bluuebunny/crossref_metadata_2025 huggingface dataset:
Beta‐adrenergic receptor stimulation enhances phosphorylation of NKCC2 at threonine96/101 via cAMP but independently of PKA (892.21)
Authors: Haque Mohammed , Ares Gustavo , Ortiz Pablo
Published: June 2021

Beta‐adrenergic receptor (β‐AR) stimulation enhances phosphorylation of NKCC2 at threonine96/101 via cAMP but independently of PKA Mohammed Z. Haque, Gustavo R. Ares and Pablo A. Ortiz The thick ascending limb (TAL) reabsorbs 30% of the filtered sodium via the apical Na‐K‐2Cl cotransporter NKCC2. NKCC2 activity and trafficking to the apical surface are enhanced by β‐AR stimulation via cAMP. NKCC2 activity may also be stimulated by phosphorylation at threonine96/101 (Thr96/101) by SPAK/OSR1 kinases and serine126 (Ser126) by PKA. However, it is not known whether SPAK and NKCC2 phosphorylation (P‐NKCC2) at Thr96/101 are enhanced by cAMP and stimulation of β‐ARs in the TAL. We hypothesized that β‐AR stimulation increases Thr96/101 P‐NKCC2 via cAMP and SPAK. To maximally stimulate β‐ARs signaling we treated TALs (20 min) with 1µM isoproterenol and the phosphodiesterase4 inhibitor rolipram (ISO). ISO enhanced Thr96/101 and Ser126 P‐NKCC2 by 79±28% and 71±28% (p<0.05, n=6). ISO also enhanced phosphorylated SPAK at Ser373 by 50±4 % (p<0.05, n=6). Inhibition of PKA (H‐89,10 µM) did not inhibit β‐AR stimulated Thr96/101 P‐NKCC2. We then tested whether cAMP enhanced Thr96/101 P‐NKCC2 by stimulating endogenous cAMP with forskolin and the phosphodiesterase inhibitor IBMX (20 min). cAMP increased Thr96/101 by 30± 8% (p<0.05, n=4) and this was not blocked by PKA inhibition; whereas Ser126 P‐NKCC2 was enhanced by 32±6 fold (p<0.05) and PKA inhibition blocked this effect by 84±4%. We conclude that β‐adrenergic receptor stimulation enhances NKCC2 phosphorylation at Thr96/101 via cAMP and SPAK/OSR1 whereas PKA mediates the effect of cAMP on Ser126.

Grant Funding Source: AHA/NIH
EGF/RhoGDIα/Rac1 pathway modulates ENaC activity and contributes to the development of salt‐sensitive hypertension (892.27)
Authors: Pavlov Tengis , Levchenko Vladislav , Ilatovskaya Daria , Palygin Oleg , Staruschenko Alexander
Published: June 2021

Epithelial sodium channel (ENaC) performs sodium reabsorption in the aldosterone sensitive distal nephron. Amongst other hormonal agents, ENaC‐mediated sodium reabsorption is regulated by the members of epidermal growth factor (EGF) family; specifically, chronic treatment with the EGF‐family members downregulates ENaC activity in vitro. We have recently shown that EGF deficiency is involved in improper ENaC activity and contributes to the development of salt‐induced hypertension in Dahl salt‐sensitive (SS) rats. Investigation of signaling pathways potentially involved in regulation of ENaC by EGF, revealed that high salt diet in SS rats decreases abundance of Rho GDP‐dissociation inhibitor 1(RhoGDIα), a well characterized modulator of Rho‐family small GTPases. RhoGDIα deficiency in cultured mCCDcl1 principal cell line increases ENaC‐mediated current formation and causes enhanced response of ENaC activity to EGF treatment. Further experiments revealed that these effects are mediated via Rac1 small GTPase. We conclude that renal EGF deficiency in the SS rats on a high salt diet provokes excessive ENaC activity via RhoGDIα/Rac1pathway and contributes to the development of salt‐induced hypertension.

Grant Funding Source: Supported by AHA, NHLBI
Electrical stimulation of the renal sympathetic nerve increases NHE3‐mediated sodium reabsorption via angiotensin II AT1 receptor (892.29)
Authors: Pontes Roberto , Crajoinas Renato , Veiga Gláucia , Girardi Adriana , Campos Ruy , Bergamaschi Cássia
Published: June 2021

The role of renal sympathetic nerve activity on sodium and water handling by the kidneys has not been fully investigated. This study aimed to evaluate the effect of acute sympathetic renal nerve electrical stimulation (RNES) on proximal tubule NHE3 regulation and the possible role of Ang II in mediating this effect. To this end, male Wistar rats (250g‐300g) were uninephrectomized and one week later were divided in three independent groups: sham (renal sympathetic nerve activity registered for 1h); RNES for 1h, (15V, 1.5Hz and 0.5 ms) and RNES treated with Losartan (10 mg/kg, i.v.). Electrical stimulation produced reduction in the levels of NHE3 phosphorylation at the residue serine 552 (100±5% vs. 66±3%), increased activity of renal cortical PKA, and a reduction in urinary output (0.0235±0.006 vs 0.007±0.002 uL/min/Kg) and urinary sodium excretion (1.48±0.2 vs. 0.58±0.1 uEq/min/kg). These anti‐natriuretic effects were completely prevented by injection of Losartan. Taken together, our findings suggest that RNES increases NHE3‐mediated sodium reabsorption and that these effects are mostly, if not all, dependent on Ang II.
Signaling mechanisms for post‐transcriptional regulation of the type IIa sodium‐phosphate cotransporter mRNA by parathyroid hormone (892.35)
Authors: Murray Rebecca , Khundmiri Syed , Clark Barbara , Lederer Eleanor
Published: June 2021

PTH is a critical regulator of serum phosphorus. We have previously shown that PTH decreases proximal tubule NpT2a mRNA expression through post‐transcriptional mechanisms, and that this effect can be reproduced by treatment with 8‐bromo‐cAMP, a PKA activator, but not phorbol myristate acetate, a PKC activator. We hypothesize that PKA is the primary mediator of NpT2a mRNA destabilization by PTH. To determine the contribution of each pathway to the PTH response, opossum kidney (OK) cells were treated with selective protein kinase inhibitors in the presence of PTH, and NpT2a mRNA expression was measured by qRT‐PCR. Pretreatment with 10µM PKC inhibitor peptide (PKCi) followed by 100nM PTH for 6h produced a 43.9% decrease in NpT2a mRNA versus PKCi alone. Pretreatment with 10µM H‐89, a PKA inhibitor, prevented the initial decrease in NpT2a mRNA by PTH but did not prevent the ultimate reduction. 8CPT‐2Me‐cAMP, a selective activator of Epac, failed to produce a decrease in NpT2a mRNA after 6h. We conclude that the initial effect of PTH on NpT2a mRNA is PKA‐dependent, whereas chronic regulation involves both the PKA and PKC pathways.

Grant Funding Source: Support for this project is provided by VA to EDL.
Renal phenotype of Sortilin‐deficient mice (892.41)
Authors: Bikulowa Kamila , Borschewksi Aljona , Dathe Christin , Willnow Thomas E. , Bachmann Sebastian , Mutig Kerim
Published: June 2021

Sortilin belongs to the family of the VPS10P‐domain receptors which mediate intracellular sorting of diverse ligands in various cell types. Sortilin is abundantly expressed in the kidney but little is known about its renal function. In this study we take advantage of sortilin‐deficient (Sort‐/‐) mice to test whether sortilin interferes with renal NaCl‐ or water reabsorption.

To evaluate the role of sortilin in the kidney, sortilin‐deficient (Sort‐/‐) mice were analyzed for their physiologic kidney performance, kidney morphology, and biochemical profile of renal transporters.

In wildtype (WT) mouse kidneys, sortilin has shown moderate expression in the distal nephron and strong expression in the principal cells of collecting duct. Sortilin deficiency had no major effects on kidney morphology. Physiologic evaluation of WT and Sort‐/‐ mice showed significantly increased urinary volume (+154%) upon sortilin disruption. Immunoblotting evaluation of distal epithelial salt transporters and water channels revealed that sortilin‐deficiency was associated with decreased levels of the Na‐Cl cotransporter (NCC) and of the phosphorylated, activated aquaporin 2.

Our results suggest that sortilin facilitates the reabsorptive function of renal distal epithelia.
Distinct mechanisms of [Ca2+]i responses to flow and hypotonicity in the distal nephron (892.5)
Authors: Mamenko Mykola , Zaika Oleg , Boukelmoune Nabila , O'Neil Roger , Pochynyuk Oleh
Published: June 2021

Changes in luminal fluid flow or osmolality elicit [Ca2+]i elevations in the distal nephron (DN) cells to regulate water‐electrolyte transport. Our group has recently identified a pivotal role of TRPV4 in mediating flow‐induced Ca2+ elevations in the DN cells, however little is known about molecular nature of the response to osmotic gradients. Here, using ratiometric Fura‐2 Ca2+ imaging in freshly isolated split‐opened DNs from wild‐type (WT) mice, we found that hypotonicity elicits a uniform and reproducible increase of [Ca2+]i in DN cells. This response is abolished by removal of extracellular Ca2+, but only moderately diminished upon depletion of intracellular Ca2+ stores. While genetic ablation of TRPV4 abrogates flow‐induced [Ca2+]i elevations, the response to hypotonicity, though significantly blunted, is retained in TRPV4‐/‐ mice. This points to additional mechanisms increasing [Ca2+]i in response to osmotic gradients. Indeed, pharmacological inhibition of Ca2+‐permeable TRPC3 channel markedly diminishes the amplitude of [Ca2+]i elevations induced by hypotonic stimuli in DN epithelium from WT mice. Moreover, the threshold of the response to decreased osmolality is elevated in TRPC3 knockouts. Thus, in contrast to flow‐induced [Ca2+]i elevations mediated by TRPV4, hypotonicity elicits a multifaceted Ca2+ response, at least in part due to coordinated activation of TRPV4 and TRPC3.

Grant Funding Source: DK095029
Modulation of sodium channels by purine nucleotides in mouse vomeronasal sensory neurons (893.1)
Authors: Vick Jonathan , Rickner Hannah , Delay Rona
Published: June 2021

The mouse vomeronasal organ (VNO) is a chemosensory structure that regulates many social and fear related behaviors. Social odorants, colloquially known as pheromones, are detected within the lumen of the VNO by bipolar vomeronasal sensory neuron (VSN)s. We have previously shown that adenosine 5’‐triphosphate (ATP), a potent neuromodulator in chemosensory systems, elicits an inward current and action potentials in VSNs through activation of ionotropic P2X receptors. However, it was unclear if G‐protein coupled P2Y receptors were involved in modulating the excitability of VSNs. Using RT‐PCR, we showed that P2Y1, 2, and 6 were expressed in the VNO. Immunohistochemical labeling with antibodies specific to P2Y1 and 2 revealed widespread expression of both receptors in the sensory and nonsensory epithelia. Using whole cell patch clamp, we observed that while ATP was without effect, adenosine 5’‐diphosphate (ADP) hyperpolarized the voltage dependence of sodium channel steady state inactivation in a subset of VSNs. Moreover, neither ATP nor ADP had any effect on the voltage dependence of sodium channel activation or fast inactivation. We hypothesize that following activation of P2X receptors by ATP, the rapid breakdown of ATP to ADP by ubiquitous ecto‐endonucleases would result in a reduction of sodium channel availability through activation of P2Y receptors.

Grant Funding Source: NIH
Tumour necrosis factor alpha and interferon gamma induce transcriptional downregulation of aquaporin 3 RNA expression through distinct mechanisms (893.28)
Authors: Peplowski Michael , Vegso Andrew , MacNaughton Wallace
Published: June 2021

Although aquaporins (AQP) are known to be involved in water movement, the role of AQP3 in the barrier dysfunction that characterizes inflammatory bowel diseases remains unknown. We hypothesized that TNFα and IFNγ were involved in the transcriptional inhibition of AQP3 RNA expression in intestinal inflammation. Methods: AQP3 expression was assessed in C57Bl/6 mice administered dextran sodium sulfate (DSS) to induce colonic inflammation. In vitro, the human adenocarcinoma cell line HT29 was treated with either TNFα or IFNγ and AQP3 pre‐mRNA and mRNA expression were assessed by real‐time RT‐PCR. Cytokine‐responsive transcriptional elements were elucidated using AQP3 promoter driven firefly luciferase expression.

Results: AQP3 was decreased early in DSS colitis, with diminished basolateral membrane staining in epithelial cells lining colonic crypts. Similarly, TNFα or IFNγ‐treated HT29 cells had decreased AQP3 pre‐mRNA expression at 2 hr, followed by decreased mRNA expression at 6‐12 hr. IFNγ‐induced transcriptional inhibition of AQP3 expression was reversed using a broad‐spectrum JAK inhibitor (JAK Inhibitor I, 10 µM), but not a JAK2 specific inhibitor (JAK2 Inhibitor II, 10 µM). Truncated AQP3 promoter constructs revealed an IFNγ‐responsive transcriptional element in the 251bp region upstream of the TATA box and a transcriptional suppressor in the 251 ‐ 666bp region upstream of the TATA box. In contrast, TNFα‐induced transcriptional inhibition of AQP3 expression was not reversed by inhibitors of the PI3K, AKT, NF‐κB, ERK/MAPK and p38 MAPK pathways (LY294002, 10 µM; Triciribine, 1 µM; BAY11‐7082, 30 µM; U0126, 10 µM and SB203580, 10 µM respectively).

Grant Funding Source: CCFC, CIHR & AIHS
Cleaved prolactin increases activity of amiloride‐sensitive sodium channels in mouse collecting duct principal cells (893.3)
Authors: Greenlee Megan , Chen Rose , Mitzelfelt Jeremiah , Eaton Douglas
Published: June 2021

In the renal collecting duct, sodium reabsorption through the epithelial sodium channel (ENaC) creates an osmotic driving force for water reabsorption resulting in increased blood volume and pressure. Investigators have previously shown that prolactin (PRL) stimulated ENaC activity in bullfrog skin cells. However, studies in mammalian systems show no effect of PRL on ENaC function. PRL is present in two forms: a 23kDa full length form (PRL) and a 14‐16kDa cleaved form (PRL‐C). We hypothesized that these two PRL molecules may exert different effects on ENaC activity. To investigate this hypothesis, we used a cell culture model of principal cells from the cortical collecting duct (mpkCCD) which express ENaC subunits and exhibit amiloride‐sensitive current. We measured transepithelial voltage and resistance across cell monolayers before and after basolateral treatment with vehicle (water) or 1‐2 ug/mL of PRL or PRL‐C and calculated transepithelial current using Ohm’s law. We found that PRL produced only a small, variable effect on ENaC activity in mpkCCD cells. However, PRL‐C approximately doubled the amiloride‐sensitive current in these cells within 24 hrs. Compared to kidney lysates, mpkCCD cells exhibit a low abundance of the PRL receptor which may explain the need for large PRL concentrations in these cells. Our results indicate that cleaved PRL stimulates ENaC activity in mpkCCD cells. In future studies, we will examine the effect of PRL and PRL‐C on single channel properties of ENaC in isolated collecting ducts and the effect of these hormones on blood pressure in vivo.

Grant Funding Source: Supported by NIH K12GM000680 and T32HL076118
Physiology and cell biology of clinical mutants of the human riboflavin transporter‐2: studies utilizing the human‐derived brain U87 cells (896.2)
Authors: Subramanian Veedamali , Kapadia Rubina , Ghosal Abhisek , Said Hamid
Published: June 2021

Riboflavin (RF) is essential for normal cellular functions of all cells including those of the brain. RF is transported into human cells via three membrane transporters, hRFVT‐1,‐2 and ‐3. Mutation in hRFVT‐2 (which is predominantly expressed in brain cells) is believed to be the cause of the rare neurodegenerative disorder, Brown‐Vialetto‐Van Laere Syndrome (BVVLS). Little,however, is known about how such mutations affect the physiology and cell biology of hRFVT‐2. We addressed this issue using the human‐derived brain U87cells as model and live cell confocal imaging.

Our results showed marked inhibition in RF uptake in U87 cells stably expressing clinical mutants L123P, L140P, G306R and L339P. Live cell confocal imaging of U87 cells stably expressing hRFVT‐2 clinical mutants showed the L123P and L339P mutants were retained within the endoplasmic reticulum, while the L140P and G306R mutants were expressed at the cell membrane though at reduced protein expression levels compared to wild‐type construct. These results show that some of the BVVLS associated mutations in hRFVT‐2 affect the transporter functionality and that this effect is mediated via alterations in membrane targeting and/or protein expression. (Supported by DVA and NIH grant DK 56061‐15).

Grant Funding Source: DVA and NIH
Characterization of a human UT‐B urea transporter antibody (897.3)
Authors: Walpole Caragh , Winter Desmond , Baird Alan , Stewart Gavin
Published: June 2021

Urea entry into the gastrointestinal tract is facilitated by the urea transporter UT‐B1 (Stewart 2011). This process is crucial to our intestinal wellbeing as it is the first step in the urea nitrogen salvaging mechanism, which is necessary for maintaining healthy gut bacterial populations. In a previous study using a bovine UT‐B antibody, a 35kDa human UT‐B1 protein was found to be highly expressed in the ascending colon (Collins et al., 2010). The aim of this study was to produce a reliable human UT‐B antibody to be used for further investigations of human UT‐B expression. Initial PCR analysis using primers designed along the UT‐B gene confirmed UT‐B1 as the major isoform expressed at the RNA level in all human gastrointestinal tissues. Three polyclonal human UT‐B antibodies ‐ one designed against the N‐terminal (UT‐B#N) and two against the C‐terminal (UT‐B#C and UT‐Bc19) ‐ were made according to the UT‐B1 amino acid sequence and extensive characterization was carried out using Western blot analysis. While both C‐terminal antibodies detected UT‐B1 in the membrane of erythrocytes as a large glycosylated signal at 45‐60kDa as expected, the UT‐B#N antibody failed to do so and was rejected. As UT‐B transporters are glycoproteins, deglycosylation experiments were performed on UT‐B1 protein using PNGaseF enzyme. While UT‐Bc19 successfully detected 36 kDa deglycosylated UT‐B1, UT‐B#C failed to do so and was also rejected. Next, β‐mercaptoethanol was used to reduce UT‐B1 from its native multimer to a monomer and UT‐Bc19 successfully detected a big shift from a smeared 40‐100kDa signal to a neat 40kDa signal. Finally, UT‐Bc19 also detected a strong 40‐70kDa glycosylated UT‐B1 signal in human ileum protein. In conclusion, these data have shown that UT‐Bc19 is the only consistent human UT‐B antibody. Future investigations of UT‐B1 expression will therefore utilise UT‐Bc19 with confidence and help establish the physiological significance of UT‐B1 in the human gastrointestinal tract.

Grant Funding Source: Supported by The Wellcome Trust and Science Foundation Ireland.
NHE3 and Clostridium difficile infection (897.5)
Authors: Engevik Melinda , Yacyshyn Mary , Yacyshyn Bruce , Haslam David , Shull Gary , Worrell Roger
Published: June 2021

Although changes in the intestinal microbiota have been linked to diabetes, obesity, Inflammatory Bowel Disease, and Clostridium difficile‐associated disease, it remains unclear how the intestinal environment, set by ion transport, affects luminal and mucosa‐associated bacterial composition. NHE3 plays an integral role in intestinal Na+ absorption, as indicated by chronic diarrhea in NHE3‐/‐ mice. In a renal cell lines C. difficile toxin B causes internalization of NHE3. We hypothesize that inhibition of NHE3, by genetic knockout or C. difficile toxin, promotes the proliferation of bacteria capable of growing in a high Na+, alkaline pH environment, altering the gut microbiota composition, which may enhance the C. difficile niche. qPCR was performed on 16S DNA extracted from NHE3‐/‐ mouse intestine and human CDI stool. Flame photometry, chloridometry and pH probe was used to measure ion composition and pH. Immunohistochemistry was used to analyze NHE3 localization and mucus oligosaccharide composition. Similar to distal colon samples from NHE3‐/‐ mice, fecal samples from humans with C. difficile infection was associated with decreased Firmicutes and increased Bacteroidetes. Fecal [Na+] was also similarly elevated being consistent with NHE3 inhibition in human CDI. Indeed, NHE3 appeared to be down regulated in biopsy’s from C. difficile infected patients, which likely contributes significantly to diarrhea. These changes also correlated with changes in host mucus oligosaccharide composition. Together these data demonstrate that loss of functional NHE3 occurs in CDI and the resulting alteration in the intestinal environment, may contribute to region‐specific changes in bacteria which serve to favor C. difficile niche development and maintenance.

Grant Funding Source: Supported by NIH DK079979 to RTW
Optimization of treatment of Hp‐associated ulcers (898.2)
Authors: Omarov T. , Abduldayeva A.
Published: June 2021

H.pylori eradication of existing treatment regimens do not lead to absolute efficiency. The purpose of the study. To study the efficacy of probiotics in the treatment of gastroduodenal ulcer.

Materials. We observed 40 patients with gastroduodenal ulcer in the acute phase. Towards to integrated eradication therapy fertal to 1 capsule 2 times a day was added and bifiform in 2 capsules 3 times a day for a month.

Results. In the period of acute ulcer decrease in the total number of E.coli was observed with normal enzymatic activity at 66.6%蠄107 CFU, Bifidobacterium Lactobacterbum and in 90.5% and 76.2% CFU蠄108 and 106, respectively. Increasing the number of E. coli with reduced enzyme activity in 47.6%蠅107 cfu, pathogenic Citrobacter 85.7%蠅106 cfu, fungi of the genus Candida in 42.8% of 蠅105 cfu .

After treatment, scarring and ulceration H.pylori eradication was observed in 100% of patients , a normal level and Bifidobacterium Lactobacterbum at 100% at a concentration of蠅108 CFU , respectively, 106, E. coli with normal enzyme activity to 90% at a concentration of CFU蠅107i Citrobacter genus Candida fungi and 80% at a concentration of蠄105 CFU and 蠄 104, respectively.

Conclusions: Probiotics in treatment of gastroduodenal ulcer increase Helicobacter pylori eradication rates, early relief of pain and dyspeptic syndromes, recovery of intestinal microflora and prevent the development of antibiotic‐associated diarrhea.
IKKβ deletion in intestinal macrophages but not intestinal epithelial cells ameliorates intestinal injury in a neonatal mouse NEC model (902.7)
Authors: De Plaen Isabelle , Liu Shirley , Managlia Elizabeth , Tian Runlan
Published: June 2021

Necrotizing enterocolitis (NEC) is thought to be due to an exaggerated inflammatory response in the premature intestine. We previously found that nuclear factor‐κB (NF‐κB) mediates the intestinal injury in experimental NEC. However, the cell‐types in which NF‐κB activation is critical are unknown. Here, we examine the respective role of epithelial cell and macrophage NF‐κB activation in a neonatal mouse NEC model, using mice lacking IKKβ, the upstream kinase responsible for NF‐κB activation in these cell‐types. Lys‐Cre+/‐ IKKβF/F, Villin‐Cre+/‐ IKKβF/F and IKKβF/F mice were exposed to a NEC protocol consisting in adult commensal bacteria inoculation/ hypoxia/cold stress/formula feeding. Pups were closely observed and euthanized at 72 hours of age or when showing signs of distress and their intestine examined histologically. Cell‐specific deletion of IKKβ was confirmed in the neonatal pups. The incidence of severe NEC (score 蠅2) was significantly decreased in Lys‐Cre+/‐ IKKβF/F pups compared to IKK βF/F controls (3/23 vs 16/23, χ2=12.9, p<0.001). However, there were no difference in the incidence of severe NEC between Villin‐Cre+/‐ IKKβF/F and IKK βF/F controls (13/28 vs 19/34, χ2=0.23, NS). Our data suggest that NF‐κB in macrophages but not intestinal epithelial cells mediates the intestinal injury in our mouse model of NEC.

Grant Funding Source: NIH (R01‐HD060876)
GPR43 activation with COX inhibition induces duodenal injury via 5‐HT pathway (905.1)
Authors: Akiba Yasutada , Kaji Izumi , Kaunitz Jonathan
Published: June 2021

The reason why NSAIDs injure the small intestinal mucosa, but spare the duodenal mucosa is unknown. The short‐chain fatty acid receptor GRP43 is expressed in 5‐HT containing enterochromaffin cells, and a selective GPR43 agonist phenylacetamide‐1 (PA1) increases duodenal HCO3‐ secretion (DBS) via 5HT4 receptor activation. We hypothesized that GPR43 activation modulates duodenal mucosal defenses via 5‐HT release in an NSAID‐induced intestinal injury model.

We measured DBS with pH and CO2 electrodes in vivo through a duodenal loop, perfused with PA1 (0.1 ‐ 10 µM) while measuring released 5‐HT in the portal vein (PV). Intestinal injury was induced by indomethacin (IND, 10 mg/kg, sc) with or without PA1 (0.1 ‐ 1 mg/kg, ig), the 5HT3 antagonist ondansetron (Ond, 3 mg/kg, ip), omeprazole (OPZ, 10 mg/kg, ig), or atropine (10 mg/kg, ig).

Luminal perfusion with PA1 dose‐dependently increased DBS accompanied by 5‐HT release into PV. IND induced small intestinal ulcers with duodenal sparing. PA1 + IND dose‐dependently induced duodenal lesions, whereas PA1 alone had no effect. PA1 + IND‐induced duodenal lesions were inhibited by Ond or OPZ, but not by atropine. Non‐duodenal intestinal lesions were reduced by Ond or atropine, but not by OPZ as previously reported.

These results suggest that although GPR43 activation enhances mucosal defenses by increased DBS via 5HT4 activation, GPR43 activation combined with COX inhibition may increase the vulnerability of the duodenal mucosa to gastric acid via 5HT3 activation.

Grant Funding Source: Supported by VA Merit Review, NIH R01 DK54221
Modeling the transcriptomics response of the late‐gestation fetal hypothalamus to estradiol and brachiocephalic occlusion (910.10)
Authors: Urbano Miguel , Chang Eileen , Rabaglino Maria , Richards Elaine , Wood Charles
Published: June 2021

Estradiol (E2) is a well‐known modulator of fetal neuroendocrine activity, and has been proposed as a critical endocrine signal readying the fetus for birth and postnatal life. To investigate the modulatory role of E2 on fetal stress responsiveness and the response of the fetal brain to asphyxic stress, we subjected chronically catheterized fetal sheep to a transient (10 min) brachiocephalic occlusion or sham occlusion. Half of the fetuses received subcutaneous pellets that released E2 (~250 μg/day, increasing plasma E2 from 62±6 to 102±12 pg/mL). Hypothalamic mRNA was analyzed using the Agilent 8x15k ovine array (019921), processed and annotated as previously reported by our laboratory. Data were analyzed by ANOVA, using JMP Genomics (SAS Institute), revealing that E2 differentially regulated (DR) 561 genes, BCO DR 894 genes and estradiol+BCO DR 1153 genes compared to BCO alone (all p<0.05). E2 upregulated epigenetic pathways and downregulated local steroid biosynthesis, but did not significantly involve genes known to directly respond to the estrogen receptor (contain ERE sequences). BCO upregulated kinase pathways as well as genes associated with lymphocyte infiltration into the brain, and downregulated neuropeptide synthesis. E2 upregulated immune‐ and apopotosis‐ related pathways after BCO, and reduced kinase and epigenetic pathway responses to the BCO. Array results are consistent with the results of more limited hypotheses previously published from these experiments and real‐time PCR validation of neuropeptide and immune‐related genes (CRH, CRHBP, POMC, AGRP, IL1β, TNFα) confirmed the array results. We conclude that increased E2 plasma concentrations invoke epigenetic pathways and appear to exacerbate brain inflammation after BCO. Actions of E2 in the fetal brain might not be dependent on ERα‐mediated gene transcription.

Grant Funding Source: Supported by HD033053 and DK076541
Fine particulate matter in the inspired air within the limits recommended by WHO alters placental structure (910.3)
Authors: Soto Sonia , Lopes Karen , Veras Mariana , Heimann Joel
Published: June 2021

Objective: To evaluate the effects of PM during the perinatal period on placenta.

Methods: Female Wistar rats (7‐9/group) were exposed to filtrated air (F) or PM (P) for 15 days. After mating, rats were divided in 4 groups and again exposed to F or P (FF, FP, PF, PP). At 19th day of pregnancy fetal placentas (fPl), decidua (Dc) and fetus were collected. Several fetal and placental parameters were evaluated. In addition, placental and maternal serum ACE activity was measured.

Results (mean±SEM ‐ p<0.05):

FF
FP
PF
PP

Mass (g)
fPl+Dc
0.63±0.010
0.61±0.009
0.57±0.0141
0.55±0.0071

fPl
0.42±0.006
0.44±0.006
0.40±0.0051,2,4
0.43±0.004

Dc
0.13±0.005
0.14±0.008
0.12±0.0051,2
0.12±0.0071,2

Thickness (mm)
fPl
4.42±0.10
4.16±0.06
3.93±0.091,4
4.40±0.11

Dc
3.15±0.10
2.77±0.091
2.92±0.09
2.95±0.11

Surface area (mm2)
fPl
117.6±2.37
90.20±2.191
101.1±2.971,2
101.7±2.281,2

(LDxTDxÏ€)/4
Dc
32.64±1.46
24.0±0.601
28.4±1.111,2
26.73±1.461

ACE activity
fPl
1.05±0.07
0.99±0.11
1.12±0.10
1.35±0.24

(nmol His‐leu/min/mL)
Dc
0.83±0.07
0.67±0.06
0.59±0.051
0.53±0.051

Serum
33.50±1.39
28.89±1.371
28.90±1.671
26.53±1.061

1vs FF,; 2vs FP; 3vs PF; 4vs PP. TD and LD=transversal and longitudinal diameter.

Conclusions: PM affects placental structure and ACE activity with a possible prejudice of transference of nutrients from mother to fetus.

Grant Funding Source: Supported by FAPESP and CNPq
l‐Leucine partially ameliorates developmental deficits in zebrafish models for Cornelia de Lange syndrome (910.7)
Authors: Xu Baoshan , Sowa Nenja , Gerton Jennifer
Published: June 2021

Cohesinopathies are genetic disorders characterized by defects in fetal growth retardation such as Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS). These diseases are caused by mutations in genes that contribute to the function of the cohesin complex such as NIPBL, Smc1A, Smc3, HDAC8, Rad21 and ESCO2. Although the essential role of the cohesin complex in chromosome segregation has been well documented, it also plays roles in DNA damage repair, chromosome conformation, and gene expression. We recently reported L‐leucine can partially rescue several defects in zebrafish models for RBS by stimulating the TOR (target of rapamycin) pathway. In this study, we investigate the TOR and p53 pathways in zebrafish models for CdLS through morpholino injection and Western blot analysis, and the effect of L‐leucine on the morphants. Our results show partial inhibition of the TOR pathway in NIPBL, Rad21, Smc3‐defective embryos, and p53 signaling was upregulated in Rad21 and Smc3 morphants, but not NIPBL morphants. L‐leucine treatment partially improved TOR activity in CdLS embryos, promoting many aspects of development, and survival. L‐leucine administration partly prevented increased apoptosis in the cohesinopathy embryos. The Rad21 morphant exhibited a significant increase in mitotic cells, which was strongly suppressed by L‐leucine. In summary, our results suggest that translational impairment may contribute to the etiology of CdLS and L‐leucine treatment may provide some benefit.

Grant Funding Source: Supported by Stowers Institute and Cornelia de Lange Syndrome Foundation Research Grants
Effect of testosterone and its antagonists on knee range of motion through RXFP1 and RXFP2 regulation in rat model (911.12)
Authors: Dehghan Firouzeh , Muniandy Sekaran , Yusof Ashril , Salleh Naguib
Published: June 2021

Objective: To elucidate the testosterone hormone influences on knee joint range of motion (ROM), and the suppressory role of its antagonists; flutamide and finasteride on relaxin receptors in the patellar tendon and collateral ligament. Methods: forty‐two ovariectomized females Wistar Kyoto (WKY) rats 8‐10 weeks, 180‐200g of weight were distributed into seven groups. Peanut oil as a control, testosterone hormone (125 & 250 μg/kg), flutamide (10 mg/kg), and finasteride (20 mg/kg) separately in presence of two testosterone doses were injected subcutaneously for three consecutive days. The antagonists were administrated 30 minutes before the testosterone injection. Following the treatment administrations, the relationships of knee ROM and RXFP1/ RXFP2 mRNA and protein in knee tissues were investigated. Results: Our data showed that knee ROM was significantly decreased in the testosterone treated groups and increased in the presence of flutamide and finasteride, independently. These two antagonists had similar effect on RXFP1 and RXFP2 expression in patellar tendon and collateral ligament. However, finasteride and flutamide blocked testosterone in both doses and increased RXFP1 and RXFP2 expression. Conclusion: This study suggests that testosterone hormone is possibly a primary mediator of RXFP1 and RXFP2 receptors to reduce knee ROM. Nonetheless, flutamide and finasteride revealed inhibitory effect on testosterone thus changed the expression of RXFP1 and RXFP2 receptors in patellar tendon and lateral collateral ligament, inequitably.

Grant Funding Source: supported by PPP grant (PV104/2012A)
cAMP control of intracellular calcium dynamics in anterior pituitary cells (911.4)
Authors: Ikenouye Larissa , Angleson Joseph
Published: June 2021

Hormone secretion form anterior pituitary cells is known to be regulated by G protein coupled receptors (GPCRs) that regulate intracellular cAMP. The objective of this study was to determine mechanisms by which elevated cAMP control intracellular calcium. We have used fura‐2 measurements of intracellular calcium concentration along with both population biochemical cAMP measurements and single cell FRET‐based cAMP measurements to investigate the relationship between [Ca2+] and cAMP in clonal rat MMQ lactotrophs. Activation vasoactive intestinal peptide receptors that are coupled to Gs led to Ca2+ influx as evident by increase in Ca2+‐oscillations. Bypass of GPCRs with the adenylyl cylclase activator forskolin also resulted in strong in increase in Ca2+ oscillations consistent with measured forskolin‐induced increase in [cAMP]. Pharmacological activation of PKA using 6‐Bn‐cAMP or activation of EPAC using 8‐cpt‐cAMP each resulted in increase in Ca2+ indicating a role for both cAMP binding proteins in control of calcium dynamics although with differing lag times likely reflecting distinct signaling pathways. The broad‐spectrum cAMP phosphodiesterase (PDE) inhibitor IBMX caused an increase in Ca2+ influx without delay. The PDE3 inhibitor milrinone caused a strong increase in Ca2+ after a delay whereas the PDE4 inhibitor rolipram caused an immediate increase in Ca2+. Roliparam also triggered apparent store release. FRET‐based measurements of [cAMP] are being used to further analyze these differences. These results suggest a possible different relationship between the various PDE sensitive pools of cAMP and Ca2+ ‐channels as well as distinct roles for PKA and EPAC in control of Ca2+ dynamics and secretion.
Citrate synthase gene comparison and use of RAPD genomic fingerprinting to study relatedness among different Aspergillus sp (912.1)
Authors: Mustafa Ghulam , Asgher Muhammad , Rahman Mehboob‐ur‐ , Jamil Amer
Published: June 2021

In the present study, we studied polymorphism in two strains of Aspergillus niger for the expression of citrate synthase (CS) gene. To elucidate the positions of mutations and to study sequence comparison, the CS gene from both wild and mutant strains was cloned and sequenced. On the basis of these partial sequences, a dendogram was made to assess homology among different strains of A. niger. Random amplified polymorphic DNA (RAPD‐PCR) analysis was also employed to study genetic variation between wild and mutant strains of A. niger and in total 31 decamer random primers were surveyed. A mutation was induced in the fungus using ethidium bromide resulted in enhanced expression of citric acid by three folds. RAPD‐PCR was executed which confirmed a significant change in the genetic makeup of mutant strain related with genotypic variability. Out of 31 decamer random primers, 15 showed polymorphism by amplifying polymorphic fragments units of total 98 and only 71.43% similarity was found between both the genomes. Keeping in view the importance of citric acid as a necessary constituent of various food preparations, synthetic biodegradable detergents and pharmaceuticals the enhanced production of citric acid by mutant derivative might provide significant boost in commercial scale viability of this useful product.

Grant Funding Source: Supported by Higher Education Commission, Government of Pakistan
The pathogenetic basis for cell therapy of post‐resuscitation disease, treatment and prevention (912.2)
Authors: Tazhibaeva D.C. , Kabdualieva N.B. , Beglarova G.E. , Ermentaeva L.N. , Baibakova M.K.
Published: June 2021

Aim of this study was to improve the effectiveness of resuscitation and to develop the basis for cell therapy and transplants in experimental studies.Material and methods. We used 357 rats. In total 429 experiments were performed. Clinical death and resuscitation was performed by L.Z. Tell’s technique (1982). Resuscitation procedures included transplantation of isolated cells from liver, spleen and thymus from heterogeneous and genetically identical rats and their fetuses. The complex of biochemical, neurological studies was performed to study the influence of cell transplants on post resuscitation disease natural history.

Results and discussion: General conclusion of our studies is that use of different cell therapy techniques could change reactivity and resistance of resuscitated animals. As it was shown previously, after resuscitation the non‐specific changes of metabolism are started. Cell therapy generally improves these changes. Metabolic processes including all branches and enzymes activity change after cell therapy to more optimal level. Most important factor of cell therapy was brain cognitive function improvement in resuscitated animals who underwent cell transplant in early after resuscitation period. Optimization of rats behavior and reflexes improvement were evident. This effect was especially marked if fetal cells from genetically identical fetuses were used.
Association of GABRG2 rs211037 polymorphism with susceptibility to epilepsy in Asians: a multicentre cohort study and meta‐analysis (912.8)
Authors: Haerian Batoul Sadat , Baum Larry , Kwan patrick , Cherny Stacey , Tan Hui Jan , Raymond Azman Ali , Mohamed Zahurin
Published: June 2021

PURPOSE:The gamma‐aminobutyric acid receptor subunit gamma‐2 (GABRG2) gene encodes GABR‐gamma2 protein, which has been implicated in susceptibility to epilepsy. Several studies have investigated whether the synonymous GABRG2 rs211037 polymorphism is a risk factor for various epilepsy types including febrile seizure (FS), idiopathic generalized epilepsy (IGE) and symptomatic epilepsy (SE), however results have been inconclusive. Therefore, we examined association of this polymorphism in FS, IGE, SE, and cryptogenic epilepsy (CE), through both a multicenter case control study and a meta‐analysis. METHODS: The rs211037 was genotyped in Hong Kong Chinese and in Malaysian Chinese, Indian, and Malay participants. Genotypes of 5101 participants, of which 1769 were from Malaysia and 3332 from Hong Kong, were included in this case‐control study. KEY FINDINGS:Of the 1179 patients, 66%, 13%, and 21% were Chinese, Indians, and Malays, respectively. Of the Chinese patients, 37% were from Malaysia and 63% were from Hong Kong. Significant association was observed between rs211037 polymorphism and susceptibility to SE (T vs. Cp=0.000003 and TT vs. CC, p=0.00001) or to epilepsy with FS (T vs. C p=0.02 and CT vs. CC, p=0.02) in overall Chinese and to IGE in Chinese from Malaysia (TT vs. CC, p=0.01), but not to CE. Meta‐analysis revealed a strong association between rs211037 with FS and SE in Asians for alleles (p=0.02, p=0.002 and p<0.00001, respectively) and for all genotype models. SIGNIFICANCE: Our data suggests that GABRG2 rs211037 polymorphism is a risk factor for susceptibility to SE and FS in Asians, particularly in Chinese.

Grant Funding Source: HIR MOHE E000025‐20001
Assessing the accuracy of femoral tunnel placement in anatomic ACL reconstruction (913.13)
Authors: Ducsharm Melissa , Banaszek Daniel , Hesse Daniel , Kunz Manuela , Reifel Conrad , Bardana Davide
Published: June 2021

Current trends in Anterior Cruciate Ligament (ACL) Reconstruction favor anatomic positioning of ACL attachment sites. Surgical inaccuracy in femoral tunnel positioning can lead to potential early graft failure and early‐onset osteoarthritis.

The purpose of this study was to evaluate accuracy of femoral tunnel positioning by orthopedic surgeons using five ACL femoral guides.

Using a human cadaveric femur, the mathematical center of the ACL femoral attachment was determined to be the anatomically ideal location for femoral tunnel placement. Five orthopedic surgeons performed arthroscopic femoral tunnel pin placement on identical artificial femurs using 5 different femoral guides (ConMedTM Linvatec Offset & BullseyeTM, Arthrex® Transportal, Stryker® VersiTomic®, Smith&Nephew CLANCYTM). Tunnel placement was compared to the ideal position using virtual Computer Tomography. A total of 125 femurs were used.

The Stryker® guide had greatest accuracy (distance from ideal footprint) at 3.9mm, and the most optimal tunnel length at 33.4mm. Only 8/125 iterations were within our set acceptable limit of 2mm from the ideal footprint.

Tremendous variability of femoral tunnel positioning was observed with each guide. Only 6.4% of trials fell within our acceptable limit. Complimentary research is needed in cadavers and humans to fully elucidate anatomic accuracy and applicability to the operating room.
Thyroid gland’s topography: preliminary results (913.5)
Authors: Docko Ana , Yendo Tatiana , Hojaij Flávio , Akamatsu Flávia , Jacomo Alfredo
Published: June 2021

The clinical evaluation and the surgical approach of the thyroid gland are standardized. However there are rare cases about ectopic thyroid tissue reported, related to abnormalities during the embryogenesis of the thyroid gland. Its prevalence is about 1 per 100 000 ‐ 300 000 people, rising to 1 per 4000 ‐ 8000 in patients with thyroid disease. In autopsy studies, the prevalence ranges from 7 to 10%. This study aims to analyze the position of the thyroid gland in 9 cadavers. The thyroid gland’s topography was classified in: on the neck, under the neck. We found 8 thyroid gland positioned on the neck and 1 under the neck, at the level of the clavicle. The knowledge of the thyroid gland’s topography and their prevalence as described in this preliminary study may contribute to avoid iatrogenic lesions in cervical surgeries.
Interconnections between the cruciate ligaments and the menisci in the knee (914.3)
Authors: Bashir Kerstin , Weninger Patrick , Hirtler Lena
Published: June 2021

The menisci are of great importance for the stabilization and for the pulling and rolling movements of the knee. The aim of this study was to describe the interconnecting fibers between the menisci and the cruciate ligaments and to show their variability.

40 tibial plateaus were included from specimen of the anatomical institute.

Lateral meniscus: It had in 93,55% connecting fibers to the footprint of the anterior cruciate ligament (ACL). 90,32 % of the lateral menisci had an osseous fixation between the ACL and the posterior cruciate ligament (PCL). Also in the posterior part there is in 51,61% a connection between the posterior horn of the lateral meniscus an the footprint of the ACL. Connections to the PCL were found in 83,87%.

Medial meniscus: The anterior horn medial meniscus had in 35,38% an overlapping area with the footprint of the ACL. In 35,48% there was either a partial or complete connection to the ACL. 87,10% of the posterior horns of the medial menisci had an insertion individual to the attachments of the ACL or the PCL. In 12,90% there were connecting fibers either towards the ACL or the PCL.

The findings of this study show the complexity of the attachments of the medial and lateral meniscus. These results should have an impact on planning the reconstruction of the menisci and their actual surgery as this states a much more intimate relationship to the cruciate ligaments of the knee than described in literature until now.
Variations in the anatomical structures surrounding the transverse process of the atlas (C1) in 15 cadavers (915.14)
Authors: Kim Pil‐Woo , Ledwitz‐Rigby Florence
Published: June 2021

The anatomical structures adjacent to the Transverse Process of Atlas (TPA) were examined by dissection in 15 cadavers age 65‐85 from 2009 to 2013. The TPA is the surgical land mark of the upper cervical area for many surgeons. The styloid process can be exposed anterior to the TPA. The internal jugular vein, Cranial Nerves (CN) XI, XII, X, IX, internal carotid artery, and sympathetic nerve are also located anterior to the TPA. In 13 cadavers, the styloid processes were elongated to 2‐3 cm lateral to medial in the middle of the TPA. In 10 cadavers, Internal jugular vein, CN XI, CNXII, CN X, and CN IX were located between the TPA and styloid process. There were 3‐5mm spaces between the TPA and styloid processes. In 3 cadavers, the styloid processes angled less, so that the CN IX was not between those bony structures. The other 2 cadavers’ styloid processes were about 1cm, so the nerves were between the TPA and the stylopharyngeus muscle. These anatomical variations amongst the individuals are relevant for surgical procedures and to understand the origin of peripheral nerve tumors that grow into the brain.
Morphometric analysis of the bony structures associated with the transcondylar approach (915.15)
Authors: LoPresti Melissa , Auquilla Leslie , Akintayo Rachel , Nwogu Nnenna , Erkuran Yilmaz Cigdem
Published: June 2021

Varying approaches to the foramen magnum are essential for access to the area, therefore understanding the bony anatomy of this region is important for the surgery. The aim of this study is to conduct a morphometric analysis of the jugular tubercle, occipital condyle, and lateral mass of C‐1, since these bony landmarks are the main obstacles hindering visualization of the clivus. Thus we have conducted a morphometric analysis in 35 adult dry skulls and C1 vertebrae. The distances on left (L) and right (R) sides were measured in millimeters. The distances were; between the anterior part of the occipital condyle to the jugular tubercle (L: 14.54± 0.36, R: 14.88 ± 0.37); from the mid part of the occipital condyle to the jugular tubercle (L: 14.43±0.33, R: 14.92±0.34), from the posterior part of the occipital condyle to the jugular tubercle (L: 9.81±0.35), R: 11.39±0.3), from the anterior part of the occipital condyle to the basion (L: 24.26±0.36, R: 24.04±0.33), from the posterior part of the occipital condyle to the basion (L: 11.36±0.25, R: 10.81±0.31) and from the jugular tubercle to the basion (L: 18.21± 0.24, R:17.56 ±0. 57). We also documented the width of lateral mass horizontally (L: 11.42±0.37, R: 11.08±0.35) and vertically (L: 19.65±0.41, R: 18.9±0.42) and the thickness of the anterior part of the occipital condyle (L: 8.82±0.18, R: 10.82±0.23), the thickness of the mid part of the occipital condyle (L: 10.42±0.28, R: 9.23 ±0.18) and the thickness of the posterior part of the occipital condyle (L: 8.42±0.16, R: 8.59±0.20). This study provides morphometric measurements of the bony landmarks for the transcondylar and its modified approaches to the foramen magnum and related structures.
Bilateral variations of the brachial plexus involving the lateral cord and median nerve: an anatomical case study with clinical implications (915.8)
Authors: Butz James , Shiwlochan Devina , Brown Kevin , Murlimanju B.V. , Prasad Alathady
Published: June 2021

In the course of routine dissection of the upper limbs of a Caucasian male cadaver, bilateral variations were observed in the brachial plexus. In the right extremity, the lateral cord was found to be piercing the coracobrachialis muscle. The musculocutaneous nerve and lateral root of the median nerve were observed to be branching inferior to the lower attachment of coracobrachialis muscle. The left extremity exhibited passage of the median nerve through the flat tendon of the coracobrachialis muscle near its distal insertion into the medial surface of the body of the humerus. A variation in the course and branching of a nerve might lead to variant or dual innervation of a muscle and if inappropriately compressed, could result in a distal neuropathy. Identification of these variants of brachial plexus plays an especially important role in both the clinical diagnosis of musculoskeletal pathology and surgical practice.