bluuebunny/crossref_metadata_2025 huggingface dataset:
SPARC: Development of a TRPA1 reporter mouse model
Authors: Bahia Parmvir K. , Kim Seol-Hee , Harsányiová Jana , Kollarik Marian , Taylor-Clark Thomas
Published: April 2020

TRPA1 is a non‐selective cation channel activated by electrophiles including environmental irritants, and pungent stimuli. It is expressed in a subset of nociceptive sensory neurons and thus plays a key role in defensive reflexes. The contribution of TRPA1 to certain physiological effects is complicated by its expression in non neuronal cell types such as fibroblasts and epithelial cells, so it is not clear to what extent TRPA1 effects are restricted to sensory afferents. We have created a mouse model that co‐expresses the red fluorescent protein tdTomato in TRPA1 expressing cells. A TRPA1‐Flp knock‐in strain was generated by incorporating a 2A‐FlpO after the last endogenous TRPA1 exon. The TRPA1‐Flp was crossed with a tdTomato reporter strain carrying a frt‐flanked STOP codon (RC::FLTG) to produce cells that produce tdTomato in TRPA1 expressing cells. From these animals, we collected sensory vagal and thoracic dorsal root ganglia to visualize tdTomato‐expressing cells in combination with immunohistochemistry. We also collected brain, brainstem, spinal cord and esophagus to visualize tdTomato‐expression to map their central and peripheral projections. tdTomato positive cells were observed primarily in the nodose ganglion and the majority of these coincided with TRPV1 expression. There was also a population of tdTomato expressing cells in the dorsal root ganglia. We confirmed, using calcium imaging, that these tdTomato positive cells are activated by the TRPA1 agonist AITC. We saw strong tdTomato expression in the nucleus of solitary tract (NTS) and in the lamina II region of the dorsal horn of the spinal cord but no expression was evident in the brain. Finally, we were able to observe tdTomato expression in peripheral fibers extending into different regions of the esophagus. In summary, we have created a transgenic knock‐in FlpO mouse model which selectively labels vagal sensory neurons with AITC‐sensitivity. This strain can be used to identify peripheral and central terminals of TRPA1 expressing nerves.

Support or Funding Information

This study is funded by National Institutes of Health Common Fund SPARC OT2 (2016–2019): “Functional mapping of peripheral and central circuits for airway protection and breathing”
Dietary Dependent Alteration of Blood‐Brain Barrier Tight Junctional Protein Expression
Authors: Reeb Chris , Sandoval Karin , Wooten Joshua , Harris Mathew , Schaller Megan , House Austin , Witt Ken A.
Published: April 2020

The impact of diet on brain health extends to the blood‐brain barrier (BBB). Western diets high in saturated fatty acids, such as lard, increase the risk for neurodegenerative disease and cerebrovascular dysfunction. Whereas, fish‐oil (FO) diets containing long‐chain polyunsaturated fatty acids can form products that reduce inflammation and have been identified to have cerebrovascular benefit. However, the impact of dietary fats on the BBB endothelial cell tight junctions remains unclear. BBB tight junction integrity is governed by key proteins, including claudin‐5, occludin, and zonula occluden‐1 (ZO1). This study examined the effect of lard and FO based diets on these key tight junctional proteins.

Male C57BL/6J mice received a diet containing either 10% lard, 10% FO, 41% lard or 41% FO over 32 weeks. Cortical brain tissue was harvested at conclusion of treatment, with protein (Western blot) and mRNA (RT‐qPCR) expression analyses performed. Body weights and blood chemistry measures were also evaluated. Two‐way ANOVAs performed, with Tukey post‐hoc test used to determine if means significantly differed, with significance set at P<0.05.

No differences in mRNA expression were observed across diets. Neither the type or percentage of fat had any impact on occludin protein expression. ZO1 protein expression was significantly higher with FO compared to lard. A significant interaction was found between the type and percentage of fat on claudin‐5 protein expression. Respective to lard, claudin‐5 protein expression significantly decreased as the percentage of fat increased from 10 to 41%. Respective to FO, claudin‐5 protein expression significantly increased as the percentage of fat increased from 10 to 41%. Respective to 10% fat, claudin‐5 protein expression was significantly lower in FO compared to lard. Respective to 41% fat, claudin‐5 protein expression was significantly lower in lard compared to FO.

Study identified ZO1 and Claudin‐5 protein expression changes within the brain are linked to diet. Data supports that diets high in FO have a greater expression of key tight junctional proteins, which suggests enhanced BBB integrity.

Support or Funding Information

Supported by the NIH, National Institute of Neurological Disorders and Stroke (grant R21NS090282).
Resting Heart Rate Variability at Sea Level Predicts Arterial Desaturation during Acute Exercise in Hypoxia
Authors: Short Kelsey Jean , Roarty Margaret C. , Amaya Jessika , Kodali Sruthi , Nylund Zachary J. , Jackson Jaren T. , McKee Kellyn E. , Harris M. Brennan , Kambis Kenneth W.
Published: April 2020

Predicting responses to acute hypoxia based on physiologic measures at sea level may be valuable in anticipating adverse responses to acute hypoxia. The purpose of this study was to examine the relationships between heart rate variability (HRV) and arterial oxygen desaturation in response to acute hypoxia exposure at rest and during exercise.

METHODS

13 subjects (6 male, 7 female) aged 20.6±0.3 years rested in a supine position for 10 min. Average resting HRV and arterial oxygen saturation (%SpO2) were calculated from the last 5 min. A VO2peak test to determine aerobic fitness (44.1±3.0 ml/kg/min) was performed on a stationary cycle at sea level. Subjects also performed a submaximal bike test for 10–15 min at 65% of HRmax achieved during the VO2peak test in normobaric hypoxia equivalent to an altitude of 3500 meters.

RESULTS

HRV (RMSSD 54.5±5.7) was significantly correlated (r=0.227, P<0.05) to the percent change (9.7±0.7) in %SpO2 from rest (97.4±0.2) to exercise (88.0±0.8) in normobaric hypoxia.

CONCLUSIONS

HRV at rest appears to be predictive of the degree of arterial desaturation during exercise in hypoxia equivalent to an altitude of 3500 meters. Current wearable technology that can measure HRV could be used to predict potential adverse responses to exercise in hypoxic conditions such as altitude.

Support or Funding Information

The Doug Morton/Marilyn Brown Endowment for Biomedical Research, The Foundation for Aging Studies and Exercise Science Research, and The Borgenicht Program.
Endothelin Receptor Antagonism Increases Cerebral Perfusion in Humans
Authors: Ward Aaron T. , Carter Katrina J. , Bolin Shawn E. , Eldridge Marlowe W. , Hagen Scott A. , Walker Benjamin J. , Lee Jeffrey W. , al-Subu Awni M. , Wieben Oliver , Schrage William G.
Published: April 2020

Microvascular cerebral perfusion (CP) is critical for adequate oxygenation of neuronal tissue in the brain. However, the mechanisms underlying proper CP regulation in humans have yet to be fully defined. Endothelin is a potent endogenous vasoconstrictor and appears to have a modest role in blood flow regulation in many peripheral vascular beds in healthy individuals. We tested the hypothesis that endothelin receptor antagonism will not influence CP in healthy young adults. Six male subjects (age: 24±4 yrs; BMI: 23±2 kg/m2) participated in the single‐blind study in which endothelin receptor antagonist (Ambrisentan, 10 mg) or placebo was administered orally. Only males participated in the current study due to potential teratogenic effects of Ambrisentan. Resting CP was quantified via 3T MRI using pseudo‐continuous arterial spin labeling (ASL) normalized to gray matter volume approximately 120 minutes following dosing. Blood pressure, heart rate, and end‐tidal CO2 were monitored. Data were analyzed via paired t‐tests and reported as mean±SD. Significance was taken as p≤0.05. Heart rate (57±8 vs. 59±7 bpm), mean arterial pressure (81±7 vs. 79±8 mmHg) and end‐tidal CO2 (34±6 vs. 37±3 mmHg) was not different between placebo and Ambrisentan, respectively (all p>0.05). Compared to placebo, Ambrisentan increased cerebral perfusion (31±3 vs. 34±2 mL/100g/min; p<0.05), representing a 8±5% increase. Regionally, Ambrisentan demonstrated a tendency to increase CP in brain regions supplied by the anterior cerebral circulation (frontal lobe: 36±11 vs. 45±5 mL/100g/min, p=0.05; parietal lobe: 35±10 vs. 43±5 mL/100g/min, p=0.08; temporal lobe: 35±10 vs. 43±6 mL/100g/min, p=0.07; basal ganglia: 35±9 vs. 41±5, p=0.09; cingulate: 39±10 vs. 47±5 mL/100g/min, p=0.08). In contrast, CP in regions supplied by the posterior circulation did not increase to a similar extent (occipital lobe: 37±9 vs. 43±7 mL/100g/min, p=0.13; cerebellum 30±16 vs. 38±6 mL/100g/min, p=0.27). Contrary to our hypothesis, these data suggest endothelin contributes to resting CP by increasing microvascular tone, an effect that may be more pronounced in the anterior brain regions. These preliminary findings contribute to resolving potential mechanisms mediating normal CP in healthy individuals and form the basis of future studies exploring the influence of elevated endothelin signaling in conditions like diabetes or heart failure.

Support or Funding Information

ADA 1‐16‐ICTS‐099
Cargo binding of an adaptor directly competes with oligomerization
Authors: Kuhlmann Nathan J
Published: April 2020

Bacterial protein degradation is a tightly coordinated process aided by protease adaptors that coordinate cell cycle and manages stress responses. In Caulobacter crescentus, cell‐cycle dependent protein degradation depends on the highly conserved ClpXP protease and several ClpXP specific adaptors that dictate hierarchal substrate degradation. While most protease adaptors deliver only a single substrate, the RcdA adaptor directly binds and coordinates the degradation of several cell‐cycle regulated substrates that share no obvious sequence or structural similarities. Here we show that cargo binding uses the same interface as that responsible for dimerization of RcdA. Cargo binding converts RcdA from a homodimer to a monomer and this effect extends to all RcdA cargo. We identify key residues in this interface with hydrogen‐deuterium exchange mass spectrometry and make mutations to generate a monomeric variant incapable of dimerization that also fails to bind cargo. Interestingly, RcdA itself is normally degraded by ClpXP unless bound to cargo, but the monomeric variant fails to be degraded, suggesting that RcdA delivers cargo or delivers itself to ClpXP. Expressing a monomeric RcdA in vivo results in irregular stalk formation and loss of normal cargo turnover. Our work shows how cargo binding competes with self‐dimerization of RcdA through direct competition for a common interface and that disruption of this interface results in defects in normal cell physiology.
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 Information

This 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 Information

This 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
Evaluating the effectiveness of flowcharts integrating pharmacology and pharmacotherapeutics content as an instructional methods in therapeutics course module.
Authors: Bose Diptiman D. , King Ethan , Zimmermann Anthony
Published: April 2020

The curriculum at Western New England University, College of Pharmacy and Health Sciences uses an integrated approach to teach the pharmacotherapeutics courses in the 2nd year of their Pharm D program. This approach incorporates didactic learning of pharmacology, medicinal chemistry, and pharmacotherapeutics of an organ system / disease state in a single module. An example of this is the integrated acute care cardiovascular disease module which covers the pharmacotherapeutics of disease states such as coronary artery disease, shock, and arrhythmias. Given the complex nature of these topics, teaching the module poses unique challenges for the learners and has historically resulted in poor course outcomes. In an effort to improve learner understanding and application of material, we restructured the course to not only improve content flow, but also introduced new teaching tools to improve learning. One such tool is the development of flowcharts that integrate concepts of pharmacology with therapeutics. Flowcharts with skeletal outlines of key points were provided to the learners on the College’s course management site. The instructors completed the charts using a doc cam system during class at the end of each disease state. A pre‐ and post‐survey was conducted to evaluate the effectiveness of these flowcharts. A pre‐survey showed that 83% (n=45) agreed that flowchart use would improve their knowledge, the post‐survey results demonstrated that the majority of learners (91%, n=60) agreed that this teaching tool did indeed help improve their overall knowledge of course content. While, 90% (n=60) agreed that flowcharts improved their understanding of pharmacology, 95% of the surveyed participants agreed that flowcharts helped them understand the role of drugs in pharmacotherapy. Furthermore, 96% (n=60) of the participants agreed that they would like to see more use of flowcharts in the other pharmacotherapeutics modules. The survey results also indicated that classroom participation improved while completing the flowcharts during class (n= 60, 84%). In addition, the course assessment outcomes in the module also improved with a decrease in course failure rates versus previous years. The results from our study indicates that the use of flowcharts improved course organization, content delivery and application in an acute care cardiovascular disease pharmacotherapeutics module. The flowcharts provide a decision‐making framework to follow that helps the learners apply the specific information required to make informed therapeutic decisions. We believe that the use of these flowcharts benefit student learning and understanding and can be used in other disease state modules
Identification of RNA Exosome Cofactors in Neuronal Cells to Probe Disease Mechanism
Authors: de Amorim Julia , Leung Sara , van Hoof Ambro , Corbett Anita
Published: April 2020

The RNA exosome, a 10‐subunit complex that mediates both RNA processing and degradation, plays a critical role in defining cellular expression profiles. This complex is ubiquitously expressed, essential, and critical for fundamental cellular functions, such as ribosomal RNA processing. Recent studies have linked mutations in genes encoding multiple subunits of the complex to tissue‐specific human disease. For example, missense mutations in the human EXOSC3 gene, which encodes an RNA exosome subunit, cause Pontocerebellar Hypoplasia type 1b (PCH1b), a disease characterized by atrophy of the pons and cerebellum. The missense mutations encode single amino acid changes in conserved regions of the EXOSC3 protein. How these amino acid substitutions confer tissue‐specific phenotypes is not known. One possible mechanism underlying the distinct disease phenotypes could be a decrease in the interaction of the RNA exosome complex with cofactors that confer specificity for RNA targets. However, most studies that identify and characterize RNA exosome cofactors have been carried out in budding yeast and thus, do not provide insight into whether tissue‐specific cofactors could exist. Our studies use immunoprecipitation from neuronal cell culture (N2A) and relevant mouse tissues to define RNA exosome cofactors. Biochemical experiments that employ cultured N2A cells were used to identify RNA exosome‐interacting proteins in both the nucleus and the cytoplasm. Preliminary results from this analysis reveal an association between the RNA exosome and a large complex of cytoplasmic tRNA ligase enzymes, which could link defects in tRNA maturation to disease pathology. We are extending these studies to explore the possibility of tissue‐specific cofactors by immunoprecipitating EXOSC3 and analyzing co‐purifying proteins from the mouse cerebellum (affected in disease) and cortex (unaffected). To complement these approaches to identify tissue‐specific RNA exosome cofactors, we are also exploring whether amino acid changes that are linked to disease alter cofactor interactions. These studies, which are being performed in N2A cells, have identified cofactors that show altered interactions with the EXOSC3 variants present in disease. These studies will provide insight into both the functional consequences of amino acid substitutions in the RNA exosome that cause disease and the role of cofactors in conferring RNA target specificity.
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 Information

Gift from Dr. John Mullen
Characterization of L‐Ascorbate Metabolism in Ralstonia eutropha H16
Authors: Naing Yadanar Than
Published: April 2020

L‐Ascorbate (Vitamin C), a primary antioxidant in plants and animals, is an abundant carbon source highly produced by plants so is an available carbon source for soil bacteria. We previously identified a novel L‐ascorbate metabolic strategy in Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699) by eight enzymes up to three transporters; the first enzyme is a membrane bound L‐ascorbate oxidoreductase (LaaO). Which face of LaaO, periplasmic or cytoplasmic, is catalytic was investigated by complementation of an laaO mutant with a cytoplasmic plant L‐ascorbate oxidase. Growth with L‐ascorbate of two of the three transporter deletion mutants was impaired but not abolished. The growth defects correlated with concentrations of L‐ascorbate or spontaneous degradation products in the media. Transcript levels of each transporter gene, fluorescence of mCherry‐transporter fusion mutants, and the L‐ascorbate and dehydroascorbate concentrations were monitored to observe the regulation of transporter genes. This work elaborates on environmental carbon cycling by a novel L‐ascorbate pathway found in hundreds of bacteria while clarifying transporter function in three different protein families.
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

Background

Polycystic 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.

Methods

Four‐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.

Results

At 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 Conclusions

In 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 Information

AHA 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 Information

NIH 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 Information

LeDucq 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

Purpose

The 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.

Methods

Gli1‐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.

Results

In 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.

Conclusion

This 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 Information

Biomedical Sciences Department Seed Grant (K. Svoboda), NIH K08DE025090 and R21 DE027928 (H. Zhao).
Chemoprevention of Aflatoxin B1‐Induced Cytokine Storm and Tumor Dormancy Escape via Dual COX‐2/sEH Inhibition
Authors: Fishbein Anna , Deng Jianjun , Hwang Sung Hee , Wang Weicang , Yang Jun , Verheul Sanne , Hallisey Victoria M. , Bielenberg Diane R. , Gartung Allison , Hammock Bruce D. , Panigrahy Dipak
Published: April 2020

Inflammation plays a critical role in stimulating tumor growth, invasion and metastasis. Hepatocellular carcinoma (HCC), the most common primary liver malignancy worldwide is usually resistant to treatment and arises most frequently in the setting of chronic inflammation. A critical underlying cause of HCC other tumor‐types includes exposure to toxic environmental carcinogens. Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus fungi, is a contaminant in a large portion of the world’s food supply. While AFB1 causes cancer presumably via genotoxic mechanisms by inducing DNA adducts, potential non‐genotoxic mechanisms of aflatoxin‐induced cancer remain poorly characterized. Cytotoxic drugs generate apoptotic cell death (“debris”), which may promote the growth of residual surviving cancer cells via inflammation. The cytotoxic aflatoxins stimulate apoptosis of multiple cell types including hepatocytes, thymocytes, and epithelial cells via increased apoptotic caspases and pro‐inflammatory cytokines (e.g. TNF‐α). As over 30–90% of individuals harbor dormant microscopic tumors, we hypothesized that AFB1 generated debris may stimulate tumor dormancy escape via pro‐inflammatory and pro‐angiogenic cytokines. Here we demonstrate that hepatocellular cancer and non‐cancer debris generated by AFB1 accelerates tumor dormancy escape by stimulating a macrophage‐derived “storm” of pro‐inflammatory cytokines and bioactive lipids. Targeting multiple inflammatory mediators simultaneously is a desired therapeutic strategy but is challenging to achieve. CYP450‐derived epoxyeicosatrienoic acids (EETs) promote the clearance of cellular debris by local macrophages and activate anti‐inflammatory programs. EETs are rapidly metabolized in the body by soluble epoxide hydrolase (sEH), but inhibition of sEH can stabilize EETs and promote the resolution of inflammation. Combined pharmacological abrogation of the cyclooxygenase‐2 (COX‐2) and soluble epoxide hydrolase (sEH) pathways (e.g. PTUPB) prevented the debris‐induced storm of both cytokines and lipid mediators. Dual COX2/sEH inhibition also stimulated macrophage phagocytosis of AFB1‐generated cell debris. AFB1 debris induced oxidative stress and reactive oxygen species were counter‐regulated by dual COX2/sEH inhibition. In animal models, the dual COX‐2/sEH inhibitor PTUPB prevented the onset of debris‐stimulated liver cancer tumor growth leading to sustained survival. Thus, carcinogen generated tumor cell debris triggers a “cytokine storm” providing a potential mechanism whereby environmental substances harbor the potential to stimulate or induce tumor initiation, growth, and/or metastasis. Enhancing clearance of cell debris via dual COX2/sEH inhibition represents a novel approach to prevent carcinogen‐induced tumor growth and recurrence.
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.

DISCLAIMERS

The 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 Information

AHA 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 Information

This work was supported by the Novo Nordisk Foundation
Cortisol as a Potential Biomarker for Suicidality
Authors: Nohr Maryssa , Mazzer Paula , Kelly Anne
Published: April 2020

Cortisol levels have been identified as one potential risk factor for suicide (O’Connor, 2016) because they have been linked to stress. Fearlessness, including impulsivity and risk taking, contribute to suicidality and cortisol may be a biomarker to aid in diagnosis. If cortisol calms fears (de Quervain et al., 2011), for at least part of the population, and, if fearlessness heightens impulsivity and risk taking as demonstrated by previous research (Smith, 2008) then cortisol may contribute to suicidality and suicide attempts. The purpose of this research is to clarify the relationship between fear, impulsivity, and risk and the role cortisol may play. Thirty males and 30 females completed the Barratt Impulsiveness Scale (Patton, Stanford, & Barratt, 1995), Domain‐Specific Risk‐Taking Scale (Blais & Weber, 2006), Death Anxiety Scale (Templer, 2006), and Fear of Pain Questionnaire‐9 (McNeil & Rainwater, 1998). Cortisol is collected by swabbing the cheek and collecting saliva. The saliva samples were then kept in a −80° C freezer until tested using an ELISA detection technique. Preliminary results suggest levels of cortisol are negatively correlated with fear of death and fear of pain. Fearlessness is expected to be negatively correlated with impulsivity and risk taking, which, in turn, are expected to be positively correlated with each other and positively correlated with cortisol levels.

Support or Funding Information

NIH P20GM103443
Population structure and Admixture of Culex pipiens complex in New York City using Genotyping By Sequencing
Authors: Seto Jeremy , Basu Saptarsi , Brekman Angelika , Bajwa Waheed
Published: April 2020

The first cases of encephalitis associated with West Nile Virus (WNV) in New York City presented in 1999. Birds serve as the primary host for WNV and transmit the pathogen through mosquitoes of the Culex pipiens complex. The Culex pipiens form pipiens (Cx. pipiens f. pipiens) are exclusively ornithophilic (avian feeding) and require blood meal in order to reproduce. Cx. pipiens f. molestus are mammophilic and feed almost exclusively on mammals. WNV transmission to mammals occurs through a succession of feeding between an infected bird and subsequent mammal. While the two forms of Cx. pipiens have preferential food sources, they are capable of hybridizing and become indiscriminate in blood meal. Biosurveillance and clinical presentation in humans has been consistent since the introduction of WNV 20 years ago. While these two forms are physically identical, they differ fundamentally in behavior. Traditionally, a single locus STR (CQ11) has been the defining disambiguator of the two forms. However, this does not illustrate any level of hybridization that would provide insight to bloodmeal preference. Understanding the amount of admixture within NYC populations can aid in the identification of these forms and in vector control processes. This study uses Genotyping by Sequencing (GBS) through Restriction site associated DNA Sequencing (RAD‐Seq) in order to identify the level of admixture within the NYC Culex populations as well as the levels of ancestral states. Admixture analysis has identified unique SNPs that illuminates some underlying behavioral differences between the two forms, as well as evolutionary divergence.