bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyperglycemia‐increased activity of blood‐brain barrier Na‐K‐Cl cotransport and Na/H exchange involves SGK1 and PKCβII (893.35)
Authors: Chechneva Olga , Klug Nicholas , Yuen Natalie , O'Donnell Martha
Published: June 2021

Hyperglycemia (HG) exacerbates cerebral edema and worsens ischemic stroke outcome although the mechanisms are unclear. We have shown that ischemic factor stimulation of blood‐brain barrier (BBB) Na‐K‐Cl cotransport (NKCC) and Na/H exchange (NHE) increases secretion of Na and water into the brain, promoting cerebral edema formation. We recently found that HG (30 mM glucose; 6, 24,48 hr and 7 d) increases cerebral microvascular endothelial cell (CMEC) NKCC and NHE abundance and activity and augments ischemic factor‐stimulated transporter activity. Serum‐glucocorticoid kinase 1 (SGK1) and protein kinase CβII (PKCβII) have been implicated in the pathogenesis of HG. Thus, here we examined whether SGK1 and PKCβII are involved in HG effects on BBB NKCC and NHE using cultured CMEC and Western blot, radioisotopic flux and microspectrofluorometry methods. HG (24 or 48 hr) significantly increased expression and activation of CMEC SGK1 and PKCβII. Inhibition of SGK1 or PKCβII with GSK650394 (10 μM) and CGP53353 (1 μM) respectively, reduced or abolished HG‐increased NKCC and NHE activity. In these studies we also found that HG (24 hr) significantly increased astrocyte NKCC activity in a manner reduced by SGK1 and PKCβII inhibitors. Our findings support the hypothesis that HG exacerbation of cerebral edema in ischemic stroke involves SGK1 and PKCβII‐induced increases in BBB and astrocyte Na transporter activities.

Grant Funding Source: Supported by NIH and ADA
Nox2‐derived superoxide does not contribute to oxidative stress‐induced vascular upregulation of T‐type channel function (893.6)
Authors: Hill Caryl , Matthaei Klaus , Drummond Grant , Howitt Lauren
Published: June 2021

Cardiovascular disease is characterised by reduced nitric oxide (NO) and increased oxidative stress. We have shown that these conditions increase the expression and contribution of T‐type channels to vascular tone through increased bioavailability of reactive oxygen species produced from NADPH oxidase (Nox). As Nox2‐generated superoxide has been causally implicated in several vascular disease models, we investigated whether Nox2 was also responsible for upregulation of T‐type channel function in cremaster arterioles in vivo, using wildtype (WT) and Nox2‐deficient mice (Nox2KO). We first validated a novel T‐type channel antagonist, TTA‐A2 (3µM; provided by Merck and Co, Inc) which demonstrated no cross reactivity with L‐type channel function in these arterioles. In both WT and Nox2KO mice, acute inhibition of NO with L‐NAME (10µM) significantly increased the T‐type contribution to vascular tone. Chronic L‐NAME treatment (40mg/kg/day) for 2 weeks significantly increased blood pressure of both WT and Nox2KO mice, as well as the T‐type channel component of arteriolar tone. Pharmacological inhibition of Nox1 (10µM ML171) and Nox4 (10µM VAS2870) together reversed the effect of chronic L‐NAME on T‐type channel function in Nox2KO mice. We conclude that Nox2‐generated superoxide is not responsible for the upregulation of T‐type calcium channels elicited by oxidative stress.

Grant Funding Source: Supported by the Heart Foundation of Australia
Computational modeling of MRP4 and BSEP substrates and inhibitors (896.10)
Authors: Swaan Peter , Köck Kathleen , Welch Matthew , Urban Thomas , Brouwer Kim
Published: June 2021

Drug‐induced liver injury (DILI) is an important cause of drug toxicity. Inhibition of MRP4, in addition to BSEP, might be a risk factor that is responsible for the development of cholestatic DILI. Recently, we demonstrated that MRP4 inhibition was associated with an increased cholestatic potential of drugs that are BSEP non‐inhibitors, while MRP4 inhibition among BSEP inhibitors did not provide additional benefit to predict cholestasis. The current study aimed to develop in silico models to delineate molecular features underlying MRP4 and BSEP inhibition.

Models were developed using 257 BSEP and 88 MRP4 inhibitors and non‐inhibitors in the training set based on various molecular descriptors as well as fingerprints (ECFP and FCFP). Models were validated using an external test set; subsequently, these models were used to predict the affinity towards BSEP and MRP4 of a database (CDD) containing over 2,200 FDA‐approved drugs. Compounds with a score above the median fingerprint threshold were considered to have significant inhibitory effects on MRP4 and BSEP. Common feature pharmacophore models were developed with the Catalyst software suite using a training set of 20 well‐characterized MRP4 substrates and inhibitors.

For MRP4 substrates and inhibitors, common feature pharmacophore models were characterized by hydrogen‐bond acceptor and donor features as possible interaction forces between MRP4 ligands and the transporter protein. Models were further used to predict over 70 potential MRP4/BSEP inhibitors by integrating hits retrieved from the CDD database with a list of 208 compounds known to cause DILI.

The Bayesian (BSEP, MRP4) and pharmacophore (MRP4) models demonstrated significant classification accuracy and predictability. Potential novel MRP4 inhibitors were predicted by virtual screening of the database based on Bayesian and pharmacophore models. Subsequent in vitro testing should further our knowledge on MRP4 and BSEP affinity and their role in DILI.

Grant Funding Source: NIH R01GM041935, R01DK61425, T32‐ES007126, and Deutsche Forschungsgemeinschaft Ko4185/1‐1
Identification and molecular characterization of 5′‐regulatory region of the human SLC52A1 (rft‐1) promoter (896.4)
Authors: Sabui Subrata , Said Hamid
Published: June 2021

The human SLC52A1 gene encodes riboflavin transporter‐1 (rft‐1) which is essential for riboflavin transport across cell membranes. Rft‐1 is strongly expressed in human placenta, small intestine, heart muscle and thymus. Nothing is known about the transcriptional regulation of the human SLC52A1. We addressed the issue by cloning the 5'‐regulatory region of the SLC52A1 gene, identified the minimal promoter region for basal activity and identified potential cis‐regulatory elements. Guided by Rapid amplification of the cDNA ends (5'‐RACE), evidence was obtained for existence of one transcription start site (TSS) at nt‐716 in intestinal epithelial cells (Caco‐2). A 384‐bp segment upstream of the TSS was found to exhibit significant promoter activity in different human derived intestinal epithelial cells (Caco‐2, HuTu80 and NCM460). Truncation of this segment revealed that core promoter activity was embedded in a 297‐bp fragment (‐146 to +149) that contains GC rich, lacks TATA element and harbors several putative transcription factor binding sites includes SP‐1, AP‐2 and NFkB. Mutating the latter sites resulted in a significant decrease in promoter activity suggesting involvement of these cis‐regulatory elements in the regulation of SLC52A1 transcription. This study reports on first characterization of the SLC52A1 promoter.

Grant Funding Source: Supported by the VA and NIH, DK56061
Systemic Porphyromonas gingivalis lipopolysaccharide exacerbates the inflammatory response post‐myocardial infarction through matrix metalloproteinase‐9 (897.6)
Authors: DeLeon‐Pennell Kristine , de Castro Bras Lisandra , Bratton Dustine , Linsey Merry
Published: June 2021

Why periodontal disease (PD) strongly correlates with increased mortality post‐myocardial infarction (MI) is unknown. Previously, we showed P. gingivalis‐lipopolysaccharide (PgLPS) exposure increases matrix metalloproteinase (MMP)‐9 in the left ventricle (LV) of wild‐type (WT) mice. Because MMP‐9 plays a critical role post‐MI, we hypothesized that circulating PgLPS would exacerbate the LV inflammatory response post‐MI through MMP‐9. We compared WT and MMP‐9 null mice (3‐7 months old; n=12 survivors/group) infused with either PgLPS (0.08 µg/g/day) or saline for 28 days before and throughout the duration of MI. PgLPS increased rupture rate (rupture/total deaths) in WT (7/7) compared to saline controls (6/15) and was attenuated in Null (1/2). Echocardiography showed LV wall thinning increased at d7 post‐MI in WT (0.61±0.05 mm) compared to d0 (0.84±0.03 mm; p˂0.05). This effect was amplified by PgLPS in WT (0.44±0.04 mm) and attenuated in Null (0.50±0.04 mm; p˂0.05 for both). Plasma proteomics showed MMP‐9, macrophage inflammatory protein‐1β, ‐1γ, monocyte chemotactic protein‐1, and ‐3 increased in WT post‐MI and intensified by PgLPS suggesting amplified inflammatory response. This effect was attenuated by MMP‐9 deletion. In conclusion, circulating PgLPS exacerbates LV inflammation post‐MI through a MMP‐9 mechanism, resulting in a greater extent of LV dysfunction.

Grant Funding Source: Supported by AHA POST14350034; HHSN 268201000036C; R01HL075360; VA 5I01BX000505; HL051971
Hydrogen sulfide protects from colitis: a possible role in stabilizing gut microbiota (898.3)
Authors: Motta Jean‐Paul , Flannigan Kyle , Agbor Terence , Blackler Rory , Workentine Matt , Da Silva Gabriela , Buret Andre , Wallace John
Published: June 2021

We hypothesised that hydrogen sulfide (H2S) modifies gut microbiota and resolves intestinal inflammation. Rats were gavaged with diallyl disulfide (DADs), and colitis was induced by dinitrobenzene sulfonic acid (DNBS). Colitis severity was assessed by macroscopical, biochemical, and histological analysis. Microbial genetic diversity was performed by denaturing gradient gel electrophoresis. Antimicrobial activity of DADs, NaHS and Na2S was evaluated by plating and growth assays. Human intestinal epithelial cell lines were used to assess expression levels of antimicrobial peptides by qRT‐PCR, western‐blotting and immunofluorescence.

Administration of DADs increased the expression of cathelicidin, separated the microbiota from the host by a thick mucus barrier, and slightly altered microbial genetic diversity. Treatment of DNBS animals with an inhibitor of H2S‐producing enzyme provoked a more severe colitis, enhanced the presence of mucosa‐associated microbial aggregates, and altered mucus barrier. These inflammatory parameters were all reduced when DNBS‐treated animals were given DADs. In vitro, H2S‐releasing compounds inhibited the growth of four strains of gut bacteria. Additionally, H2S altered the level of expression of cathelicidin and trefoil factor‐3 in human epithelial cells.

The findings shed a new light on the therapeutic potential of H2S delivery during the course of colitis.

Grant Funding Source: Izaak Walton Killam PDF fellowship / University of Calgary Eye's High PDF fellowship
Intestinal stem cells rely on signals from underlying tissue for expansion following cytotoxic damage (899.1)
Authors: Seiler Kristen , von Furstenberg Richard , Schenhals Erica , Smith Brian , Henning Susan
Published: June 2021

Objective: Characterize signals required for epithelial regeneration after damage from Doxorubicin (Doxo). We hypothesize these signals arise from underlying tissue. Methods: Jejuni from Doxo‐treated mice were harvested for histology, assessment of ISC proliferation by flow cytometry, crypt culture, and microarray. Results: Crypt depth increases 4 days after Doxo; flow data confirm increased ISC proliferation at this time: 14±5 % of CD24lo cells are EdU+ in controls vs. 37±4%, 4d after Doxo (p=0.02). Interestingly, Lgr5‐EGFPhi cells are 7‐fold decreased at this time. In culture, crypts harvested from Doxo‐treated mice are equally proliferative as those of control mice. Addition of de‐epithelialized intestinal tissue (DEIT) from mice 4d after Doxo yields an increased average bud number per enteroid [1.45±0.31 buds vs. 5.10±0.96 buds (p <0.05)]; it also induces proliferation of crypts from treated animals [1.16±0.30 buds vs. 4.83±0.29 buds (p<0.0001)]. Microarray analysis of DEIT shows differentially expressed genes and pathways, potentially driving this observed effect. Conclusions: These data suggest a critical role of underlying tissue in regulating ISC behavior after damage, and warrant further investigation of mechanisms.
Notch signaling regulates proliferation and differentiation in the adult mouse intestine (899.4)
Authors: Zayan Nichole , Carulli Alexis , Samuelson Linda
Published: June 2021

Human gastrointestinal tumors can exhibit increased Notch signaling; accordingly, intestinal Notch over‐activation was examined in genetic mouse models ubiquitously expressing the Notch intracellular domain (NICD) in the intestinal epithelium. Based on previous studies, we hypothesized that Notch over‐activation would induce increased proliferation, decreased secretory cells, and increased absorptive cell numbers. NICD expression was activated in bitransgenic mice (Villin‐CreERT2 ; Rosa‐FSF‐NICD‐nGFP) by 5 daily doses of tamoxifen with intestine collected on day 6. We found that epithelial proliferation was increased and the goblet cell population was decreased in the NICD intestine. However, contrary to our hypothesis, the alkaline phosphatase‐positive enterocyte population appeared decreased, which was confirmed by reduced mRNA levels of the enterocyte marker sucrase‐isomaltase. Based on these data, we concluded that Notch over‐activation increases proliferation and decreases all differentiated cell types, not just secretory cells. This finding suggests that constitutive Notch activation in the intestine leads to a hyperproliferative state, consistent with activation of this pathway in human tumors.

Grant Funding Source: Supported by APS Fellowship (NEZ) and NIH grants R01‐DK078927 (LCS) and F30‐DK095517 (AJC).
The pathogenic role of poly(ADP‐ribose) polymerase 1 in experimental colitis (902.11)
Authors: Larmonier Claire , Shehab Kareem , McFadden Rita‐Marie , Hill Faihza , Alizadeh Darya , Thurston Robert , Ghishan Fayez , Kiela Pawel
Published: June 2021

Poly(ADP‐ribose) polymerases (PARPs), including its most abundant isoform PARP1, catalyzes the transfer of ADP‐ribose units from NAD+ to a number of target proteins. In vivo studies have demonstrated that genetic ablation or pharmacological inhibition of PARP1 ameliorates experimental colitis pathophysiological changes. However, the studies have failed to characterize the mechanism by which PARP1 inhibition is protective in colitis. We evaluated the effect of PARP1 deletion in T cell‐mediated colitis in the following scenarios: adoptive naïve CD4+CD45RBHigh T cell transfer from WT or Parp1‐/‐ mice into Rag2‐/‐ and Rag2‐/‐ x Parp1‐/‐ DKO mice and DSS‐induced colitis in WT, Parp1‐/‐, Rag2‐/‐, and Parp1‐/‐Rag2‐/‐ DKO mice. Compared to WT mice, Parp1‐/‐ mice were protected from DSS‐induced colitis. However, WT and Parp1‐/‐naïve T cells were equally colitogenic in Rag2‐/‐ recipients. DKO mice transferred with WT T cells or treated with DSS showed only marginal improvement. Our preliminary data indicate that Parp1 is dispensable in naïve and effector T‐cells, while Parp1‐/‐ mice are protected from colitis resulting from DSS‐induced epithelial damage. Moreover, the lack of significant protection in T cell transfer or DSS models of colitis in DKO mice suggests a pathogenic role of Parp1 in the innate immune cells and regulatory T cells during colitis.

Grant Funding Source: NIH Grant 1K01DK099268‐01
Zinc recovers altered intestinial ion‐transport and barrier function caused by Shigella infection in T84 cells (902.9)
Authors: Kazi Mirajul , Paramita Sarkar , Sheikh Irshad , Chakraborty Subhra
Published: June 2021

Zinc (Zn) has been shown to improve diarrhea. However, mechanism linking Zn to improve inflammatory diarrhea caused by Shigella sp. is not known. We tested that Zn restore paracellular and transcellular ion transport by modulating tight junction protein expression and chloride secretion, altered by Shigella infection. Human colonic epithelial cells, T84 were grown onto transwell inserts for measurements of permeability to dextrans, transepithelial electrical resistance (TER), short‐circuit current (Isc), and dilution potential (DP) in Ussing chamber. Cells infected with Shigella flexneri 2a caused reduction of TER by 40%, DP [4 ± 1.2 vs. 1.5 ± 0.3 mV] which was prevented in presence of Zn. An increase in paracellular flux of 4kDa dextran [2.1± 0.6 vs. 0.8 ± 0.2 µg/3.5hr/cm2] with respect to 70kDa was evident while in the presence of Zn, flux of 4kDa dextran was reversed by 52%. Immunofluorescence study revealed removal of claudin 1 and 2 from tight junction complex correlated with the loss of TER and paracellular flux. Studies with Ussing chambers demonstrated reduced cAMP stimulated electrogenic ion transport in infected cells. Zn application prevented this effect. Our results suggest that in T84 cells, Shigella infection caused (1) altered barrier function (2) altered Cl secretion. Zn application reversed these transport functions. These conclude that Zn may have beneficial role in inflammatory diarrhea.

Grant Funding Source: Supported by Dept. of Biotechnology, Govt. of India
The effect of wheat products on the colonization of the microbiota in the intestines of zebrafish (903.2)
Authors: Savarese Mary , Tousignant K. Davis , Uno Jennifer
Published: June 2021

Celiac disease is sensitivity to the wheat protein, gluten, characterized by intestinal inflammation and villous atrophy of the small intestine in genetically susceptible individuals. Inducing factors of celiac disease include ingestion of gluten, antibiotic exposure, infections, and a dysbiosis of the microbiota. Zebrafish have emerged as a viable model to study gastrointestinal diseases; however, they have yet to be used to directly study celiac disease. The aim of this work was to assess the potential use of zebrafish as a model for studying celiac disease. Fish were placed on a wheat‐based or brine fish regime. Antibiotics were used to knock‐down the microbiota in order to examine the reestablishment of the gut flora. All fish intestines were pooled into groups of 3, bacterial DNA was isolated and primers were used to amplify the 16S gene of specific bacterial groups. Bacterial levels were calculated as a percentage relative to total 16 S bacteria. Results show no significant difference in total bacterial expression however there was a significant decrease in Bifidobacterium (n=3, p< 0.05) in the wheat fed relative to the brine shrimp fed fish. These results confirm current studies indicating decreased expression of Bifidobacterium in bacterial DNA isolated from celiac patients. These data indicate the potential use of zebrafish to further the understanding of gut inflammation associated with wheat allergies.

Grant Funding Source: Supported by Elon University
The effects of Rosmarinus officinalis L. aqueous extracts on gastric acid secretion in isolated rats stomach (904.4)
Authors: Celebi Fikret , Gelen Volkan , Cınar Dursun , Sengul Emin
Published: June 2021

The Effects of Rosmarinus officinalis L. Aqueous Extracts on Gastric Acid Secretion in Isolated Rats Stomach.

Volkan Gelen, Emin Sengül, Fikret Çelebi, D.Ali Cınar.

Department of Physiology, Faculty of Veterinary Medicine, University of Atatürk, Erzurum, Turkey Rosmarinus officinalis L. (Rosemary) has been shown to have antioxidant, anti‐inflammatory and antiulces activites in different conditions.The present work was performed to investigate the possible effects of rosemary aqueous extracts on gastric acid secretion in isolated rats stomach.The stomachs of adult male Sprague‐Dawley rats were removed.The stomach was placed in a 20‐ml organ bath containing a serosal nutrient solution.Gastric acid secretion was measured from mucosal content in the isolated rat stomach.The results obtained in vitro experiments showed that doses of 300 µg/ml, 600 µg/ml and 1200 µg/ml of rosemary aqueous extracts have increased basal gastric acid secretion (except, 150 µg/ml).The present results suggest that rosemary may has a relieving effect on indigestion, probably due to increasing basal gastric acid secretion.
Resistance exercise improves the iron status without increasing the iron absorption in iron‐deficient rats (907.1)
Authors: Fujii Takako , Nakashima Ayumi , Tanaka Chihiro , Matsuo Tatsuhiro , Okamura Koji
Published: June 2021

We have previously reported that resistance exercise improved the iron status in iron‐deficient rats (Fujii 2011). The current study investigated the mechanisms underlying this exercise‐related effect. Male four‐week‐old rats were divided into a sedentary (S) or an exercise (E) group, and all rats were fed an iron‐deficient diet (12 mg iron/kg) for eight weeks. The rats in the E group performed climbing exercise (5 min x 6 sets/day, 3 days/week). The hematocrit and hemoglobin levels were significantly higher in the E group than in the S group. The plasma iron and transferrin saturation and total iron content in the body measured using the whole body homogenates of the rats tended to be higher in the E group. The mRNA expression of hepcidin tended to be higher in the E group, while the mRNA expression of duodenal cytochrome b, divalent metal transporter 1 and ferroportin 1 did not differ between the groups. The apparent absorption of iron was significantly lower in the E group than in the S group. Therefore, the resistance exercise‐induced improvement of the iron status does not appear to be due to an enhancement of iron absorption.
Resistance exercise decreases iron absorption without deteriorating the iron status in rats (907.2)
Authors: Okamura Koji , Asai Tsuyoshi , Kondo Emi , Okumura Yuka , Fujii Takako , Matsuo Tatsuhiro
Published: June 2021

To investigate the effects of resistance exercise on the dietary iron balance and the iron status, rats were divided into a sedentary (S) or an exercise (E) group and given different diets in terms of energy and iron sufficiency for three weeks. The energy‐deficient groups consumed 70% of the energy that was consumed by the energy‐sufficient groups. The iron‐deficient groups consumed a diet containing half of the iron (20 mg Fe/kg diet) of a standard diet. The E groups showed significantly higher fecal iron excretion compared to the S groups, thus resulting in a significantly lower apparent absorption, regardless of the energy and iron sufficiency of the diet. The iron status in the blood was not significantly lower in the E groups than the S groups. The iron content in the liver, heart and spleen did not differ between the E and S groups. The renal iron content of the rats that consumed the diet with sufficient energy but deficient iron was significantly higher in the E group than in the S group, while no such differences were observed in the kidneys in the groups that consumed other diets. These results suggest that, although resistance exercise decreases the dietary iron absorption, it does not appear to worsen the iron status in rats.
HFE deficiency attenuates impulsivity induced by intranasal manganese (907.7)
Authors: Ye Qi , Kim Jonghan
Published: June 2021

HFE deficiency attenuates impulsivity induced by intranasal manganese Qi Ye and Jonghan Kim Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA While excessive manganese (Mn) alters emotion and mood, olfactory Mn transport is enhanced in Hfe deficiency, which is the major cause of iron overload hemochromatosis. Hence we investigated the effect of HFE on Mn‐related impulsivity using Hfe‐knockout (Hfe‐/‐) and wild‐type (Hfe+/+) mice after intranasal instillation of MnCl2 (5 mg/kg) daily for 3 wks. Mn‐instilled mice spent more time in open arms than water‐instilled mice in an elevated plus maze, indicating an increased impulsivity (p=0.024, n=10‐13). Upon Mn exposure, however, Hfe‐/‐ mice entered the open arm later than Hfe+/+ mice (p=0.008), indicating that Mn‐induced impulsivity was corrected by Hfe deficiency. Since dopamine transporter (DAT) in the prefrontal cortex is involved in impulsivity, we conducted western blot and found that Mn instillation increased DAT levels in Hfe+/+ mice (1.3‐fold increase; p=0.004) but not in Hfe‐/‐ mice. To further investigate if oxidative stress induced by metal exposure is associated with altered emotion, activities of superoxide dismutase (SOD), an anti‐oxidative enzyme, was evaluated. SOD activity was elevated in Hfe‐/‐ mice(p<0.001), but reduced in Hfe+/+ mice (p=0.046) upon Mn exposure. Taken together, our results suggest that individuals with HFE deficiency could be less susceptible to airborne Mn‐induced impulsivity, likely due to altered dopamine signaling and enhanced protective activity of SOD.

Grant Funding Source: NIH R00 ES017781
GIP regulates glucose transporer‐2 to augment jejunal glucose absorption in obese mouse prior to the onset of diabetes (908.5)
Authors: Coon Steven , Rajendran Vazhaikkurichi , Singh Satish
Published: June 2021

Background: Jejunal glucose absorption is mediated by apical SGLT1 in normal‐, and by GLUT2 in obese mice. GIP, an incretin hormone, activates SGLT1 in normal mice. Aim: To determine if GIP regulates glucose absorption via GLUT2 in the obese jejunum. Methods: Short circuit current (Isc, a measure of SGLT1 activity) and glucose flux (measure of SGLT1 & GLUT2) were obtained under voltage clamp conditions at 0, 1, 2 and 5 mo in jejunums from mice fed a high‐fat diet. Results: In obese mice, GIP significantly activated SGLT1‐mediated glucose absorption for up to 2 mos, but was drastically decreased by 5 mos (2 vs. 5 mos = 725 vs. 49 µA/cm2). In contrast, the rate of GIP‐activated glucose absorption was steadily increased for up to 5 mos on a high‐fat diet (2 vs. 5 mos = 1.4 vs. 2.3 µEq/h.cm2). Western‐blot analyses of biotinylated proteins revealed that GIP exposure enhanced GLUT2 expression in apical membranes of jejunum at 5 mos. Conclusions: GIP both activates and recruits sub‐apical GLUT2 to apical membranes to augment glucose absorption in the jejunum of obese mice.

Grant Funding Source: Supported by NIH CTSI grant number UL1TR000157
Exploring the complexity of multidrug resistance in cancer (91.1)
Authors: Gottesman Michael
Published: June 2021

For the past 40 years, our laboratory has been studying the molecular basis of multidrug resistance in cancer. The use of new, specifically targeted anti‐cancer drugs offers the promise of revolutionizing cancer treatment, but the development of drug resistance continues to be an important clinical problem. Most studies of drug resistance have been conducted in cultured cancer cell lines which have enabled the cataloguing of several hundred genes whose increased or decreased expression or mutation results in resistance to many different drugs known as multidrug resistance (e.g. energy dependent multidrug efflux pumps such as ABCB1, P‐glycoprotein). Unfortunately, cultured cancer cells are in general poor models for the more complex mechanisms of resistance that occur in vivo. By interrogating the expression of 380 known drug resistance genes in clinical samples from patients with ovarian cancer, hepatocellular carcinoma, and acute myelogenous leukemia, whose clinical response to chemotherapy is known, we have shown that alterations in expression of many different established drug resistance genes correlates with clinical response (or lack thereof). These studies indicate that precise molecular knowledge for each cancer type, and for each person's tumor, will be needed to make predictions about tumor response, and to design alternative treatment regimens.
Modulation of proangiogenic and antiangiogenic balance by cystathionine β‐synthase and cystathionine γ‐lyase in human placenta (911.10)
Authors: Ni Xin , Hu Tianxiao , He Ping , Gu Hang
Published: June 2021

Imbalance release of proangiogenic and antiangiogenic factor from placenta causes maternal endothelial dysfunction which is main manifestation of preeclampsia(PE). Hydrogen sulfide (H2S) “the third endogenous gaseous signaling transmitter”, has been shown to have a pro‐angiogenic effect. We investigated whether H2S modulate the release of proangiogenic and antiangiogenic factors in human placentas. It was found that H2S synthesis enzymes cystathionine β‐synthase(CBS) and cystathionine γ‐lyase(CSE) were expressed in human placenta. The levels of CBS and CSE positively correlated with the levels of agiogenic factor vascular endothelial growth factor(VEGF) and inversely correlated to antiangiogenic factor soluble fms‐like tyrosine kinase 1(sFlt‐1) in placentas. H2S precursor L‐cysteine significantly increased VEGFA release whereas decreased sFlt‐1 release in cultured placental explants andd placental trophoblasts. The effects of H2S precursor could be reversed by siRNA of CBS and CSE. Moreover, H2S decreased miR‐200c which target VEGF and increased miR‐133b which target sFlt‐1. The level of miR‐200c was significantly increased whereas miR‐133b decreased in PE placentas. Our results suggest that H2S is an important factor that maintains the balance of proangiogenic factor VEGFA and antiangiogenic factor sFlt‐1 in placenta. Reduced H2S generation in placentas leads to imbalance release of proangiogenic and antiangiogenic factor from placenta.

Grant Funding Source: Natural Science Fundation of China No.81370734
Assessment of LH, FSH and prolactin levels of patients for IVF treatment in selected fertility clinics in Port Harcourt metropolis (911.14)
Authors: Onwuka Francis , Onwuka Adaora , Efenovwe Maureen
Published: June 2021

The study was designed to determine the levels of endocrine hormones such as LH, (FSH) and prolactin in women attending In vitro fertility clinics. Sixty (60) women were used for the study. Fasting blood samples were collected on day two of their menstrual cycle, and serum hormone (FSH, LH and Prolactin) concentrations analyzed by ELISA method with automated micro plate reader. The subjects were married for at least two years; and were within the child bearing age of 35‐44years. Out of 60 patients examined, 29 (48.3%) had normal hormonal concentrations, 31(51.7%) had elevated hormone levels represented as follows:3(5%) had elevated levels of LH,FSH and prolactin,1(1.7%) had elevated level of LH and prolactin,6(10%) had elevated levels of FSH only with normal concentrations of LH and prolactin, 2(3.3%) had elevated levels of FSH and LH with normal prolactin level, 23(38.3%) had elevated levels of prolactin,19(31.7%) showed elevated levels of prolactin with normal levels of LH and FSH. The results obtained were compared with standard values. The result showed a high incidence (38.3%) of hyperprolactinaemia which has been identified by several other researchers as one of the major causes of infertility .From this result, hormonal evaluation is important in the diagnosis and treatment of infertility cases. .

Grant Funding Source: none
Different effects of isoflavones genistein and daidzein on the endometrial epithelial tight junction barrier (911.15)
Authors: Poonyachoti Sutthasinee , Kiatprasert Pongpat , Buathong Norathee , Srisomboon Yosawee , Deachapunya Chatsri
Published: June 2021

Genistein (Ge) and daidzein (Di), the active isoflavones primarily found in soybean, have various beneficial health effects. The present study aims to investigate the effects of these isoflavones on tight junction (TJ) barrier integrity in immortalized endometrial epithelial cells (IMM). Expressions of the TJ integrity markers, zona occludens‐1 (ZO‐1) and claudins (cldn)‐1, ‐3, ‐4, ‐7 and ‐8 were analyzed by quantitative reverse transcription and real‐time polymerase chain reaction (RT‐PCR) and transepithelial electrical resistance (TER) by voltohmmeter. Treatment with Ge (10 nM or 1 μM) for 48 h increased the TER, and this was correlated with the mRNA expression of cldn‐3 and/or ZO‐1. However, low dose of Ge (0.1 nM) decreased both the TER and the expression of cldn‐3 and/or ZO‐1. Di did not alter the expression of the TJ proteins. Our results suggest that genistein increases barrier function possibly via the expression of cldn‐3 and ZO‐1. This represents an important protective role of genistein against endometrial barrier disturbance.

Grant Funding Source: Chulalongkorn University Research Fund and Srinakharinwirot University Research Fund
Clinically asymptomatic hypothyroidism secondary to hypercortisolism in Wistar rats (911.3)
Authors: Novais Renata , Fonseca Fabrícia , Laureano‐Melo Roberto , Araújo Iracema , Seara Fernando , Mecawi Andre , Silva Alba , Reis Luis
Published: June 2021

Hypothyroidism is the result of inadequate production of thyroid hormone or inadequate action of thyroid hormone in target tissues. Hypothyroidism can be seen secondary to hyperadrenocorticism with an overlapping clinical signs. We intend to demonstrate that clinical symptoms of hypothyroidism secondary to hyperadrenocorticism can be undetectable even if there is already evidence of hormonal disease. Male Wistar rats (~ 280g, n = 7) were submitted to daily subcutaneous administration of 200µg of adrenocorticotropic hormone (0.2ml ACTH 1‐24 synthetic 1mg/ml) or vehicle (NaCl 0,9% 0.2ml) for 7 days. At the end of the experiment, were obtained the values of plasmatic thyroid hormones (TH): T3 (Triiodothyronine) and T4 (Thyroxine) by chemiluminescence method; serum corticosterone, TSH (Thyroid Stimulating Hormone) and hypothalamic and pituitary type 2 deiodinase (D2) by radioimmunoassay technique. ACTH group obtained significant elevated level of corticosterone (p < 0.05) and low levels of HT (p < 0.0001). Despite, TSH levels showed no significant difference between groups, ie, compatible with hypothyroidism secondary to hypercortisolism. Additionally, D2 levels showed no significant differences between groups, demonstrating that those tissues did not represent important sources of reducing HT in our protocol. Although it has been demonstrated hormonal difference between the groups, no clinical signs were detected in ACTH group. These data suggest that the lack of symptoms of hypothyroidism and hyperadrenocorticism in this protocol can be attributed to early of the syndrome, once hormone values could be able to confirm both diseases.

Grant Funding Source: Supported by FAPERJ, CNPq and CAPES
Regional and temporal regulation of prostaglandin E2 receptor expression: a role for decidual activation in term labor (911.7)
Authors: Vinturache Angela , Mosher Andrea , Wood Stephen , Slater Donna
Published: June 2021

Objective: To assess if expression of prostaglandin E2 receptors (EP1, EP2, EP3 and EP4) are spatially or temporally regulated in decidua with labour onset.

Methods: Decidua biopsies from caesarean section deliveries, of labouring (TL) and non‐labouring (TNL) women and from both upper (US) and lower segment (LS) regions of the uterus were collected (n=12/group). Real‐time RT‐PCR was used to quantify levels of EP1‐4 gene expression.

Results: All four EPs are expressed in US and LS decidua. Expression of EP1, EP2, and EP4 is higher in LS compared to US in TNL decidua, but did not change with labor initiation. In contrast, EP3 was significantly higher than the other EP receptors in both US and LS in TNL decidua. The EP3 mRNA levels decrease in LS but not US decidua with labor initiation.

Conclusions: The presence of four EP receptor subtypes within the decidua in labouring and non‐labouring women suggests that prostaglandin E2 plays an active biological role during pregnancy and labour via differential EP interaction. Our observation that EP3 levels change with labor onset suggests that EP3 are the predominant mediators of PGE2 activity in the decidua at term. These findings on differential EP regulation in decidua with labour are important for the understanding of the underlying mechanisms that govern uterine contractility and quiescence.

Grant Funding Source: Supported by Canadian Institute of Health Research and Alberta Innovates‐Health Solutions
Differential expression of fibroblast growth factors and their receptors in hair follicles of fine‐wool sheep (912.10)
Authors: Liu Lumei , Liu Nan , Harrison Scott , Han Jian
Published: June 2021

Fibroblast growth factors (FGFs) and their receptors (FGFRs) constitute signaling pathways for many developmental and repair processes in mammalian tissues, including those of keratinocytes in hair follicles. FGF family members are encoded by 22 genes and regulate the proliferation of keratinocytes, but the specific genes of this family affecting the overall growth cycle have not been identified. The objective of this study is to screen for FGFs in hair follicles of fine‐wool sheep by microarray and proteomic assays. Hair follicles were sampled in triplicate from neck and groin regions during anagen and telogen growth phases. These regional and growth phase comparisons were examined with gene expression microarrays, and then screened further by two‐dimensional gel electrophoresis and MALDI‐TOF/TOF mass spectrometry. Results showed that the FGF10, FGF18 and FGFR3 genes differ in expression across both the regional and growth phase comparisons. Each of these three genes was then characterized by multiple sequence alignment where sets of orthologous genes across eight species with hair follicle development were compared to a distal species without hair follicle development. Structural gene regions associating with hair follicle development were identified. This enables future studies to be conducted for validating molecular function of these gene regions in hair follicle development.
Regulation of renal Cry2 expression by the circadian clock protein Per1 (912.12)
Authors: All Sean , Richards Jacob , Gumz Michelle
Published: June 2021

Increasing evidence has demonstrated that the circadian clock plays a critical role in the regulation of renal function and blood pressure. We have shown that the circadian protein Per1 regulates expression of several genes involved in the regulation of renal sodium reabsorption. We have previously demonstrated that Per1 may act through repression of the canonical circadian repressor Cry2, preventing its repression of Per1 target genes (Richards et al. AJP Reg 2013). In this study, we show that Per1 knockdown in vitro or in vivo results in increased levels of Cry2. Recent work by our lab has also demonstrated that reduced levels of Per1 in vivo is associated with decreased levels of plasma aldosterone and increased levels of Cry2 in the adrenal gland (Richards et al. AJP Renal 2013). In order to enter the nucleus, Per1 must be phosphorylated by casein kinase 1 δ/ε (CK1δ/ε). Our previous work has demonstrated that CK1δ/ε inhibition in vitro recapitulates the effect of Per1 knockdown, including up regulation of Cry2 (Richards et al. AJP Renal 2012). However, the effect of in vivo CK1δ/ε inhibition on Cry2 has not been tested. Wild type 129/sv mice were injected with vehicle or the CK1δ/ε inhibitor, PF670462. Mice were euthanized at midnight and kidneys were harvested and dissected into renal cortex and inner medulla. RNA and nuclear protein were isolated. Western blot analysis confirmed that CK1δ/ε inhibition resulted in decreased nuclear Per1 levels. QPCR demonstrated, for the first time, increased Cry2 mRNA in both renal cortex and inner medulla in CK1δ/ε inhibitor treated animals versus vehicle treated animals. These results are consistent with our previous in vitro data in which Per1 represses Cry2.

Grant Funding Source: Supported by NIH, American Heart Association, and American Society of Nephrology
Assessing nuclear receptor isoforms using a standardized one‐step real time protocol (912.4)
Authors: Sutherland Catherine , Bistulfi Gaia
Published: June 2021

Nuclear receptors (NRs) are transcription factors able to regulate transcription based on environmental cues. They are fundamental for development and differentiation, and impaired NR function is associated with cancer and developmental diseases. There are more than fifty known NR families, each one comprising several genes encoding different isoforms. The relevance of NRs’ isoforms is unknown, but it is likely that they play a major role in modulating each other’s activity. The objective of this project was to develop an affordable, efficient, reliable method to quantify NR genes’ isoforms. We used one‐step real time PCR, which is more sensitive than the traditional two steps protocol, where retrotranscription of RNA into cDNA precedes real time analysis. To allow for absolute quantification of gene isoforms we recurred to a cRNA standard curve method, which accounts for different primer sets’ efficiencies. This method allowed us to compare expression levels of different amplicons specific to gene isoforms. Our results show that the method was quick, reliable and efficient in determining isoforms copy number, opening the way to studies focused at clarifying NRs’ isoforms function.
Genes revealed by Bayesian graphical model analysis in pathways mediating hypertension are differentially expressed in the renal medulla of Dahl salt‐sensitive rats (912.6)
Authors: Evans Louise , Peterson Christine , Stingo Francesco , Woo Ahn Kwang , Liu Pengyuan , Vannucci Marina , Laud Purushottam , Yang Chun , Liang Mingyu , Cowley Allen
Published: June 2021

A 1.37Mbp region of the Brown Norway rat Chr13 confers protection from salt‐sensitivity to Dahl salt‐sensitive (SS) rats. Of five candidate genes identified in this region, only Rfwd2 was highly expressed in the renal medulla. A pathway‐based Bayesian graphical model analysis utilizing RNA‐seq and GeneChip data was used to identify relationships between Rfwd2 and genes in pathways related to blood pressure. GeneChip data was used to assess whether the genes with a high marginal posterior probability of interdependence with Rfwd2 were differentially expressed in the renal medulla of SS vs. salt‐resistant congenic SS.13BN26 rats fed 0.4% (LS) or 4% NaCl (HS) diet for 7‐days. Of the 9 genes with the highest absolute partial correlations with Rfwd2, 4 were quantifiable by GeneChip analysis and significantly different (P<0.05) between SS and SS.13BN26 rats: F8 (Factor VIII) (lower in SS on LS/HS); Csnk1d (Casein kinase 1 delta) (higher in SS rats on LS/HS); Pik3ca (Phosphatidylinositol‐4,5‐bisphosphate 3‐kinase) (lower in SS rats on LS, suppressed in both on HS diet); Acly (ATP citrate lyase) (increased with HS in both). These analyses have allowed the unbiased identification of genes associated with our candidate gene providing new insights into salt‐sensitivity.
Investigation on the metastatic changes to the liver as a consequence of metastatic breast cancer: one cadaver’s story (913.10)
Authors: Gallegos Christian , Nusse Gloria
Published: June 2021

Every semester we receive an average of 4 cadavers with a different cause of death for each one. Cadaver case number UCSF‐13‐237 had metastatic breast cancer as her cause of death meaning there was more going on inside of her than what was explicitly described on the cause of death report. Once the abdominopelvic and thoracic cavities were opened, the liver was easily identified that covered 1/3 of the abdominal cavity. The comparison between a normal liver embalmed to the secondary liver cancer from case number UCSF‐13‐237 will be used to deduce the weight difference, the parameters of the liver and the gross visual anatomy of the changes in the blood vessels. The comparison of the two livers will show the before and after images of the affects secondary liver cancer has on the liver.
A deep fascia exists in the midface: the missing plane coming from the temporal region (913.4)
Authors: Andretto Amodeo Chiara , Innocenti Alessandro , Casasco Andrea , Barker Daniel , Keller Gregory
Published: June 2021

Objective of the study: to demonstrate that the deep temporal fascia is connected to the muscles of the infraorbital prezygomatic area and that a deep fascial layer exists in the midface.

Methods: 24 hemifaces of 14 caucasian cadavers were dissected. Skin was incised over the temporal and prezygomatic area. The temporoparietal fascia and the Smas layer were dissected and reflected. The superficial sheet of the deep temporal fascia was incised and elevated.

In 4 specimens full thickness biopsies were harvested at the level of the zygomatic bones, in the area between the temporal region and the prezygomatic region and observed after cryopreservation.

Results: In 12 specimens dissections showed a continuous layer of fascia coming from the temporal area: the superficial sheet of the deep temporal fascia reached directly the prezygomatic area. In 16 specimens the superficial sheet of the deep temporal fascia inserted at the level of the zygomatic as classically described and continued, after inserting, over the prezygomatic area.

In all specimens this deep fascial layer devided the fat deep to the orbicularis oculi muscle into 2 layers (suborbicularis oculi and preperiosteal fat) and the deepest one was on the same plane of the temporal fat pad.

Histologic examination confirmed the surgical findings: a continuous layer coming from the temporal region reaches the prezygomatic area and devides the fat deep to the orbicularis oculi muscle into 2 layers.

Conclusions: A deep fascial plane connected to the superficial sheet of the deep temporal fascia exists in the midface and devides the fat deep to the orbicularis oculi muscle into 2 layers. It can either continue directly from the temporal area or insert over the zygomatic bone and then reach the prezygomatic area. The temporal fat pad situated between the superficial and the deep temporal sheet can be compared to the deep fat layer below the orbicularis oculi muscle.
Experimental model of Achilles tendon injury in rats (914.10)
Authors: Akamatsu Flávia , Teodoro Walcy , Saleh Samir , Silva Alexandre , Hojaij Flávio , Martinez Carlos , Andrade Mauro , Jacomo Alfredo
Published: June 2021

Background: Injuries to tendons are among the most common injuries to the body. To determine whether a proposed animal tendon injury model is appropriate, some conditions must be met. First, the correct anatomic structure must be modeled and the desired pathologic condition must be simulated.

Purpose: To describe an experimental model to study Achilles tendon healing.

Methods: Forty male Rattus norvegicus albinus, Wistar lineage adult male weighing 250 to 300 g were used for this experiment and thirty were surgically submitted to bilateral partial transverse section of the Achilles tendon. The right tendon was treated with RF whereas the left tendon served as control. On the third postoperative day, the rats were divided into four experimental groups consisting of ten rats each which were treated with monopolar RF (Tonederm™) adjusted to 650 kHz and 2w, for two minutes twice a week and a group of normal animals without any intervention, until they were sacrificed on the 7th, 14th and 28th days, respectively. Tendons were weighed and collagen quantification was evaluated by hidroxyprolin content.

Results: Significant reduction in collagen content on day 7, 14 and 28 was related to control experiment to normal tendon (7 days, p<0.01; 14 e 28 days, p<0.05).

Conclusion: The experimental model has been effective and available to be used to study Achilles tendon healing.