heart rate (HR) under circumstances of indomethacin (IM)‐induced gastric and small intestinal injury is potentially lethal factor and our previous experimental findings confirmed this fact. Preconditioning is one of perspective approaches to protect the heart and other organs against adverse influences. We demonstrated previously that glucocorticoids released during mild stress contribute to protection of the gastric mucosa against severe stress through maintaining various links of the general body homeostasis, including blood pressure (BP). Here we studied the effects of stress preconditioning on HR and BP under circumstances of gastric and small intestine injury induced by IM in conscious rats. Preliminary fasting (24 h) rats were subjected IM (35 mg/kg, sc) administration with preconditioning stress (cold‐restraint, 10 °C, 30 min and keeping at room temperature 1 h before IM administration) or without it. HR and BP were measured by non‐invasive tail cuff method before IM injection and 4, 24 and 48 h after IM administration in stressed and non‐stressed animals. The gastric and the small intestinal mucosa were examined for injury 4 h (formation of the gastric erosions) and 24 and 48 h (formation of the small intestinal injury) after IM administration. Plasma corticosterone levels were also measured. IM administration induced formation of gastric erosions (4 h after its injection) and their healing for 48 h was followed by a development of the small intestinal injury. Formation of gastric erosions caused an elevation of corticosterone levels 4 h after IM; their following healing resulted in a decrease of IM‐induced corticosterone levels 24 and 48 h after IM injection till baseline level. IM‐induced gastrointestinal injury was accompanied by a gradual decrease of systolic BP and increase of the HR for 48 h after IM. Preconditioning stress reduced IM‐induced gastric erosions as well as small intestinal injury 4 and 24 h after its injection, respectively, and recovered HR and systolic BP 48 h after IM. Thus, the data suggest that preconditioning stress may reduce the vulnerability of the gastric and the small intestinal mucosa to ulcerogenic action of IM and stabilizes the hemodynamic parameters.

Support or Funding Information

The study was supported by grant of Russian Science Foundation (RSF) N 19‐15‐00430.
Polyethylene‐glycol chitosan hydrogel accelerates traumatic wound healing in diabetic mice
Authors: García Angel Eduardo Bárcenas , Merino Miriam Veronica Flores , Tolosa Ibeth Mayerly Gaitan , Muñoz Daniela Rodríguez , López Miguel Angel Camacho , Pantoja Rigoberto Oros
Published: April 2020

Wounds are considered a public health problem worldwide; it represents a significant economic impact on health systems. Diabetic lesions, microvascular dysfunction, trauma and burns, are some factors related to poor repair. Nowadays in regenerative medicine, biocompatible biomaterials (e.g., hydrogels) have proven been effective in regenerative processes. Therefore, preformed synthetic matrices could be a therapeutic alternative to accelerate wound repair.

Objective

to evaluate the Polyethylene‐glycol chitosan hydrogel (hg‐PEGDA‐Q) effect on traumatic wounds. Two CD1 mice (10 weeks, n = 5) were used: healthy control (CT) and with alloxan‐induced Diabetes Mellitus (DM) (200 mg / kg). Under anaesthesia, two traumatic dorsal wounds separated by 1 cm (diameter (⌀) 5 mm, area 19.63mm2) were caused to each mouse: a) without treatment (WT) (proximal wound) and b) with hydrogel PEGDA‐Q (⌀ 5 mm, thickness 2 mm, distal wound). The macro and microscopic evolution were evaluated on 12 postoperative day. For analyse the macroscopic images were rescaled through a vernier and ImageJ software (1: 1 mm scale). hg‐PEGDA‐Q matrix was synthesized by sequential method and photopolymerization.

Results

in CT group, hg‐PEGDA‐Q decreased the diameter and area with respect to untreated wound (hg‐PEGDA‐Q vs. WT) (⌀ 2.71 ± 0.64 mm vs. 3.71 ± 0.27 mm; area 5.79 ± 1.67 mm2 vs. 9.23 ± 1.97 mm2). In DM group, the same behaviour was observed with hydrogel (DM hg‐PEGDA vs. DM‐WT) (⌀ 3.04 ± 0.64 mm vs. 3.90 ± 0.29mm; area 6.29 ± 2.35 mm2 vs. 12.01 ± 3.66 mm2). Regarding initial diameter (5 mm, day 0), in control group, wound retraction (WR) was (hg‐PEGDA‐Q 45% vs. WT 25%), while in diabetic group WR was (hg‐PEGDA‐Q 39% vs. WT 21%). Comparing the initial area (19.63 mm2 at day 0), in control group the WR was (hg‐PEGDA‐Q 70% vs. WT 52%), while in DM group the WR was (hg‐PEGDA‐Q 67 % vs. WT 38%). Histomorphological analysis: hg‐PEGDA‐Q generated complete reepithelialization and dermo‐epidermal changes suggesting an advanced wound healing process compared to control group without treatment. DM group treated with hg‐PEGDA‐Q changes were similar, however consolidation was lower. In contrast DM WT group, reepithelialization and wound consolidation were deficient. Results indicate PEGDA‐Q hydrogel scaffolding network could be a therapeutic alternative to accelerate healing processes in normal and diabetic conditions
High‐Fat Induces Cardiac Remodeling via Dysbiosis of the Gut Microbiome
Authors: George Akash K. , Singh Mahavir , Stanisic Dragana , Malonee Carissa J. , Molnar Jack , Homme Rubens P. , Tyagi Suresh C.
Published: April 2020

Although consumption of a high‐fat diet (HFD) causes metabolic changes, the mechanisms remain unknown. We hypothesize that HFD instigates dysbiosis, and cardiac muscle remodeling by inducing matrix metalloproteinases (MMPs) that lead to an increase in the white fat contents in the body, and an intervention with a probiotic (PB) reverses these phenomena, in part, by increasing lipolysis (PGC‐1α, and UCP1), fat‐browning, and decreasing lipogenesis. To test this hypothesis, we used wild type (WT) mice and fed them with HFD for a period of 16‐weeks with or without a PB in the drinking water. Cardiac injury was measured by estimating the CKMB activity. The results demonstrated that CKMB activity was robust in the HFD mice. Interestingly, CKMB activity was normalized in the PB treated mice group. Free fatty acids (FFAs), and methylation levels were increased but the butyrate entity was decreased in the HFD mice group, suggesting ensuing of the “dysbiotic 1‐carbon metabolism” phenotype. Further, expression levels of PGC‐1α, and UCP1 were measured by Western blottings, and the MMP activity was estimated by zymography. The collagen histology was also performed on tissue samples collected from the mice. Contractions of isolated single myocytes were recorded employing an “Ion‐Optic” system, and the cardiac functions were scored by Echocardiography. The vasculature in the mice‐hearts was analyzed by BaSo4 X‐ray angiography, and the blood flow rate was studied using the Laser Doppler imaging system. Together, our findings suggest that mice fed with HFD gained significant weights, and they showed an increase in the systemic blood pressure, along with alterations of the vascular structures, and blood flow rates. Interestingly, some of the dysregulated effects were normalized by PB treatment. Furthermore, the extent of fibrosis, and MMP‐2 activity were quite robust in the HFD fed mice, and again the treatment with PB was able to mitigate cardiac tissue remodeling. Myocytes’ calcium‐dependent contractions were disrupted by consumption of the HFD, and treatment with PB was again able to mitigate the deleterious effects. We conclude that HFD induces dysbiosis of the “1‐carbon metabolism”, and treatment with PB creates eubiosis, and browning of the body fat.
Anatomical Basis of Epileptogenesis in a Mouse Model of Traumatic Brain Injury
Authors: Golub Victoria , Reddy Samba
Published: April 2020

Post‐traumatic epilepsy (PTE) is characterized by progressive spontaneous recurrent seizures (SRS) following traumatic brain injury (TBI). PTE is poorly controlled by current medications and the molecular pathophysiology of PTE is unclear. A critical step for preventing PTE is to determine a key pathway that drives disease progression during the “latent period” after TBI, termed epileptogenesis. The potential mechanisms of underlying post‐traumatic epileptogenesis include neuroinflammation, degeneration of neuronal cells, and alterations in the hippocampal neurogenesis and mossy fiber sprouting. In this study, we sought to demonstrate a causal anatomical relationship between neuroinflammation and/or neurodegeneration and the onset of PTE in mice. TBI was induced in mice by a controlled‐cortical impact paradigm and were then monitored by video‐EEG recording for 120 days with a deep electrode placed within the contralateral hippocampus. At days 1, 3, 7, 30, 60, 120 post‐TBI, brains were fixed for immunohistochemistry and analyzed for principal neurons (NeuN+), interneurons (PV+), astrogliosis (GFAP+), microgliosis (IBA1+), and neurogenesis (DCX). Alterations in hippocampal and hemispheric architecture were visualized with quantitative analysis. Pronounced atrophy was evident within the ipsilateral hemisphere in the form of large lesions which extended from the cortex to the hippocampus by 120 days. In the contralateral hippocampus, inflammatory markers of microgliosis and astrogliosis reached peak levels within the first week before steadily declining throughout the 120‐day observation period. In contrast, neurodegeneration of NeuN+ cells and PV+ interneurons was not evident at these early stages, but cell loss became unmistakable starting 30 days post‐TBI. By this point, nearly 30% of PV+ interneurons were lost, with the dentate gyrus serving as the greatest region of cell loss (~40% loss). These studies were repeated using a moderate 1mm injury to emphasize the relationship between injury severity and neurodegeneration. Our findings are reminiscent of preferential loss of inhibitory interneurons in epilepsy patients and suggest that early neuroinflammation may catapult neurodegeneration within the damaged areas, which appears to be a critical mechanism for the onset of epileptogenesis. Specifically, progressive loss of PV+ GABAergic interneurons within the dentate gyrus correlates with progression of SRS from 30 to 60 days, coinciding with the onset of SRS at 39 days post‐TBI.

Support or Funding Information

**Funded by DOD Award #W81XWH‐16‐1‐0660**
Creatine Supplementation Exhibits Sex Differences in White Adipose Tissue and Increases Mitochondrial Markers in Female Rats
Authors: Ryan Chantal Rose , Dunham Tyler , Murphy Jensen , Roy Brian D. , MacPherson Rebecca E.K.
Published: April 2020

Activation of white adipose tissue (WAT) thermogenesis, known as WAT browning, has emerged as an attractive approach to treat obesity and obesity‐related diseases. Traditionally BAT or browning WAT has been defined by the expression of uncoupling protein 1 (UCP1), a mitochondrial protein that uncouples respiration from ATP production. However, recent work has highlighted creatine metabolism or futile cycling as a potential thermogenic pathway. Creatine‐driven futile cycling is a catabolic process which results in ATP oxidation by mitochondrial creatine kinase, resulting in the phosphorylation of creatine and increases in ADP concentrations that drive thermogenic respiration. The purpose of the present study was two‐fold: 1) to determine the effects of creatine supplementation on markers of WAT browning, and 2) to determine if creatine supplementation exhibits sex differences. Thirty‐two Sprague‐Dawley rats (16 male, 16 female) were randomly assigned to one of four experimental groups: control, 2.5g/L, 5g/L and 10g/L of creatine monohydrate (CM) and 1% sucrose via drinking water. Rats had ad libitum access to their drinking water and a standard chow diet (AIN‐93G) throughout the study. Following the 8‐week intervention, brown interscapular adipose (BAT) and white subcutaneous inguinal (iWAT) fat depots were collected. Western blotting analysis demonstrated no significant changes in BAT samples for both males and females. Males had lower cytochrome C than females with 10g/L of creatine monohydrate (p<0.05). In iWAT, mitochondrial markers (COXIV, PDH, citrate synthase, and cytochrome C) and GAMT exhibit no changes in males, however, a decrease in UCP‐1 at 5g/L compared to control was observed (p<0.05). Females exhibit a lower control PDH content than males (p<0.01) in iWAT, accompanied by an increase in PDH levels at all doses of CM compared to control (p<0.05). Furthermore, in females COXIV and cytochrome C presented significant increases with 5g/L and 10g/L, respectively (p<0.05). Both males and females did not present with body weight differences despite dose differences. This study presents novel work demonstrating that female WAT exhibits less mitochondrial markers than males under control conditions, however it demonstrates the potential for CM supplementation to increase female adipose thermogenicity to a similar level observed in males.

Support or Funding Information

Supported by NSERC, Canada
PVN‐projecting NTS Neurons is Involved in Sympathetic Long‐Term Facilitation After Acute Intermittent Optogenetic Stimulation
Authors: Paundralingga Obed , Shimoura Caroline Gusson , Cunningham Joseph Thomas
Published: April 2020

Caudal Nucleus Tractus Solitarius (NTS) neurons receive inputs from cardiorespiratory visceral afferents and send projections to the paraventricular nucleus of the hypothalamus (PVN). Acute intermittent optogenetic (AIO) stimulation of caudal NTS induces sympathetic long‐term facilitation (LTF), a progressive increase of sympathetic nerve activity. The contribution of PVN‐projecting NTS neurons to sympathetic LTF is unknown. This study used an intersectional viral approach to test the effects of acute intermittent optogenetic (AIO) stimulation of PVN‐projecting NTS neurons in sympathetic LTF. Adult male Sprague‐Dawley rats were anesthetized with isoflurane and injected rAAV2/Ef1a‐DIO‐hchR2(H134R)‐mCherry in the NTS and a retrograde AAV9.hSyn.HI.eGFP‐cre.WPRE.SV40 in the PVN four week prior to LTF experiments. This method was used to induce the CRE dependent expression of channel rhodopsin in PVN‐projecting NTS neurons. To measure LTF, we recorded splanchnic sympathetic nerve activity (SSNA), arterial pressure, and heart rate (HR) in anesthetized (urethane (800mg/kg) and α‐chloralose (80 mg/kg) cocktail delivered by intraperitoneal injection), paralyzed with gallamine triethiodide (20 mg/ml, 0.25 ml/h, iv) and mechanically ventilated adult rats. The caudal NTS was stimulated to induce AIO with a train of 10 stimuli each consisting of pulse of light of 10‐ms duration delivered at 20 Hz for 1 min every 6 min. Twenty minutes after AIO, SSNA amplitude increased by 25.36 ± 24.16% (n=3), and mean arterial pressure (MAP) increased by 7.43 ± 12.88 mmHg (n=3). During AIO induction, the acute SSNA and MAP varied with some rats demonstrating increases (n=2) or decreases (n=1) of SSNA, MAP, and HR. In other animals (n=3) where AIO decreases SSNA (‐58.96 ± 36.18%) and MAP (‐14.37 ± 8.57 mmHg), we tested the possible contribution of the baroreceptors in these decreases by bilateral muscimol (1–10 pmol/100nL) injections in the Caudal Ventrolateral Medulla (CVLM). AIO stimulation after inhibition of CVLM caused an increase in SSNA (1.293 ± 17.65%) and smaller decrease in MAP (−2.46± 6.2 mmHg). These results suggest that the splanchnic sympathetic LTF induced by acute intermittent stimulation of NTS neurons might be mediated by the PVN‐projecting neurons, although the acute responses may be excitatory or inhibitory. Further study is needed to clarify the mechanisms responsible for the differences in the cardiovascular and SSNA responses to acute, single optogenetic stimulation of PVN‐projecting NTS neurons. Despite the observed differences in the acute responses, the results indicate that sympathetic LTF can be induced by the optogenetic activation of NTS neurons that project to PVN.

Support or Funding Information

NIH Grant P01 HL088052
Improved fractionation method demonstrates phosphorylated Akt is localized to the cytoplasm in LβT2 cells
Authors: Garcia Alexis , Molinar-Inglis Olivia , Nicholas Dequina , Keyes Jeremiah , Adame Jose , Cisneros Metztli , Lawson Mark , Zhang Jin , Trejo JoAnn
Published: April 2020

Cellular processes controlling proliferation, migration, differentiation and adherence require signal transduction processes. Subcellular compartmentalization of signaling proteins controls their temporal and spatial activity, and can be assessed by subcellular fractionation. However, most fractionation protocols are not effective for certain cell lines as the integrity of the samples produced are often compromised by cross‐contamination. Here, we report a newly optimized fractionation protocol that yields clean cytoplasmic, nuclear, and membrane fractions. The optimized fractionation assay was validated utilizing the LβT2 gonadotrope cell line that allowed us to test compartmentalized signaling. It is known that insulin is required for glucose metabolism but the physiological relevance of insulin in the reproductive LβT2 cell line remains unclear. We conducted signaling time course assays that confirmed Akt, an integral component in glucose metabolism, is phosphorylated in insulin‐stimulated LβT2 cells. Assessment of Akt’s subcellular localization via the optimized fractionation protocol revealed phosphorylated Akt is primarily found in the cytosolic fraction. These findings further raised the question as to whether Akt activation was dependent on being membrane‐bound to endosomes. Endosomal markers confirmed the presence of endosomes in the cytosolic fraction and immunoprecipitation revealed phosphorylated Akt remained in the cytosolic fraction only. Currently, we are using confocal microscopy to verify that our cytosolic, nuclear, and membrane markers are not mislocalized and assaying phosphorylated Akt on endosomes. In conclusion, we found that Akt is phosphorylated and localized in the cytoplasm in LβT2 cells. Future work is aimed towards understanding the role of phosphorylated Akt in glucose metabolism in LβT2 cells.