bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hepatic deletion of MET aggravates acetaminophen hepatotoxicity and impairs liver regeneration in mice
Authors: Bhushan Bharat , Koral Kelly , Stoops John W. , Mars Wendy M. , Apte Udayan M. , Michalopoulos George K. 
Published: April 2020

MET is a receptor for hepatocyte growth factor (HGF) and is considered critical for liver regeneration after partial hepatectomy (Phx). Combined disruption of MET along with epidermal growth factor receptor (EGFR) signaling results in compete elimination of regenerative response after Phx, highlighting importance of these growth factor receptors in liver regeneration. A few studies also indicate a role of MET in cell survival signaling in hepatocytes. However, the role of MET in liver injury and compensatory regeneration after acetaminophen (APAP) overdose, a clinically significant model of acute liver failure (ALF), is not known, which was investigated in the current study. APAP (300 and 600 mg/kg) treatment in mice caused remarkable activation of MET in a dose‐dependent manner. Interestingly, MET activation was observed even at a very early stage (3 hr post‐APAP), prior to any observable necrosis or compensatory regenerative response, and was sustained during peak liver injury. Similar dose‐dependent activation of ERK1/2 signaling (one of the downstream mediators of MET) was also observed after APAP treatment. Next, albumin‐driven CRE approach was utilized for liver‐specific deletion of MET in order to study a causative role of MET in APAP‐induced liver injury and regeneration. WT or MET‐KO mice were treated with 300 mg/kg APAP and investigated at 0, 1, 3, 6, 12 and 24 hr post‐APAP. Liver‐specific MET deletion aggravated APAP‐induced liver injury as investigated by measuring serum ALT and AST levels, which was further corroborated by quantifying necrotic areas. Although, liver injury was not significantly different during early initiation phase, it was remarkably aggravated in MET‐KO mice during the progression phase of APAP hepatotoxicity (12 and 24 hr). Further, hepatocyte proliferation and activation of cell cycle machinery were strikingly lower in MET‐KO mice, indicating compensatory liver regeneration was compromised in these mice. While spontaneous recovery and 100% survival was observed in WT mice, significant mortality (67%) was observed in MET‐KO mice. Lastly, macrophage‐specific deletion of MET utilizing LysM‐CRE did not alter liver injury or regeneration after APAP overdose in mice, indicating macrophage‐specific MET is not involved. In conclusion, our study revealed a novel protective role of MET in liver injury and in promoting compensatory liver regeneration during APAP‐induced ALF.Support or Funding InformationThis study was supported by the Menten Endowment Foundation, University of Pittsburgh
In‐vivo synthesis of GM1 Ganglioside through CRISPR‐Cas9 mutation in CHO‐K1, Chinese hamster ovary cells (Cricetulus griseus)
Authors: Venegas Mata Cindy S. , Mitchell Jon C. 
Published: April 2020

GM1 ganglioside has been shown to have therapeutic properties against, Parkinson’s disease, Alzheimer’s, dementia and with dramatic results in Huntington’s Disease (Holler, et al., 2016). Studies also show recovery in CNS related injuries with GM1 therapy (Karpiak et al., 1986). New studies continue to confirm GM1 gangliosides therapeutic properties and may increase the need for GM1 ganglioside. Currently, large scale, synthesis of this ganglioside involves isolation from a natural product. New synthesis methods, such as chemical synthesis, yields an insignificant quantity for alternative large‐scale production (Aureli et al., 2015). Research presented here attempts to address a new method of large‐scale production by in vivo synthesis of GM1 ganglioside through a CRISPR‐Cas9 induced mutation in Chinese Hamster Ovary cells (CHO‐K1) by introducing the mutation of the GLB1 gene that causes a substantial accumulation of GM1 in cells known as GM1 gangliosidosis. In‐vivo synthesis provides relief from natural product isolation and may be cost effective to chloroform: methanol protocols while providing similar results.Support or Funding InformationThis material is based upon work supported by the National Science Foundation/EPSCoR Cooperative Agreement #OIA‐1849206 and by the State of South Dakota, SD‐GOED, NSF I‐Corps Team program and Dr. Larry and Sue Holler, GlycoScience Research Incorporated
In Vitro Assessment of Acute Neuro‐inflammation in a Model of the Blood Brain Barrier
Authors: Phillips Cassandra , Ortega-Alvarez Ramon , Ojuka Dak , Victor Anne-Deyssa , Fama Kathryn , Toth Charles 
Published: April 2020

The purpose of this experiment was to develop an in vitro model of the blood brain barrier that incorporated brain endothelial microvascular cells (BMECs) and neuronal co‐cultures composed of neurons and astrocytes. All cell types were derived from human induced pluripotent stem cells (iPSCs) in accordance to the differentiation protocols utilized. The neurons and astrocytes were differentiated separately, and then seeded together when both cultures reached maturity. A 12‐well transwell plate format was used for the barrier model; the BMECs were seeded in the transwell inserts and the co‐cultures were seeded in the bottom wells. The inserts were coated with collagen and fibronectin, and a poly‐L‐ornithine and laminin ECM was used for the bottom wells. A fluorescein assay was used as a preliminary investigation of barrier function with only the BMECs in the transwell inserts. Barrier function will also be evaluated with immunofluorescence by staining for the ZO‐1 tight junction marker, which is associated with endothelial monolayers. In addition to assessing barrier function, the model will be used to investigate acute neuro‐inflammatory responses. Inflammation will be induced with small molecules such as poly (I:C) and LPS, and then barrier integrity will be evaluated using a fluorescein assay. Tryptophan metabolites will be utilized to analyze the role of the microbiome in neuro‐inflammation. An in vitro model of the blood brain barrier will be a valuable tool to observe and assess neuro‐inflammatory responses because a human stem cell model is more physiological relevant.Support or Funding InformationGift from Dr. John Mullen
Renal Androgen and Renin Angiotensin System mRNA Expression in Polycystic Ovary Syndrome
Authors: Ye Stephanie A. , Everman Steven , Torres Fernandez Edgar D. , Romero Damian G. , Yanes Cardozo Licy L. 
Published: April 2020

BackgroundPolycystic Ovary Syndrome (PCOS), the most common endocrine disorder in women, is characterized by androgen‐excess and ovarian dysfunction. PCOS is associated with obesity, insulin resistance (IR) and increased blood pressure (BP). Currently therapeutic options to treat PCOS‐associated cardiovascular risk factors are limited. We have previously characterized a rat model of PCOS in which chronic androgen‐excess causes increased body weight (BW), fat mass (FM), IR, BP, and activation of some intrarenal Renin Angiotensin System (RAS) components. Still, the mechanisms responsible for increased BP in women with PCOS remain unclear. In the present study, we tested how chronic androgen‐excess changes the renal mRNA expression profile of the androgen receptor (AR) and RAS components in a model of PCOS.MethodsFour‐week old female SD rats were randomized to subcutaneously receive dihydrotestosterone (DHT, 7.5 mg/90 days) pellets (DHT) or sham surgeries (control) for 90 days (n= 6–10/grp). At the end of the experimental period, food intake (FI), BW, FM measured by Echo‐MRI, and BP using radiotelemetry were assessed in conscious, freely moving animals. Renal mRNA expression was assessed in cortical and medullary tissues separately by RT‐qPCR.ResultsAt the end of the experimental period, DHT rats exhibited higher FI, BW, FM and BP compared to control. In DHT rats, renal cortical mRNA expression of AR, angiotensinogen, AT1R, renin receptor, cathepsin A, aminoendopeptidase A, dipeptidyl peptidase 3, neprilysin, neurolysin, prolylendopeptidase, insulin‐like growth factor 2 receptor were significantly increased. In addition, renal medullary mRNA expression of angiotensin converting enzyme (ACE), angiotensin converting enzyme type 2 (ACE2), angiotensinogen, angiotensin 2 receptor type 1 (AT1R), membrane alanyl aminopeptidase, dipeptidyl peptidase 3, aminopeptidase A, kallikrein 1, neprilysin, neurolysin, prolylendopeptidase, and thimet oligopeptidase were significantly increased. Renal medullary mRNA expression of renin and aminopeptidase B were significantly decreased in DHT rats.Summary and ConclusionsIn summary, hyperandrogenemia leads to upregulation of AR in renal cortex and medulla. The mRNA expression of renal cortical and medullar ACE/AT1R axis components was upregulated in DHT rats. Additionally, mRNA expression of the ACE2 was upregulated mainly in the kidney medulla of DHT rats which could be a compensatory mechanism. This study highlights the effect of androgen‐excess modulating the intrarenal RAS and AR. Treatments focused on targeting RAS system components could be promising therapeutic tools to ameliorate the cardiovascular and renal abnormalities observes in women with PCOS.Support or Funding InformationAHA 0830239N (L.L.Y.C.) and 12SDG8980032 (D.G.R.), EFFERG (L.L.Y.C.), and NIH R21 DK‐113500 (D.G.R.) and P20 GM‐121334 (L.L.Y.C.)
GABAergic synaptic inhibition in the hypothalamic paraventricular nucleus (PVN) is strengthened by acute activation of glutamatergic inputs from the median preoptic nucleus
Authors: Yamaguchi Junya , Andrade Mary Ann , Truong Tamara , Toney Glenn M. 
Published: April 2020

Pre‐sympathetic neurons (PSNs) in the hypothalamic PVN are normally maintained in a state of near discharge quiescence reflecting the resting dominance of GABAergic inhibition. Our studies indicate that synaptic inhibition is actively maintained in the PVN, in part, by EAAT3‐mediated uptake of L‐glutamate (Glu) into local GABAergic synaptic terminals arising from the surrounding peri‐nuclear zone (PNZ). Notably, this Glu‐GABA strengthening (GGS) mechanism relies on apparent increases both in GABA synthesis and packaging into synaptic vesicles. Studies have so far demonstrated that PVN GGS can be elicited either by exposure of brain slices to bath Glu (100 μM) or high K+ (+10 mM). Studies performed in vivo indicate that Glu nanoinjection into the PVN increases GABAergic inhibitory control of renal sympathetic nerve activity (SNA). To investigate whether endogenous synaptic Glu release can drive PVN GGS, glutamatergic EPSCs were evoked in PVN PSNs from the median preoptic nucleus (MnPO). To accomplish this, we created an angled horizontal slice preparation containing the MnPO, PVN and PNZ. Single pulse (0.5 ms, 0.5 to 1.0 mA) electrical stimulation of MnPO evoked amongst PVN PSNs (n=2) mostly unitary EPSCs (ampl.: −139 ± 5 pA, tau: 3.27 ± 0.01). In slices prepared from vGAT‐ChR2(H134R) expressing mice, GABAergic IPSCs were evoked by blue light stimulation (20 ms, 0.4 mW, 0.067 Hz) of PNZ GABA neurons before and after glutamatergic EPSCs were electrically evoked from MnPO (1 Hz, 10 min). The amplitude of photostimulated GABAergic IPSCs at baseline (−152 ± 47 pA) was increased (−220 ± 83 pA) within ~3 minutes following MnPO stimulation (n=2). PNZ eIPSC amplitude was not increased (before: −89.5 ± 9.7 pA, after: −86.5 ± 10.3, pA) when MnPO stimulation was performed in the presence of the pan‐specific Glu uptake inhibitor TBOA (100 μM). Findings indicate that excitatory inputs to PVN from the forebrain lamina terminalis can acutely strengthen GABAergic inhibition through an EAAT3 uptake‐dependent mechanism consistent with PVN GGS. Grading of PVN synaptic inhibition in pace with excitation could help prevent excitotoxic injury of PSNs during prolonged physiological challenges (e.g., water deprivation) and in chronic diseases (e.g., neurogenic hypertension) accompanied by persistently elevated SNA.Support or Funding InformationNIH HL088052 (GMT)
Four weeks of dietary potassium restriction causes distal convoluted tubule remodeling
Authors: Su Xiao-Tong , Saritas Turgay , Yang Chao-Ling , Welling Paul A. , Ellison David H. 
Published: April 2020

Previous studies have described a ‘renal potassium switch’ within the distal nephron that turns on the thiazide‐sensitive Na‐Cl cotransporter (NCC) in the distal convoluted tubule (DCT) in response to low potassium intake and off in response to high potassium intake. Studies using genetically modified mice indicate that decreased NCC activity is associated with decreased DCT length and mass; increased NCC activity is associated with increased, DCT length and mass. The aim of our study was to test whether dietary potassium intake causes the DCT remodeling physiologically.Male C57Bl/6 mice were provided either control potassium diet or low potassium diet for four weeks and blood and kidneys were harvested. To determine the length of the DCT in three dimensions, we used Ethyl‐cinnamate‐based optical clearing, combined with whole‐mount immunolabeling, confocal microscopy and three‐dimensional morphometric analysis.Mice on low potassium diet for four weeks were severely hypokalemic (plasma potassium <2 mM) compared with mice on control diet (4.1 mM). Western‐blot analysis of the whole kidney confirmed that total and phosphorylated NCC were higher in mice on low potassium diet, compared to mice on control diet. By immunolabeling with pThr53‐NCC antibody, we visualized the DCTs in optically cleared kidney slices. Three‐dimensional morphometric analysis suggested that four‐weeks of low potassium diet (487±16 μm, n=5) increased DCT length by 13% compared to NK diet (431±3 μm, n=3).Our results indicate that the DCT remodels physiologically to maintain potassium homeostasis. Additional animals are currently being studied.Support or Funding InformationLeDucq Fondation, KidneyCure
Timed Induction of Gli1 (P3 To P30) Was Expressed In Mouse Trabecular Meshwork
Authors: Svoboda Kathy K. , Thomson Robert , Zhao Hu K. 
Published: April 2020

PurposeThe objective of this project is to determine if Gli1 positive cells contribute to the anterior eye structures including the trabecular meshwork and Schlemm’s canal using an inducible Gli1‐CreERT2; tdTomatoflox (Gli1‐tdTomato) mouse model. Gli1 is downstream of the Sonic hedgehog (Shh) signaling pathway. This pathway is mediated by the Gli transcription factors (Gli1, Gli2, and Gli3) that deferentially activate and repress the expression of specific eye development genes. In addition, Pitx2, FOXC1, and FOXC2 are critical transcription factors for anterior eye development. A second objective is to establish if FOXC1, FOXC2, and Pitx2 are co‐expressed in the Gli1 positive cells.MethodsGli1‐tdTomato expressing mice were induced at different stages with tamoxifen. Eyes were examined that were induced on various embryonic and post‐natal days, P3, P7, and P30 and sacrificed several days to weeks later. Mouse tissues were fixed and prepared for frozen or paraffin sectioning. Tissue antigen retrieval was done using citric acid buffer. Indirect immunohistochemistry for FOXC1, FOXC2, and Pitx2 was completed. Some samples were counterstained with phalloidin. Sections were analyzed with confocal microscopy for spatial distribution of the Gli1+ labeled cells and FOXC1, FOXC2, and Pitx2.ResultsIn Gli1‐tdTomato mice induced on P3, P7, and P30, the periocular mesoderm, trabecular meshwork including Schlemm’s canal, eyelids and optic nerve were positive for Gli1 several days to 3 weeks later. In contrast, the cornea, lens and sclera did not have positive cells for Gli1. Expression levels for FOXC1, FOXC2, and Pitx2 were generally higher in mice harvested at younger ages. After postnatal day 30, the expression decreased with little to no expression of FOXC1 and FOXC2 while Pitx2 expression remained relatively constant in anterior angle tissues.ConclusionThis inducible Gli1‐tdTomato mouse model may be an excellent system for determining the role of Gli1 positive cells in trabecular meshwork and Schlemm’s canal development. The elegance of this model is it can be activated at specific developmental stages and Gli1+ cell derivatives can be tracked and other detection probes (phalloidin, nuclear stains, Immunohistochemistry etc), can be incorporated to determine cell characteristics or over‐all tissue structure. Our results provide evidence that Gli1+ cells are contributing to the development of the anterior angle tissues including trabecular meshwork and Schlemm’s canal.Support or Funding InformationBiomedical Sciences Department Seed Grant (K. Svoboda), NIH K08DE025090 and R21 DE027928 (H. Zhao).
The Other 51%: An Updated Characterization of the Female Rat’s Estrous Cycle
Authors: Jennings Laura , Wong Benjamin , McCranor Bryan 
Published: April 2020

The estrous cycle in female rats is analogous to the menstrual cycle in humans, lasting 4–5 days on average, and is composed of 4 main stages (proestrus, estrus, metestrus and diestrus) and 4 transitional stages. Each stage differs in length and is characterized by distinctive cell types with unique morphologies, which helps in the histological identification of each specific stage. Generally due to added expense and/or increased variables, animal research has historically been conducted on male animal models, and those findings are applied across the whole population.The most notable limitation in the utilization of the female animal model is the added unknown variability caused by hormonal fluctuation on the experiment. The main aim of this study was to create estrous synchronization of each experimental cohort, limiting the introduction of these variables. Although group housing has been shown to induce spontaneous synchronization (in mice), the inability to select for a specific stage, as well as limited success in rats, required an alternative method. We used luteinizing hormone‐releasing hormone analog (LHRHa) one week prior to exposure to induce estrous synchronization. Estrous stage was assessed via vaginal lavage and crystal violet staining. Slide comparisons of lavage cells collected prior to and after chemical manipulation showed differences in quantity and/or morphology of cells in each stage. In our study, we targeted a successful synchronization to the diestrus stage at 168 hours post‐injection. In our assessment of over 140 animals, we found that synchronization was successful 49.3% of the time. Overall, the use of LHRHa to induce estrous synchronization maximized experimental time management and limited estrous‐cycle‐dependent physiological and metabolic changes, enabling biochemical data to be collected and interpreted with minimal extraneous variables. These data are extremely beneficial to the drug discovery process, as researchers now look for more gender‐balanced models to assess new therapeutics.DISCLAIMERSThe views expressed in this poster are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense, and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and the Animal Welfare Act of 1966 (P.L. 89–544), as amended.
Effects of transient ischemia‐reperfusion on in vivo mitochondrial fission and fusion in the murine cerebral vasculature
Authors: Rutkai Ibolya , Chandra Partha K. , Cikic Sinisa , Mostany Ricardo , Busija David W. 
Published: April 2020

Mitochondria play important roles in the regulation of cerebral circulation. The effects of ischemia with and without reperfusion on mitochondrial morphology have been well documented in different cell types of the brain. However, the time course of these events, an important determinant of mitochondrial targeting, has been understudied due to technical limitations.The aim of this study was to determine how mitochondrial fission and fusion dynamics are affected by ischemia‐reperfusion, in endothelial cells that form the basis of the neurovascular unit.A thinned skull cranial window was implanted in young, male and female (3–6 month old; n = 5) offspring of B6;129S‐Gt(ROSA)26Sortm1(CAG‐COX8A/Dendra‐2)Dcc/J and B6.Cg‐Tg(Tek‐cre) 1Ywa/J mice expressing mitoDendra 2 fluorescent protein in endothelial mitochondria. Two‐photon microscopy was used to visualize the brain vasculature, via RhodamineB Dextran labelled blood plasma and mitoDendra2 simultaneously at 875 nm. After 2 imaging sessions taken 60 minutes apart, animals were allowed to recover. Then, mice were exposed to 30 minutes of ischemia via middle cerebral artery occlusion followed by 4 hours of reperfusion. Additional time‐lapse images were taken between the 2nd and 4th hour of the re‐perfusion period. Three stacks of 50 images, 1 μm apart, were taken of different regions of interests, containing arteries, veins, and capillaries using a 40X objective and a 1X digital zoom; whereas 3X digital zoom was used to take images for the analyses of mitochondrial fission and fusion events. Ischemia‐reperfusion resulted in a prominent vasoconstriction of the pial vasculature (15% decrease in vessel diameter; n = 66 diameter measurements) as well as the disintegration of green mitochondria signal, suggestive of fission. The in‐depth analysis of time‐lapse images (8–12 stacks of images per channel per imaging session per mouse using a 3X digital zoom) is currently ongoing.Overall, we expect to correlate in vivo mitochondrial changes derived by fission and fusion with vascular events in response to ischemia‐reperfusion in large, penetrating arteries and veins as well as in capillaries.Support or Funding InformationAHA 17SDG33410366; U54 GM104940; HL‐093554
Selection of Stabilized Variants of Chymotrypsin Inhibitor 2
Authors: Teilum Kaare , Nielsen Louise Hamborg , Gavrilov Yulian , Olsen Johan , Roche Jennifer , Nielsen Alex Toftgaard , Lindorff-Larsen Kresten 
Published: April 2020

Designing proteins with stability higher than the wild type is not always straightforward. Methods for rational design often fail to predict stabilizing mutations and the effect of substituting interacting pairs of amino acid residues. One reason for this is that the sets of protein variants and experimental data used to optimize the computational methods primarily consist of single site substitutions of hydrophobic residues with Ala. Very often multiple substitutions with other residues than Ala are necessary for stabilizing a protein.Here we have used an in cell folding sensor and FACS sorting to select stable variants of chymotrypsin inhibitor 2 (CI2) from a library of random mutations. We show that the readout from this sensor system is a good proxy for in vivo protein stability. Of 28 selected variants, computational tools only predict the effects of the mutations well for a few. In particular, we find a super stabilized double variant of CI2 where each of the single variants have a neutral effect on stability. These observations could not be predicted with current computational tools. To rationalize the effect of this pair of mutations on the structure and dynamics of CI2 we have determined the structures of all states in the mutant cycle and in addition characterized their dynamics by NMR spectroscopy. Our analysis points to subtle changes in both structure and dynamics that collectively results in the increased stability. The results may help future methods for improved predictions of the effects of substituting pairs of interacting residues.Support or Funding InformationThis work was supported by the Novo Nordisk Foundation