bluuebunny/crossref_metadata_2025 huggingface dataset:
Fumarase Overexpression Abolishes Hypertension Attributable to eNOS Haploinsufficiency in Dahl Salt‐Sensitive Rats
Authors: Xue Hong , Geurts Aron , Usa Kristie , Wang Feng , Phillips Jenifer , Henderson Lisa , Baker Angeles , Tian Zhongmin , Liang Mingyu
Published: June 2021

Background

Human blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through a malate‐aspartate‐arginine pathway.

Objective

We examined the role of the fumarase‐NO pathway in the development of hypertension in the genetic background of the Dahl salt‐sensitive (SS) rat, a model of human salt‐sensitive hypertension and renal injury used widely.

Methods and Results

SS rats with heterozygous mutation of endothelial NO synthase (eNOS or Nos3) (SS‐Nos3+/−) were bred with SS rats with a hemizygous Fh transgene. SS‐Nos3+/− rats without the Fh transgene (SS‐Nos3+/−/Fh0/0) developed substantial hypertension with a mean arterial pressure (MAP) of 138.5 ± 3.8 mmHg on a 0.4% NaCl diet, and 183.0 ± 3.7 mmHg after 14 days on a 4% NaCl diet. MAP decreased remarkably to 123.1 ± 1.4 mmHg on 0.4% NaCl, and 143.3 ± 1.5 mmHg on 4% NaCl in SS‐Nos3+/− rats with a Fh transgene (SS‐Nos3+/−/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS‐Nos3+/−/Fh0/1 rats compared to SS‐Nos3+/−/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression. However, the decrease in NO metabolite in the renal outer medulla of SS‐Nos3+/−/Fh0/0 rats was reversed in SS‐Nos3+/−/Fh0/1 rats. Levels of L‐arginine, but not the other 12 amino acids analyzed, were significantly higher in SS‐Nos3+/−/Fh0/1 rats than in SS‐Nos3+/+/Fh0/0 rats.

Conclusions

Fumarase has potent effects in restoring NO production in renal outer medulla and blunting the development of hypertension attributable to eNOS haploinsufficiency.

Support or Funding Information

This work was supported by US National Institutes of Health grant HL116264‐5244 (M.L.)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolic Insults Drive the Development of Glomerular Sclerosis and Proteinuria in Salt‐Sensitive Hypertensive Nephropathy
Authors: Rinschen Markus , Palygin Oleg , Siuzdak Gary , Staruschenko Alexander
Published: June 2021

Hypertensive nephropathy causes more than one third of end stage renal cases, but its molecular mechanisms are not fully understood. Here, we performed a multi‐layered omic analysis of a well‐established animal model of hypertensive nephropathy, the Dahl salt‐sensitive (SS) rat. Specifically, we performed a time‐course untargeted and targeted metabolomics as well as quantitative phosphoproteomic and proteomic analysis of glomerular and whole kidney cortex. For this experiments three groups of age matched 8 weeks old SS rats were fed either a low salt (0.4% NaCl) or high salt (4% NaCl; 7 and 21 days of HS). Analysis of microalbuminuria, as a marker of kidney injury, revealed that SS rats had a progressive kidney damage following HS treatment (Alb/Cre ratio was 4.6 ± 0.8, 10.7 ± 1.5 and 12.2±2.0, for SS rats fed LS and HS for 7 and 21 days, respectively). Metabolomics, phosphoproteomic and proteomic analyses were performed either in the kidney cortex homogenate or pure fraction of glomeruli. Glomeruli were isolated by consecutive sieving as we previously reported. Metabolomic analysis of the glomeruli revealed a decrease in energy fuels before the onset of proteinuria, including glucose‐6‐phosphate, alpha‐Ketoglutarate, and branched chain amino acids, suggesting a wide‐spread energy depletion. In addition, proinflammatory molecules such as free fatty acids were increased before onset of proteinuria. Notably, these changes did not occur in the kidney cortex where glomeruli fraction was negligeable, suggesting glomerular vulnerability as a key event in hypertensive nephropathy. Phosphoproteomics revealed that podocyte‐specific nutrient‐sensing 5′ AMP‐activated protein kinase (AMPK), as well as regulatory‐associated protein of mTORC1 Raptor, were activated before the onset of structural damage. Structural changes as shown by complement deposition and fibrosis proteins were detectable upon onset of heavy proteinuria. Furthermore, comparative proteomics across glomerular insult models revealed that increased expression of alpha‐Ketoglutarate depleting enzymes is common in glomerular injury. Oxidative protein modifications was also detected in the extracellular matrix of the glomeruli, explaining the energy depletion. In the kidney cortex, reduced uptake of amino acids was observed among other metabolic changes. These data suggest metabolome‐driven insults in the glomeruli of the kidney that proceed the insult in the other parts of nephron. Therefore, this study provides a novel framework for the study of hypertensive nephropathy.

Support or Funding Information

Supported by the DFG.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel method for establishing claudin knockout clones using TALENs
Authors: Tokuda Shinsaku , Yu Alan
Published: June 2021

In the multicellular organisms, epithelia function as a barrier between external and internal environment. Tight junctions (TJs) are one of the cell‐cell junctions between epithelial cells and regulate the permeability of the paracellular pathway. Claudins, a large family of transmembrane proteins, are major component of TJ strands. Most epithelia express multiple different claudins, and the expression pattern of claudins are thought to provide diverse permeability of TJs in each epithelium. However, it is difficult to measure the permeability of each claudin with the current technique, and the permeability property of each claudin is still poorly understood. To clarify the permeability property of each claudin, we attempted to knockout claudin genes using transcription activator‐like effector nucleases (TALENs) in MDCK cells. In the previous study, we succeeded in establishing claudin‐2 knockout clones and claudin‐4 knockout clones in MDCK cells. However, the efficiency of the knockout is low and a lot of effort was required to establish knockout clones. To improve the efficiency of establishing claudin knockout clones, we made a construct in which forward and reverse TALENs were connected with internal ribosome entry site (IRES). Using the constructs, we succeeded in establishing claudin‐2, −3, −4 and −7 knockout clones. In conclusion, we developed the method for establishing claudin knockout clones. The analysis of these claudin knockout clones are thought to provide an important insight in the permeability property of each claudin and TJ physiology.

Support or Funding Information

JSPS Overseas Research Fellowships

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High mobility group box‐1 increases epithelial sodium channel activity and inflammation in small airway epithelial cells
Authors: Grant Garett J , Coca Camila , Helms My N
Published: June 2021

In Cystic Fibrosis (CF) patients, inherited defects in anion transport leads to hyperactive epithelial Na+ channel (ENaC) activity. ENaC dysfunction in CF lung is known to cause airway surface liquid dehydration. Thick and dry mucus, in turn, leads to an inflammatory lung phenotype that often occurs without detection of infection in CF patients. Many investigative groups have reported that airway tissue and fluid from murine models exhibiting the CF lung phenotype (Scnn1b‐Tr mice), as well as CF patients, have increased levels of high mobility group box‐1 (HMGB‐1). HMGB‐1 is a cytokine mediator of inflammation and is a biomarker for severity of CF lung disorder. Herein, we hypothesize that HMGB‐1 plays an important role in the pathogenic regulation of ENaC, inflammation, and fibrosis in sterile airways. To begin testing this hypothesis, we measured transepithelial sodium current (Isc) and single channel ENaC open probability (Po) in human small airway epithelial cells (SAEC) in the presence or absence of 1 mg/mL human HMGB‐1 peptide. Electrophysiological measurements show that HMGB‐1 significantly increased amiloride‐sensitive ENaC Isc from −9.7±1.4 μA/cm2 to −18.4±2.9 μA/cm2 in confluent monolayers of SAEC (n=12; p=0.01) within 30 min. Subsequent amiloride treatment of the same SEAC monolayers decreased Isc 71%. Single channel measurements were conducted in primary SAEC obtained from 4 separate donor lungs in the cell‐attached configuration; HMGB‐1 increased ENaC Po from 0.18±0.02 to 0.35±0.04 (n=17; p<0.01) in single channel analysis. Mean absolute total cell counts were significantly higher in bronchoalveolar lavage fluid (BALF) from mice intraperitoneally (IP) injected with HMBG‐1 (10 mg in 1 mL saline/kg) vs vehicle injected mice. Total immune cell counts were 27.4×103± 8.8×103 (in HMGB‐1) vs. 5.2×103±1.3X103 (in vehicle) injected mice (n=8 counts from 2 independent studies; p<0.05). Masson's Trichrome labeling of IP injected mice show that HMGB‐1 significantly increases pulmonary fibrosis (see Fig 1). Flow cytometric analysis of HMGB‐1 and vehicle injected mice showed significant increase in IL‐1β, IL‐10, IL‐6, IL‐27, IL‐17A, IFN‐β, and GM‐CSF (n=3; p<0.05; see Fig 2). Together, our data suggests that HMGB‐1 plays an important role in the CF lung phenotype; additional studies are underway to strengthen these observations. Impact: Successful completion of our work can lead to novel therapies needed to counteract ENaC dysfunction and inflammation in the CF lung.

Support or Funding Information

This work was supported by NIH 1 R01 HL137033‐01A1 awarded to MH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inner Medullary Urea Transporters Contribute to Development of Renal Fibrosis in Mice With Unilateral Ureteral Obstruction
Authors: Rianto Fitra , Ellis Carla L , Hassounah Faten , Wang Xiaonan H. , Klein Janet D. , Sands Jeff M.
Published: June 2021

Fourteen percent of the population of the United States is affected by chronic kidney disease (CKD). Although the mechanisms that are responsible for CKD remain unclear, there are many clinical and animal studies showing that low protein diets reduce the long term effects of CKD. One possible explanation for these benefits is the restricted urea load of the low protein diet suggesting a role for the urea transporters in this process. Unilateral ureteral obstruction (UUO) is a well known model to produce fibrogenic responses such as increased capillary macrophages, pro‐fibrogenic proteins, interstitial matrix, myofibroblasts, etc. in CKD rodents. We hypothesized that the urea transporters contribute to the development of renal fibrosis. We used UUO mice receiving a maximal urea load (high protein diet) to investigate contributions of urea transporters to renal fibrosis. C57Bl6 mice ± genetic ablation of UT‐A1 and UT‐A3 urea transporters (UT‐A1/A3 KO) underwent UUO surgery (ligation of the left proximal ureter). Four days after surgery mice were given a high (40%) protein diet for 10 further days. Mice were killed and obstructed kidneys collected for protein and histochemical analysis. All of the obstructed kidneys showed evidence of hydronephrosis after 14 days. BUN was unchanged by UUO: WT control: 44.2 mg/dl, WT UUO: 46.8 mg/dl, KO control: 43.4, KO UUO: 47.1. TGF‐b was increased in both WT and UT‐A1/A3 KO mice with UUO (302% and 389% respectively). WT with UUO showed a 320% increase in α‐SMA vs mice without UUO. UT‐A1/A3 KO mice with UUO showed a blunted 271% increase in α‐SMA vs unobstructed UT‐A1/A3 KO mice. Vimentin, another marker of fibrosis, was significantly increased 555% by UUO (vs no UUO). UUO in UT‐A1/A3 KO mice did not increase vimentin. H&E staining revealed similar infiltration of inflammatory cells in both UUO WT and UUO UT‐A1/A3 KO mice. Trichrome staining showed higher collagen levels in UUO WT vs UUO UT‐A1/A3 KO mice. Immunohistochemical staining for αSMA and vimentin revealed higher levels in UUO WT vs UUO UT‐A1/A3 KO mice. These data suggest that urea transporters do not prevent inflammation caused by UUO. However, the overall development of fibrosis in UUO mice fed a high protein diet is attenuated by the absence of urea transporters suggesting that urea reabsorption by urea transporters contributes to renal fibrosis. If urea transport inhibitors replicate the effects of genetic knock out, they may represent a promising future therapy.

Support or Funding Information

Relypsa Nephrology and Cardiology Fellowship Grant;NIH RO1 DK41707

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hypogonadal Hypertension in Male Sprague‐Dawley Rats Is Reversed by Testosterone Replacement Therapy, which Suppresses Renin‐Angiotensin System Expression
Authors: Hanson Andrea E. , Garcia Nikolas W. , McKenna Joshua M. , Perusquia Mercedes , Stallone John N.
Published: June 2021

Testosterone (TES) and other androgens are efficacious vasodilators acutely, both in vitro and in vivo; however, their long‐term effects on arterial blood pressure (BP) are unclear. We previously reported that castration (CsX) produced hypertension in normal male rats, and that TES replacement therapy (TRT) was antihypertensive and normalized BP. Thus, long‐term effects of endogenous TES and exogenous TRT on BP and renin‐angiotensin system (RAS) function were studied in intact (InT) and castrated (CsX) male Sprague‐Dawley (SD) and Testicular‐feminized male (Tfm, androgen receptor defective) rats (12–13 wk old). Weekly measurements of systolic BP (tail cuff plethysmography) revealed a progressive rise in BP over 10 wks in CsX (108 ± 0.9 vs. 139 ± 1.2 mmHg), while BP remained stable in InT (109 ± 3.1 vs. 115 ± 0.5). During the next 5 weeks, half of CsX received TRT (CsX+TES‐enanthate‐replaced; 1.75 mg/kg, 2X/wk). BP gradually declined to normal in CsX+TES replaced rats (115 ± 1.2), while BP remained elevated in CsX (141 ± 1.2) and normal in InT (113 ± 0.3). In separate CsX‐SD rats, treatment with Losartan (LST; 250 mg/L drinking water) prevented development of hypertension at 10 wks (95 ± 0.8 CsX+LST vs. 139 ± 1.2 in CsX). During the next 5 weeks with TRT, BP declined in CsX+TES (113 ± 1.3) and remained lower in CsX+LST (99 ± 0.4). In Tfm, CsX resulted in a similar rise in BP (108 ± 0.6 vs. 139 ± 0.4 mmHg), and TRT reduced BP to a similar extent (106 ± 1.5). Real‐time PCR (rt‐PCR) of kidney from InT, CsX, and CsX+TES rats revealed that CsX increased expression of renin (45.5%), AT1 receptor (AT1R; 38%), and angiotensin converting enzyme (ACE; 239%) mRNA, while TRT normalized expression of renin, AT1R, and ACE mRNA to levels of InT rats. In contrast, CsX reduced expression of angiotensinogen (Angt) mRNA (59%) while TRT restored Angt mRNA to 78% of InT rats. 24 hr urine output (UO) in InT‐SD was 48.3 ml/kg at baseline, 34.3 at 10 wks, 38.4 at 12 wks, and 34.8 at 15 wks. UO in CsX‐SD was 47.6, 30.3, 25.2, and 27.7; UO in CsX+TRT was 46.1, 29.2 36.2, and 29.5. These data suggest that: 1) endogenous androgens (TES) exert antihypertensive effects in male SD and Tfm rats; 2) the antihypertensive effect of TES appear to involve a diuretic effect on the kidney, which is non‐genomically mediated; and 3) these antihypertensive effects may involve TES‐induced reductions in RAS expression in the kidney.

Support or Funding Information

State of Texas

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fecal Bypass Macronutrients Impact Stool Quality in Dogs and Cats while Species Differentially Impacts Nutrient Digestibility
Authors: Jackson Matthew Irick , Wernimont Susan M. , Carnagey Kristen , Jewell Dennis E.
Published: June 2021

Objectives

Evaluate the impact of animal and food characteristics on apparent digestibility and stool quality in dogs and cats.

Methods

Healthy dogs and cats were housed in environments promoting social interaction, having access to natural light that varied with season and opportunity to exercise. Animals were fed to maintain healthy body weight and health was monitored through regular clinical and physical checkups. Across two decades, 11,963 studies examining apparent nutrient digestibility were conducted using dry and canned foods. Studies were carried out under IACUC approved protocols. Proportional digestibility for dry matter, fat, nitrogen‐free extract, and fiber was calculated as difference between analyzed amounts in the food versus feces. Stool quality was evaluated using a standardized 5‐point system. Relations between animal characteristics (age, stool quality) and digestibility, as well as interactions between macronutrients that bypassed digestion, were evaluated with linear mixed models. Results significant at p≤0.05 are reported.

Results

In dogs, there was a negative association between age and linear coefficients for fiber, dry matter, carbohydrate, and fat digestibility but a positive association between age and quadratic coefficients for carbohydrate and dry matter digestibility. In cats, there was positive association between age and linear coefficients for protein and energy while there were negative associations between age and the linear coefficient for fiber and the quadratic coefficient for protein. Maximum protein digestibility occurred at approximately 14 years in dogs and at 8 years in cats; maximum crude fiber digestibility in dogs and cats occurred at 1 year. In both species, high fecal protein and low fecal fiber predicted decreased stool quality, and fecal fiber was positively associated with fecal protein and fat.

Conclusions

Aging in dogs and cats differentially impacted nutrient digestibility; protein and energy digestibility had a positive linear association with age in cats, while dry matter, carbohydrate, energy and fat digestibility had a negative linear association with age in dogs. In both species, fiber digestibility linearly decreased with increasing age. Undigested fecal fiber was associated with improved stool quality while fecal protein was detrimental. Intriguingly, the impact of fecal bypass macronutrients on stool quality was significantly greater in cats than dogs. This is the largest investigation to date evaluating associations between age and macronutrient digestibility in dogs and cats, and supports the development of diets to optimize health throughout the lifespan.

Support or Funding Information

Hill's Pet Nutrition, Inc. Topeka, Kansas, USA

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of a Probiotic and a Specifically Immunized Egg Powder on Sleep and Overall Health, Well‐being, and Gastrointestinal Symptoms in a Subclinical Population
Authors: Trigg Dorothy , Shaw Rachael , Asad Taheya , Jones Ashleigh , Skelton Michele
Published: June 2021

The purpose of this study was to examine the effects of a specifically immunized egg powder taken with a probiotic on sleep quality and patterns of sleep using the Pittsburgh Sleep Quality Index (PSQI) and health related quality of life as it applies to the gastrointestinal tract using the Gastrointestinal Quality of Life Index (GIQLI) in healthy subjects with subclinical GI complaints. In a randomized, double‐blind placebo‐controlled study, sixteen subjects were divided into two groups. One group received a probiotic plus 2 grams of a specifically immunized egg powder (treatment group) while the other group received a placebo. The PSQI and GIQLI were given to subjects prior to supplementation and immediately after a 4‐week supplementation phase. There were no significant differences in PSQI scores between groups. However, a significant improvement in sleep quality was measured in the treatment group (p < 0.05). There were no significant differences in GIQLI scores between groups. However, a significant improvement in the physical subset score of the GIQLI was measured in the treatment group (p < 0.05). Additionally, there was a strong statistical trend for improvement in the social subset score of the GIQLI and the total score on the GIQLI in the treatment group (p < 0.06). Preliminary analysis suggests that a combination of a probiotic and a specifically immunized egg powder may contribute to improved sleep and overall gastrointestinal health and well‐being in healthy subjects with subclinical GI complaints.

Support or Funding Information

IgY Nutrition

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of 6‐weeks of Time‐Restricted Feeding in Normal Weight Middle‐Aged and Older Adults
Authors: Nagy Erzsebet E , Rossman Matthew J , Mazzo Melissa R , Denman Blair A , Jankowski Lindsey R , Richey James J , Johnson Sarah A , Wang Yang , Peterson Courtney M , Chonchol Michel B , Seals Douglas R , Martens Christopher R
Published: June 2021

Calorie restriction (CR) improves multiple indicators of healthspan and physiological function in humans. However, CR is contraindicated in normal‐weight middle‐aged and older (MA/O) adults because it induces deleterious adaptations, such as reductions in body mass (including skeletal muscle mass) and bone mineral density (BMD). Time‐restricted feeding (TRF), which involves consuming all calories within an 8–10‐hour window and fasting for the remainder of the day, has the potential to maximize the benefits of CR while minimizing these possible adverse effects in normal‐weight MA/O adults. We performed a randomized controlled crossover clinical trial to evaluate the feasibility, tolerability and lack of adverse changes in body composition and BMD following six weeks of TRF vs. normal feeding (NF) in normal‐weight MA/O adult men and women (n=22, 67±6 years). A secondary aim was to explore the potential efficacy of TRF for improving select indices of healthspan and physiological function. Adherence to TRF was excellent with all subjects successfully reducing their eating window from 11.7 ± 0.3 hours/day to within 8 hours/day (7.6±0.1 hours/day, p<0.05). Compared with NF, all subjects maintained their regular daily energy intake during TRF, with no significant changes in macronutrient composition or overall diet quality, as assessed using the Healthy Eating Index (HEI; NF= 63±3 vs. TRF= 61±2, p>0.05). TRF was well‐tolerated with no increases in self‐reported desire to eat, satiety, or stomach fullness (all p>0.05 vs. NF) and interestingly, lower feelings of hunger (p<0.01 vs. NF). Importantly, TRF did not affect body mass, BMD or body composition (all p>0.05 vs. NF). TRF improved two indices of cardiorespiratory fitness, including a reduced heart rate during submaximal exercise (p<0.05) and increased distance traveled during a 6‐minute walk test (NF=570±12 m vs. TRF=585±11 m, p<0.05). Glucose tolerance, assessed during a 2‐hour oral glucose tolerance test in a subset of subjects (n=16), also improved with TRF (NF=15,500±300 mg/dL x min vs. TRF=14,500±200 mg/dL x min, p<0.05). These findings suggest that TRF is feasible and well‐tolerated in normal‐weight MA/O adults and is not associated with adverse changes in body composition or BMD. Moreover, TRF may improve cardiorespiratory fitness and glycemic control, two important indicators of healthspan and future risk of chronic disease, disability, and/or frailty. Thus, TRF may be a practical alternative to conventional CR for improving healthspan in normal‐weight MA/O adults.

Support or Funding Information

Supported by Aging T32 AG000279

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unexpected Lethal Consequences of Disrupting Angiotensinogen in Agouti‐Related Peptide (AgRP)‐Expressing Cells
Authors: Sapouckey Sarah A , Deng Guorui , Pearson Nicole A , Gibson‐Corley Katherine N , Grobe Justin L
Published: June 2021

Genetic disruption of angiotensin (ANG) type 1A receptors (AT1A) in cells which express Agouti‐related peptide (AgRP) abolishes thermogenic sympathetic nerve activity (SNA) and resting metabolic rate (RMR) responses to leptin and high fat diet (HFD). Although it is known that glia secrete angiotensinogen (AGT), in silico reanalysis of published single‐cell RNAseq datasets describing transcriptomes of cells from mouse hypothalamus (GSE74672) and RNAscope fluorescent in situ hybridization independently confirm that AgRP neurons of the hypothalamic arcuate nucleus (ARC) in adult wildtype mice express AGT mRNA. We therefore sought to explore the functional significance of this expression pattern. Why, if AgRP neurons are bathed in AGT in the interstitial space, would they synthesize AGT? Mice were generated which lack AGT specifically in AgRP cells (AGTAgRP‐KO mice) using the Cre‐Lox approach (sire: AgRP‐Cre+, AGTF/WT x dam: AgRP‐Cre−, AGTF/F). Of the first 116 offspring, this resulted in expected overall distributions across sex (53% female), Cre (47% Cre+) and AGT (46% AGTF/F) genotypes, but an underrepresentation of targeted AGTAgRP‐KO mice at weaning (n=5 females + 1 male; Χ2=52.0, df=3, p<0.001), suggesting pre‐weaning or in utero lethality of the genotype. Before and at 8 weeks of age, female AGTAgRP‐KO mice exhibited normal body mass (n=5, 17.3±0.4 vs n=12 littermates 17.3±0.3 g); 5 weeks of 45% HFD caused significant weight gain in all mice (p<0.05), but AGTAgRP‐KO mice gained less (body mass: 18.3±0.4 vs 19.7±0.3 g, p<0.05, and fat mass by NMR: 1.6±0.4 vs 2.3±0.2 g, p<0.05), which was not due to suppression of food intake (p=NS) or digestive efficiency as assessed by bomb calorimetry (p=NS). Histological examination of the kidney suggests the presence of interstitial fibrosis, inflammation, glomerulosclerosis and tubular dilation in AGTAgRP‐KO mice. Interestingly, Cre activity is present within the medulla of both the adrenal gland and kidney but not liver of AgRP‐Cre mice. Preliminary tests suggest reduced total aldosterone (n=5, 3.6±1.7 vs n=9 littermates 6.9±1.1 ng/d) and corticosterone (n=6, 0.4±0.1 pg vs n=9 littermates 0.9±0.2 ng/d) in the urine, but no changes in AGT gene expression in the liver or kidney (n=3,4 p=NS) of AGTAgRP‐KO mice. We conclude that (i) AGT is expressed by AgRP cells of the ARC, (ii) the AgRP‐Cre construct drives Cre activity in these cells, but also in layers of the adrenal gland and kidney, (iii) disruption of AGT in cells targeted by AgRP‐Cre unexpectedly causes a developmental lethality that is more penetrant in males, and (iv) in surviving females, the disruption of AGT in AgRP‐expressing cells causes resistance to weight gain through increased energy expenditure.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hybrid Rat Diversity Program (HRDP): A Rat Resource for Systems Genetics
Authors: Dwinell Melinda , Takizawa Akiko , Lazcares Lynn , Saba Laura , Tabakoff Boris , Shimoyama Mary
Published: June 2021

Genetic susceptibility to disease, sensitivity to environmental elements, and pharmacogenomics are critical components of the concepts of precision medicine. Rats are a major model for studying complex disease mechanisms, behavioral phenotypes, environmental factors, and for drug development and discovery. Inbred rat strains control for genetic background and allow for repeated, reproducible molecular, cellular, and whole animal phenotyping. Through the development of the Hybrid Rat Diversity Program, a powerful panel of inbred rats with genomic, physiologic, and behavioral phenotype data will serve as a catalyst for research in many fields. The Hybrid Rat Diversity Panel (HRDP) will consist of 96 inbred rat strains to maximize power to detect specific genetic loci associated with complex traits while maximizing the genetic diversity among strains. The 96 strain panel consists of 33 genetically diverse inbred strains and two panels of recombinant inbred panels: FXLE/LEXF (33 strains, Japan) and HXB/BXH (30 strains, Czech Republic). Embryo resuscitation and breeding are underway at the Medical College of Wisconsin (MCW). Whole genome sequencing for all 96 strains will be performed by the Genomic Sciences and Precision Medicine Center (MCW) sequencing core to achieve 30X coverage (Illumina NovaSeq S4). Liver and brain transcriptome analysis will be done for the entire HRDP panel through PhenoGen (http://phenogen.ucdenver.edu) at the University of Colorado (Tabakoff and Saba). Genomic, phenotype, and strain information will be made available through the Hybrid Rat Diversity Panel portal at the Rat Genome Database (http://rgd.mcw.edu). MCW will partner with the Rat Resource & Research Center (RRRC, http://www.rrrc.us) at the University of Missouri for strain distribution. The HRDP will provide a genetically stable population of rat strains with fully sequenced genomes, transcriptomes for brain and liver, and general phenotypic characterization to be used for systems genetic studies and fine mapping of complex traits.

Support or Funding Information

National Institutes of Health, Office of the Director, R24OD024617

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Reexamination of Breast Cancer Gene Expression Data Using (and Comparing) Advanced Predictive Modeling Techniques
Authors: Hummel Ruth M , Genung Daniel
Published: June 2021

Using the human breast cancer data from the MAQC‐II Project (GEO Accession GSE20194), we re‐explore the association between gene expression and Estrogen Receptor Status across advancing stages of the disease. We specifically compare various predictive modeling techniques, such as Radial Basis Machines and Partition Trees, as well as various predictor reduction techniques, including K‐Means Clustering and LASSO, and we discuss the merits of each. We use k‐fold cross‐validation to improve upon a simple single training/validation split in comparison of model performance and consistency.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dysregulation of metalloproteinase complex ADAM10‐MMP14 plays the key role in preservation of adherens junction integrity at renal cystogenesis
Authors: Xu Frank , Kong Tianqing , Lu Tzongshi
Published: June 2021

Background

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common lifeth‐reatening genetic diseases and is characterized by early cystogenesis in kidney, which eventually results in end‐stage renal disease. We previously reported that the inhibition of A Disintegrin and metalloproteinase domain‐containing protein 10 (ADAM10) can block the cystogenesis in renal tubular epithelial cells through the preservation of E‐Cadherin. In addition, recent studies also indicate that another major matrix metalloproteinase, Matrix metalloproteinase‐14 (MMP14) promotes cystogenesis in kidney epithelial cells, which was blocked by its inhibitors. In our study, we investigate these two major sheddases association and their roles in preservation of adherens junctions in renal cystogenesis

Methods

Immunoprecipitation, immunostaining and 3‐D cell culture technologies are used to analyze the interaction between metalloproteinase ADAM10 and MMP14 in Madin‐Darby Canine Kidney (MDCK) cells.

Results

Our data shows that ADAM10 and MMP14 form a stable complex on the membrane of MDCK cells at hemopexin domain of MMP14. The activation of MMP14 is required for ADAM10 in shedding of E‐Cadherin. Either an enzyme‐inactive mutant MMP14E240A (Glu240 to Ala) or the deletion of the catalytic domain of MMP14 (MMP14ΔCAT) significantly decreases the sheddase activity of ADAM10. In addition, knockdown of MMP14 decreases the cleavage of ECadherin by ADAM10, and knockdown of ADAM10 enhances the activation of proMMP2 by MMP14. Furthermore, ectopic expression of MMP14 increases the proliferation of renal epithelial cells, alters the cell‐cell adhesion and promotes the cystogenesis of renal epithelial cells in 3‐dimensional collagen‐1 gels. Ectopic expression of the MMP14E240A mutant promotes strong contact inhibition of the cells, and results in tubular rather than cystic growth in collagen gels.

Conclusion

Our study shows for the first time that ADAM10 form a unique, stable complex at hemopexin domain of MMP14 on the surface of epithelial cells, and the ADAM10‐MMP14 complex regulates their sheddase activity on many cell junctional proteins. Furthermore, ADAM10 is a negative regulator of MMP14 that ADAM10 inhibits the shaddase activity of MMP14. The functional interaction between ADAM10 and MMP14 as a complex could play a pivotal role in the cystogenesis of renal epithelial cells. Our data provide a potential therapeutic target in ADPKD through the modulation of ADAM10‐MMP14 complex.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High School Physiology Innovations: The Arduino Blood Pressure Monitor
Authors: Anjur Sowmya
Published: June 2021

Students in my Physiology and Disease (PAD) which is a Biology elective at the Illinois Mathematics and Science Academy (IMSA), Aurora, Illinois, measure heart rates and blood pressure to study the correlation between these parameters. IMSA is a residential magnate school for students gifted in math and science. I have evolved my PAD class to be based upon student centered learning, which has proven to be the best way to challenge students to take responsibility for their own learning. Several opportunities are provided to students for hands‐on experiences, such as building a working model of the pumping heart and measuring body parameters such as heart rate, lung capacity and blood pressure before and after exercise. Students normally measure their blood pressure using sphygmomanometers while exercising. To enhance student learning and improve integration between different disciplines, an interdisciplinary and collaborative project was introduced where students will modify the previously introduced Arduino heart rate monitor to make the Arduino blood pressure monitor. This will help them understand the connections between programming and measurement. This project build upon previous integration of different disciplines such as biology, computer science and physics, encouraging collaboration between students proficient in any or all of these disciplines. It is hoped that students will understand the working of the heart better by creating the Arduino blood pressure monitors.

Student understanding of material as evidenced by their assessment scores improved significantly with the introduction of the mechanical heart model building assignment. At this time, students in my classes are currently completing the blood pressure model prototypes and their feedback for this project will be collected along with any suggestions for improvement or modification. Evaluation of this assessment includes appraisal of the quality, accuracy and creativity of student monitors. It is hoped to continue building upon this non‐traditional method of modeling for other physiology concepts next semester, to further enhance student learning.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using innovative technologies to increase student engagement in an online Anatomy and Physiology course
Authors: Jones Heather M
Published: June 2021

Online education has become increasing popular among students with the recent advancements in multimedia technology. Many college, university, and professional school curricula have been transformed into online formats to meet the rising demand from learners. One of the pitfalls of converting a live course into an online course is not considering differences in how the student will interact with the content and incorporating active learning into the curriculum. This work will outline how three separate technologies which were added to an online first year Pharmacy School Anatomy and Physiology course to increase student retention as assessed by performance on summative examinations. The three methods that were incorporated were Screencast‐o‐matic© screen capture technology, Easygenerator© course design, and the use of Socrative©. Screencast‐o‐matic© is an online video capture system which was used in conjunction with a touch enabled laptop to allow for real‐time annotation of PowerPoint presentations. The Easygenerator© platform is a course design program which incorporated formative assessment directly into course content using an easy to navigate platform. Finally, Socrative© is a web‐based question response system which allowed students located all over the country to respond live during synchronous lecturing sessions. Implementation of these technologies in the online course resulted in significant increase in examination averages on the first two unit assessments. The third unit examination also showed improvement in mean score, although it was not significant for all years. There was also a high approval of the technologies. The Easygenerator© activities received an average overall rating of 9.17 (out of 10) with 60 students evaluating the content. In conclusion, the addition of interactive technologies into an online Anatomy and Physiology course has not only reduced student frustrations with the content, it has improved student performance.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐term culture of human pluripotent stem cells on coverslips coated with peptide‐poly(styrene‐co‐vinyl benzoic acid) copolymer
Authors: Narayanan Karthikeyan , Wan Andrew C.A. , Ying Jackie , Rajendran Vazhakkurichi M
Published: June 2021

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the potential to grow indefinitely (self‐renewal) in culture and differentiate into any cell type in adult human body. The hESCs and hiPSCs represent important resources for regenerative medicine and for disease modeling. Therefore, growing stem cells in undifferentiated state is important. The human pluripotent stem cells are expanded traditionally using either mitotically inactivated mouse embryonic fibroblasts (feeder cells) or Matrigel (MG). Both feeder cells and MG are prepared using mouse tissues that have several disadvantages. These manufactured expensive biological materials that have limited scalability also have high batch‐to‐batch variability. In addition, the animal‐derived (xenogenic) materials have potential to spread inter‐species pathogens. Thus, in order to replace these animal based biological materials, great efforts have been invested to develop synthetic materials that could be used for long‐term pluripotent stem cell growth and proliferation. As a consequence, materials such as poly [2‐(methacryloyloxy) ethyl dimethyl‐(3‐sulfopropyl) ammonium hydroxide] (PMEDSAH) and UV treated peptide‐acrylate materials have been used to culture hESCs. Since these synthetic materials are polymerized directly on the surface of cell culture dish, its application in broad sector and large‐scale expansion of stem cells for downstream applications has limitation. Thus, in the present study, we used a copolymer system containing poly (vinyl benzoic acid‐co‐styrene) (PVBA‐St), which could be coated onto various cell culture surfaces, such as glass, polystyrene cell culture plate etc. We synthesized PVBA‐St via nitroxide‐mediated polymerization to obtain polymer with narrow polydispersity index (PDI) and controlled molecular weight. In this study, we tested the stem cell growth on glass coverslips coated with peptide (extracellular matrix related) conjugated‐PVBA‐St. Up on initial screening, three active peptides were identified suitable for pluripotent stem cells expansion and self‐renewal. The long‐term culture of three pluripotent stem cell lines (H7, HUES‐7 and DF699) further confirmed the applicability and robustness of the copolymer system.

Support or Funding Information

Funding was provided by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). VMR was supported by funding from National Institutes of Health grant (NIH/NIDDK RO1DK104791)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Millennial Students Study Alone Using Mixed Educational Resources
Authors: Kilarkaje Narayana , Al‐Hussaini Heba , Ettarh Rajunor
Published: June 2021

It is widely believed that the millennial generation has a preference for teamwork, interactive learning experiences, and visual media to classical educational methods. To investigate whether this hypothesis is correct or not, we examined the study preferences newly joined medical students soon after their matriculation at Kuwait University College of Medicine (KUMS). Two hundred and twenty first‐year medical students at Kuwait University College of Medicine were surveyed by questionnaire shortly after starting in medical school. They were asked to respond to questions relating to learning habits and preferences. Students were asked for responses regarding 1) whether or not they request feedback after learning/studying, 2) whether they study alone or in the groups, and 3) whether they prefer printed or electronic materials resources to study. A one‐sample chi‐square test was used to estimate statistical significances in differences between the groups. The age‐range of the students was 17–21 years (mean 18.8 ± 0.6 SD). The number of students who usually request after‐study feedback was comparable to those who do not (47.7% versus 48.2%). Ninety‐seven percent of students prefer to study alone compared to 30% of respondents who prefer to study in a group. The overlap between these two categories (29.1%) represents those respondents who study alone and also in groups. Seventy‐five percent of respondents prefer online‐based resources compared to 66.4% of respondents who usually study using paper‐based resources. Forty‐two percent of respondents study using paper and online resources. The overlap between these two categories (42.7%) represents those respondents who use both types of resources. We conclude that the students admitted to KUMS show a preference for a ‘studying alone habit’ which they might have adopted before joining the medical school. They show no preference for either paper or electronic educational resources. We suggest that instructional activities may enhance learning if a mixed learning environment approach is employed that supports printed and electronic resources, as well as individual and group learning.

Support or Funding Information

Nil

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Learning with Concept Maps versus Learning with Case Based Study in Neuroanatomy‐A comparison
Authors: Anand Mahindra Kumar , Chhabra Prabhjot Kaur
Published: June 2021

Introduction

Meaningful learning skills are very important for medical students as they need to keep abreast of the rapid changes in science and the practice of medicine. Thus, in order to have competent doctors, there is a need to train medical students in ways that help them to become life‐long meaningful learners. Hence, one of the major goals of medical educators is fostering meaningful and self‐directed learning among medical students. Concept maps are educational tools that encourage meaningful learning. It helps in developing synthesis and analytical skills among students. Case based study in medical education is another tool to foster meaningful learning. It helps in developing analytical and decision making skills. It also facilitates learning by doing.

Present study was undertaken to compare learning with concept maps and learning with case based study in neuroanatomy to achieve following objectives:

Sensitize 1st year MBBS students to concept mapping and case base learning as teaching‐learning tools

Compare learning with concept maps versus learning with case base learning.

Obtain feedback from students in regards to learning with concept maps and case base study.

Material and Method

A randomized cross sectional prospective study was conducted on 150 medical students of 1st year after due permission from IRB. Students were divided into two groups A and B. Group A studied “Internal capsule, basal ganglion and spinal cord” by developing “concept maps”. Group B leant through case base learning method. Pre and post‐tests were conducted for both groups with pre validated questionnaire. After posttest, group A was exposed to case based learning and group B was exposed to concept maps. Feedback was obtained from all students in regards to learning with concept maps and case based study through a 5 point Likert scale. Student's t test was applied for statistical analysis.

Result

There was significant improvement in knowledge of both group A and B students (p value was <0.0001). There was no significant difference in gain of knowledge between two groups (p value was 0.0010). 90% students found the case based learning was interesting and engaging. 60% students found that learning with case study helped them to understand topic better. 90% students found that development of concept maps was time consuming.

Discussion and Conclusion

Case based learning is a better learning tool to engage students in active learning process. It also facilitates better understanding of given topic in comparison to development of concept maps.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.