bluuebunny/crossref_metadata_2025 huggingface dataset:
Sex specific effect of antenatal glucocorticoids on nitric oxide production stimulated by angiotensin peptides in renal proximal tubule cells (1085.6)
Authors: Bi Jianli , Chen Kai , Su Yixin , Chappell Mark , Rose James
Published: June 2021

We have shown that renal proximal tubule cells (RPTCs) from male offspring exposed to antenatal betamethasone (B) increase Na+ uptake during Angiotensin II (ANG II) stimulation more than RPTCs from vehicle exposed animals while Na+ uptake by cells from females is not altered by B. The ability of angiotensin 1‐7(ANG 1‐7) to decrease sodium uptake was blocked in RPTCs from males but not in RPTCs from females exposed to B . Sodium uptake may relate to nitric oxide (NO) production in RPTCs. Therefore, this study aimed to assess the effect of antenatal glucocorticoid on nitric oxide production stimulated by angiotensin peptides in RPTCs in adult sheep exposed to B.

We obtained kidneys from 1‐1.5 year old male and female sheep exposed to either B (n=5) or Vehicle (V) (n=5) at 80‐81 days gestation. RPTCs were cultured in DMEM/F12 and stimulated with ANG II or ANG1‐7 with or without blockers. Nitric oxide production in cells was measured by DAF‐FM diacetate, nitric oxide indicator. ANOVA was used for data analysis.

ANG II suppressed NO production and the suppression was blocked by candesartan (in males: F=4.2, P=0.05; in females: F=5.9, P=0.02). In presence of PD the suppression was greater in male antenatal B than male V exposure sheep (F=17.8, P=0.006). Compared with females, suppression of NO production by ANG II is greater in male B exposed sheep (F=10.7, P=0.005). Increase of NO production by ANG 1‐7 was greater in RPTCs in male V than B treated sheep (F=5.5, P=0.03). There was no difference in the increase of NO production by ANG 1‐7 in female B vs. V sheep. The increase was blocked by D‐Ala. NO production by ANG 1‐7 was greater in female than male B exposed sheep (P=0.02). The NO production was blocked by L‐NAME. The results suggest antenatal B alters NO changes in response to ANG II and ANG 1‐7 in male but not in female RPTCs. The suppression of NO production by ANG II is mediated by AT1 receptor and NO production induced by ANG 1‐7 may involve in Mas receptor. The differences in NO may mediate sex related differences in Na+ uptake by these cells.

Grant Funding Source: Supported by HD‐47584 and HD‐17644.
Acid alpha‐glucosidase gene replacement therapy to the diaphragm remodels ventilatory function in severe Pompe disease (1091.8)
Authors: Smith Barbara , Martin Daniel , Mah Cathryn , Lawson Lee Ann , Falk Darin , Fuller David , Islam Saleem , Collins Shelley , Byrne Barry
Published: June 2021

Pompe disease is a neuromuscular disorder due to mutations in the gene that encodes the acid alpha‐glucosidase (GAA) enzyme, which results in lysosomal glycogen accumulation in striated muscle and motor neurons. The severe infantile form leads to cardiac hypertrophy, respiratory failure and early mortality. An approved enzyme replacement therapy reduces the cardiomyopathy and increases survival, yet many patients eventually require mechanical ventilation (MV). Our objective is to investigate an alternative treatment, by correcting the defective gene in an open‐label, Phase I/II clinical study of AAV‐GAA therapy to the diaphragm. We hypothesized that retrograde transport of AAV‐GAA would promote diaphragm glycogen clearance to restore inspiratory motor function. To date, five children (ages 2‐15) with full‐time MV dependence have received intramuscular rAAV1‐CMV‐GAA delivery into the diaphragm and completed follow‐up ventilatory testing. Maximal pressure inspiratory generation did not improve one year after dosing, but maximum unassisted tidal volume increased significantly (median: 22% gain, range: 11‐77%). Moreover, all subjects generated improved spontaneous breathing without MV assistance (median: 586%, range: 192‐2901% improvement over baseline levels). We conclude these findings may be related to vector transduction properties, fiber atrophy from chronic MV, and properties of the postoperative muscle conditioning prescription.

Grant Funding Source: K12 HD055929; CRB‐HLBI1‐S‐10‐00223
Three‐dimensional structure and association with blood vessels of chemosensitive and nonchemosensitive locus coeruleus neurons from neonatal rats (1092.15)
Authors: Putnam Robert , Graham Cathy , Li Ke‐yong
Published: June 2021

We studied the 3 dimensional structure of LC neurons from neonatal rats. Neurons were identified as either chemosensitive or nonchemosensitive based on their firing rate response to hypercapnia, studied using whole‐cell patch clamping. Chemosensitive neurons have less rounded soma than nonchemosensitive neurons and they have dendrites arising from two poles to produce a fusiform appearance. The dendritic branches extend a greater distance from the soma of chemosensitive than nonchemosensitive neurons. Chemosensitive LC neurons have at least one projection that makes a closer approach to the floor of the 4th ventricle than nonchemosensitive neurons, but on average this closest approach is still greater than 50 microns. Nothing about the 3 dimensional structure of chemosensitive neurons appears to be correlated with the neuron being chemosensitive. Interestingly,however, the dorsal and intermediate zones of the LC have a very high percentage of chemosensitive neurons while the ventral zone has mostly nonchemosensitive neurons. Based on Image J analysis of LC regions stained for neurons (Neu‐N) and blood vessels (isolectin), there is a higher density of blood vessels in the chemosensitive dorsal and intermediate regions of the LC compared to the nonchemosensitive ventral region, suggesting greater perfusion of chemosensitive neurons. Finally, we found that capillaries make direct contacts with the soma of chemosensitive neurons but not with the soma of nonchemosensitive neurons. Our findings are consistent with chemosensitive signaling being based in the soma of LC neurons and provide anatomical information for the construction of multi‐compartment computational models of chemosensitive neurons. We are currently comparing our findings in LC neurons with chemosensitive and nonchemosensitive neurons from the nucleus of the solitary tract (NTS).

Grant Funding Source: Supported by NIH Grant HL56683
Chronic rapamycin treatment decreases markers of autophagy and mitophagy in a sex‐specific manner (1094.8)
Authors: Drake Joshua , Bruns Danielle , Miller Richard , Hamilton Karyn , Miller Benjamin
Published: June 2021

The lifespan extending effect of rapamycin treatment is greater in female than male mice. Previous data from our lab demonstrated that chronic rapamycin inhibited mTOR substrates related to protein synthesis in a sex and tissue dependent manner. Therefore, we further hypothesized that sex differences may also exist in the mTOR regulation of autophagy and mitophagy. UM‐HET3 mice fed rapamycin (14ppm; Rap) for 12 weeks were compared to controls (Con). Tissues were homogenized and mitochondrial (mito) and cytosolic (cyto) fractions were isolated by differential centrifugation. In skeletal muscle of male, but not female mice, phosphorylation of ULK1‐S757 was increased in Rap and LC3 II:I ratio was decreased in Rap cyto. In heart, phosphorylation of ULK‐S555 was increased in Rap cyto and mito while S757 was increased in Rap cyto independent of sex. Additionally, Rap mito LC3 II:I ratio was decreased independent of sex compared to Con in heart. In the liver, Rap mito Parkin was decreased compared to Con independent of sex. These results suggest that chronic rapamycin paradoxically decreases markers of autophagy and mitophagy in heart and liver independent of sex and, in skeletal muscle of male mice. Rapamycin resistance in mTORC1‐dependent markers of autophagy/mitophagy may give new insights into the regulation of lifespan by rapamycin.

Grant Funding Source: Supported by: 53‐8983‐0, 1K01AG031829‐01, P30AG013283, R01AG042569
Hsp90 acetylation regulates mineralocorticoid receptor subcellular dynamics and aldosterone‐induced promoter transactivation (1097.15)
Authors: Alvarez de la Rosa Diego , Jimenez‐Canino Ruben , Lorenzo‐Diaz Fabian , Giraldez Teresa
Published: June 2021

The mineralocorticoid receptor (MR) is a nuclear receptor that transduces the biological effects of the corticosteroid hormone aldosterone, playing a key role in the regulation of transepithelial Na+ reabsorption, extracellular volume and blood pressure. MR forms cytosolic heterocomplexes with heat‐shock protein 90 (Hsp90), Hsp70 and other proteins such as immunophilins. Interaction with Hsp90 is essential to maintain MR in a ligand‐binding competent conformation and to regulate nucleocytoplasmatic shuttling. Acetylation of residue K295 in Hsp90 regulates its interaction with androgen and glucocorticoid receptors. In this work we hypothesized that Hsp90 acetylation may provide a regulatory step to modulate MR cellular dynamics and activity. We used Hsp90 acetylation mimic mutant K295Q or non‐acetylable mutant K295R to examine MR ligand‐dependent and independent nucleocytoplasmatic shuttling and gene transactivation function in cell models with different endogenous levels of Hsp90 expression. Furthermore, we manipulated endogenous Hsp90 acetylation levels by controlling expression or activity of histone deacetylase 6 (HDAC6), the enzyme responsible for deacetylation of Hsp90‐K295. Taken together, our data demonstrates that Hsp90 expression level and acetylation‐dependent activity are essential for regulating MR subcellular localization, ligand‐induced nuclear translocation and gene transactivation.

Grant Funding Source: Supported by grants BFU2010‐16625 and CSD‐2008‐00005 from MINECO, Spain
Dopamine D1‐like receptors (D1R and D5R) differently regulate phospho‐PKC in HEK‐293 cells heterologously expressing human D1R or D5R (1097.6)
Authors: Yu Peiying , Yang Jian , Yang Jian , Yang Sufei , Jose Pedro
Published: June 2021

Dopamine cellular signaling via the D1 receptor (D1R) involves a cross talk between protein kinase A (PKA) and protein kinase C (PKC) in HEK‐293 cells heterologously expressing human D1R (hD1R). We tested the hypothesis that dopamine D1‐like receptors (D1R and D5R) differentially regulate phospho‐PKC isoforms in HEK‐293 cells heterologously expressing hD1R or hD5R. In hD1R but not hD5R cells, short‐term (15, 30, and 60min) stimulation with the D1‐like receptor agonist, fenoldopam, (1μM) increased phosphorylated (corrected by total PKC, relative to vehicle treatment) PKCθS676 (1.79±0.21, 15 min; 1.54±0.10, 30min; and 1.40±0.10, 60min, n=6), PKCµS744 (1.65±0.25, 15 min), and PKCµS916 (1.60±0.09, 15min) (n=5‐6). The effect on PKCµS916 and PKCµS744 decreased with the time; no other phospho‐PKC isoforms were affected. In hD5R but not hD1R cells, long‐term (8 and 24hr) stimulation with fenoldopam (1µM) caused an increase in PKCβ2S660 and PKCηS674; there were no other phospho‐PKC proteins affected. The long‐term fenoldopam‐mediated increase in PKCβ2S660 and PKCηS674 proteins was blocked by the D5R (in the absence of D1R) antagonist Sch23990 (5µM) (PKCβ2S660: fenoldopam=1.66±0.14 vs. control=1.06±0.04, Sch 23390=0.89±0.25 and Sch+fenoldopam=1.11±0.143; PKCηS674: Fen=1.45±0.05 vs. Con=1.04±0.04, Sch=0.71±0.20 and S+F=1.02±0.06) (P<0.05, n=4, Newman‐Keuls test). The D5R‐mediated stimulation (1μM/24 hr) of PKCβ2S660 and PKCηS674 proteins was also blocked by a PKC inhibitor bisindolylmaleimide II (1µM) and two PKA inhibitors, H89 (10µM) and Rp‐cAMP (50µM). The D5R‐mediated (1μM/24 hr) increase in PKCβ2S660 and PKCηS674 was also blocked by phosphoinositide 3‐kinase inhibitor LY294002 (1µM). We conclude that short‐term stimulation of D1R increases phospho‐PKCηS676 and PKCµS proteins while long‐term stimulation of D5R increases PKCβ2S660, and PKCηS674 proteins via PKA and PI3K signaling pathways.

Grant Funding Source: HL023081, HL074940, DK039308
Influence of TNF‐α on vasodilation of isolated rat pulmonary artery rings (1106.17)
Authors: Gassama Abubacarr , Ray Clare , Turner Alice , Egginton Stuart , Kumar Prem
Published: June 2021

Inflammation promotes endothelial damage leading to vascular smooth muscle cell (VSMC) hyperplasia, vasoconstriction, and vascular remodelling resulting in an increased pulmonary arterial pressure¹. As TNF‐α decreases the bioavailability of NO in systemic vessels, we investigated the effects of TNF‐α on vasodilation in isolated pulmonary arteries (PA).

PA segments were isolated from terminally‐anaesthetised Wistar rats and vessel rings were pre‐incubated in DMEM (+/‐ TNF‐α, 1000U/mL) for 2 or 6 hours before mounting on a wire myograph. U46691 (400nM) was used to generate pre‐tone and cumulative dose‐response curves generated for carbachol (CC) and sodium nitroprusside (SNP). Data was normalised to maximal KCl contraction and significance taken as P<0.05.

Responses to KCl and U46691 were not significantly different between control and TNF‐α treatments. CC and SNP relaxed pre‐constricted rings but TNF‐α treated rings were significantly less responsive at all doses of either CC or SNP. No difference was noticed between 2 or 6 hours of TNF‐α treatments.

This data suggests that TNF‐α pre‐treatment had no effect on the VSMC vasoconstrictor ability but does alter the dilator response to NO.
Aerobic exercise increases stress resistance in arteries of old mice (1106.9)
Authors: Gioscia‐Ryan Rachel , Battson Micah , Sindler Amy , Cuevas Lauren , Zigler Melanie , Seals Douglas
Published: June 2021

Impaired ability of the aging vasculature to respond to stress may contribute to endothelial dysfunction, assessed as reduced endothelium‐dependent dilation (EDD). Mitochondrial (mito) dysregulation (e.g., excess mito reactive oxygen species (mtROS) production) with aging may increase susceptibility to stress. Aerobic exercise improves endothelial function with advancing age, but whether exercise affects vascular stress resistance is unknown.

Max carotid EDD to acetylcholine (ACh) was reduced in old (O, n=7; 26‐28 mo) versus young (Y, n=7; 6‐8 mo) c57 mice (74 ±5% vs. 94 ±2%, p<0.05), but restored in old mice with 12 weeks access to voluntary run wheels (OVR, n=8; 89 ±3%, p<0.05 vs. O). Inducing mtROS with rotenone (0.5 µM, 40’ incubation) further impaired EDD in O (‐44 ±8%, p<0.05 vs. ACh alone), but not Y and OVR (‐7 ±2% and ‐3 ±15%). Incubating arteries with a simulated “Western” diet (sWD: 80 mM glucose, 300 µM palmitate, 40’) reduced max EDD in both O and Y (‐26 ±4% and ‐28 ±5% vs. ACh alone, both p<0.05), but OVR were resistant (‐6 ±5%). Simultaneous incubation with mito‐specific antioxidant MitoQ prevented sWD‐induced impairments, indicating mtROS mediation.

Increased stress resistance in OVR was accompanied by amelioration of age‐related increases in aortic mito‐specific superoxide production (EPR; O: 1.5 ±0.4, p<0.05 vs. Y: 1.0 ±0.3 and OVR: 0.9 ±0.4 AU) and expression of phosphorylated (activated) adaptor protein p66SHC (Western blot; O: 11.3 ±6.1, p<0.05 vs. Y: 1.0 ±0.6 and OVR: 2.5 ±2.6 AU), a master mtROS regulator.

Our results suggest that aerobic exercise increases resistance to acute stress in aging arteries, possibly via favorable changes in vascular mito oxidative stress and homeostasis.

Grant Funding Source: Supported by NIH AG013038, AG000279, HL107120
The gut microbiota suppresses insulin‐mediated fat accumulation via GPR43 (1107.4)
Authors: Kimura Ikuo , Ozawa Kentaro , Inoue Daisuke , Ayukawa Kumiko , Tsujimoto Gozoh
Published: June 2021

Recent evidence suggests that the gut microbiota affects host nutrient acquisition and energy regulation and is thereby related to obesity, insulin resistance, and diabetes in the host. During feeding, the gut microbiota produces short‐chain fatty acids (SCFAs) as important energy sources utilized by the host. SCFA receptor GPR43‐deficient mice exhibited obesity, whereas adipose‐specific GPR43 overexpressing mice exhibited leanness under normal conditions; both of these mice did not exhibit either phenotype under germ‐free conditions or after antibiotic treatment. Furthermore, SCFA‐mediated GPR43 activation suppressed adipose insulin signaling, leading to inhibition of fat accumulation in the adipose tissue, and unincorporated lipids and glucose were utilized primarily in muscles. These findings established GPR43 as a sensor for excessive dietary energy, thereby controlling body energy expenditure while maintaining metabolic homeostasis.
Stable PTPN2 knockdown in human intestinal epithelial cell lines increases claudin‐2 expression and activation of MEK/ERK and PI3K signaling (1111.7)
Authors: Krishnan Moorthy , McCole Declan
Published: June 2021

Stable PTPN2 Knockdown in Human Intestinal Epithelial Cell Lines increases Claudin‐2 expression and activation of MEK/ERK and PI3K signaling
Moorthy Krishnan and Declan F McCole, Division of Biomedical Sciences, University of California, Riverside, Riverside, CA 92521.

Genome‐wide association studies identified protein tyrosine phosphatase non‐receptor type‐2 (PTPN2) as a susceptibility gene for inflammatory bowel disease (IBD), Diabetes and Celiac disease. Increased permeability of the intestinal epithelium is believed to be an important factor contributing to IBD. The aim of this study was to generate a stable PTPN2‐deficient intestinal epithelial cell line to investigate the role of PTPN2 and its protein product, T‐cell protein tyrosine phosphatase (TCPTP), in protecting epithelial barrier function. We developed Caco2‐BBE and HT‐29 intestinal epithelial cell lines (IECs) stably expressing short hairpin RNA for PTPN2. Western blotting and densitometry indicated that TCPTP protein expression was reduced by 50 ± 0.3% in PTPN2‐depleted Caco2‐BBE and HT‐29 cells, while there was a 3.5 ± 1.3 and 4.2 ± 1.5 fold increased expression of claudin‐2 in Caco2‐BBE and HT‐29 cells respectively. Furthermore, knockdown of PTPN2 resulted in decreased transepithelial electrical resistance (TER) (76 ± 3% and 50 ± 4%) in Caco2‐BBE and HT‐29 cells respectively) compared to control, and this correlated with the observed increase in claudin‐2 expression. Interestingly, Western blot and immunofluorescence studies showed that basal phosphorylation of ERK1/2 and Akt was increased in PTPN2 KD cells. In conclusion, we report an inverse relationship between PTPN2 (TCPTP) and claudin‐2 expression associated with increased activation of MEK/ERK and PI3K pathways in stable PTPN2‐deficient human IECs.

Grant Funding Source: Supported by NIH R01DK091281
Esophageal squamous cell carcinoma model of 3D organotypic tissue culture system with genetic modification of epithelial growth factor receptor and p120‐catenin (1119.3)
Authors: Yang Xuebin , Lehman Heather , Bruggeman Richard , Nie Xingcao , Stairs Douglas
Published: June 2021

Objectives: To evaluate whether our genetically (EGFR & p120ctn) modified 3D OTC esophageal epithelium resembles human ESCC.

Methods: Four sets of OTC (5 per set) were obtained: control, p120ctn knockdown (p120ctnD), EGFR upregulation (EGFRU), and combined p120ctnD and EGFRU (p120ctnD&EGFRU). Morphology was analyzed by H&E using ImageJ. Proliferation was quantified by Ki‐67 immunohistochemistry (IHC). Keratins (K) were studied by gene microarray and IHC in OTC, 10 human normal esophageal and 24 ESCC specimens.

Results: In our OTC, p120ctnD showed mild disorganization, mildly increased cellularity and significantly increased proliferation (20±4% vs 31±4%, p<0.05) compared to the control. EGFRU showed moderate disorganization, marked increased cellularity (104±31 vs 192±64, p<0.05) and proliferation (32±7%, p<0.05). Interestingly, p120ctnD&EGFRU contained apparently transformed epithelium resulting in cellular invasion into the matrix. It had significantly increased cellularity (292±72), nuclear size (13.2±0.1 µm vs 11.1±0.8 µm) and proliferation (45±9%) (each p<0.05). p120ctnD&EGFRU had decreased RNA expression of K1, K4, K10 and K15. IHC studies showed similar keratin patterns between normal human esophagus and OTC control and between human ESCC and p120ctnD&EGFRU.

Conclusions: Our genetically modified OTC esophageal epithelium resembles human ESCC with similar morphologic changes and keratin patterns.
Hyperosmotic stimulation elicits a purinergic‐glutamatergic coupling in PVN presympathetic neurons (1130.8)
Authors: Candido Ferreira Neto Hildebrando , Antunes Vagner , Stern Javier
Published: June 2021

We have previously shown that ATP applied in the PVN induces an increase in sympathetic activity, an effect attenuated by the antagonism of P2 and/or glutamatergic receptors. Here, we evaluated precise mechanisms underlying the ATP‐glutamate interaction in the PVN, and assessed whether this interaction contributed to osmotically‐driven PVN presympathetic neuronal activity. Whole‐cell patch clamp recordings obtained from PVN‐RVLM neurons showed that ATP (100 µM, 1 min, bath applied) induced an increase in firing rate (62±26%, 14/20), an effect blocked by kynurenic acid (1 mM) or PPADS (10 µM). While ATP did not evoke changes in synaptic activity, glutamate currents evoked by focal application of L‐glu (1 mM, 100 ms, n=9) were increased in amplitude (194±73%) and area (241±116%) by ATP. Glutamate potentiation did not involve NMDA receptors, since focal application of NMDA (50 µM, 100 ms, n=13) was not changed by ATP. However, AMPA receptor‐evoked currents (50 µM, 50 ms, n=15) were potentiated by ATP (amplitude: 100% vs 132±7%; area: 100% vs 149±8%), an effect blocked by PPADS (n=12) and intracellular BAPTA (n=14). Finally, a hyperosmotic stimulus (mannitol 1%, 355 mOsm, 1 min, n=9) potentiated AMPA responses (amplitude: 136±12%; area: 155±23%), an effect blunted by PPADS (n=6). Our data suggest that P2 and AMPA receptors in PVN‐RVLM presympathetic neurons participate in the responses evoked by hyperosmotic stimulation.

Grant Funding Source: Supported by FAPESP (#2010/17997‐0; 2010/05037‐1; 2012/12444‐8) and NIH
Conserved effects of squalene synthase inhibitors on gene expression changes in primary cultured rat and mouse hepatocytes (1148.2)
Authors: Rondini Elizabeth , Duniec‐Dmuchowski Zofia , Dombkowski Alan , Kocarek Thomas
Published: June 2021

Squalene synthase inhibitors, such as squalestatin 1 (SQ1), are cholesterol‐lowering drugs that block the first committed step in sterol biosynthesis. Unlike statins (e.g., pravastatin), inhibition of squalene synthase causes accumulation of non‐sterol isoprenoids that can function as signaling molecules that modulate the activities of various nuclear receptors including the constitutive androstane receptor (CAR), farnesoid X receptor (FXR), and peroxisome proliferator‐activated receptor (PPAR). To characterize the effect of isoprenoids on hepatocyte physiology further, we used microarray analysis to compare the gene expression responses in rat and mouse primary cultured hepatocytes that were treated with either SQ1 or pravastatin. In both species, genes involved in cholesterol biosynthesis were significantly up‐regulated by either drug consistent with the depletion of cellular cholesterol. Compared to pravastatin, treatment with SQ1 differentially affected 1796 genes in rat hepatocytes and 3689 in mouse hepatocytes (蠅 ±1.5‐fold change). Among these, we identified 112 conserved genes that were differentially higher and 54 lower in SQ1‐treated hepatocytes. Functional annotation identified that conserved responses were associated with induction of fatty acid metabolism and inhibition of cell cycle processes. The magnitude of gene expression changes was species‐dependent, likely due to differences in isoprenoid metabolism. Our findings indicate in addition to sterol depletion, squalene synthase inhibitors may uniquely influence cellular energy metabolism and cell cycle regulation.

Grant Funding Source: This work was supported by NIH grant HL050710.
Identification of redox‐ and glucose‐sensitive Txnip interacting proteins using BioID (1153.14)
Authors: Forred Benjamin , Neuharth Skyla , Kim Daein , Roux Kyle , Vitiello Peter
Published: June 2021

Many cell signaling processes are dependent on redox‐dependent interactions through the thioredoxin (Trx) family. Thioredoxins regulate the reduction‐oxidation state of protein substrates through antioxidant enzymatic activities. Thioredoxins have a conserved catalytic site that undergoes reversible oxidation to cysteine disulfide through the transfer of reducing equivalents from the catalytic site cysteine residues to a disulfide substrate. While Txnip has been identified as a critical modulator of several disease pathologies, including β‐cell death during hyperglycemia, cardiac hypertrophy, and bronchopulmonary dysplasia, the molecular mechanisms underlying these processes are relatively unknown. The objective of these studies was to establish a cell model to identify Txnip interacting proteins in an effort to understand molecular events underlying the aforementioned disease pathologies. A proximity‐based biotin‐dependent labeling system (called BioID) was fused to Txnip and stably‐transfected into HEK293 cells to identify novel interacting proteins. Proteomic analysis of BioID lysates identified 50 interacting proteins and mutation of a redox‐senstive cysteine (C247S) in Txnip disrupted several interactions. Furthermore, we demonstrate that this cell model can identify dynamic Txnip interactions through treatment with endogenous Txnip regulators such as hyperglycemia. HEK293 cells treated with increasing glucose concentration upregulated Txnip expression in a dose‐dependent manner. Comparative BioID analysis revealed several glucose‐dependent Txnip interacting proteins such as its E3 ubiquitin ligase, ITCH. Continued studies will focus on understanding the biological relevance of the identified interactions to better understand cellular alterations resulting in disease pathologies.

Grant Funding Source: Supported by NIH/NIGMS (1P20GM103620) and NSF/REU (1262744)
Matrix metalloproteinase‐2 is localized to the mitochondria‐associated membrane in the heart (1154.4)
Authors: Hughes Bryan , Fan Frank , Cho Woo Jung , Schulz Richard
Published: June 2021

Intracellular matrix metalloproteinase‐2 (MMP‐2) is activated by pro‐oxidants to proteolyze intercellular targets. MMP‐2 has recently been reported in mitochondria, a critical source of cellular oxidative stress. However, the methods used to determine MMP‐2 subcellular localization could not differentiate between MMP‐2 in the mitochondria or the mitochondrial‐associated membrane (MAM), a subdomain of the endoplasmic reticulum (ER). We hypothesized that mitochondrial MMP‐2 may be situated in the MAM and therefore investigated the subcellular distribution of MMP‐2. Immunogold electron microscopy revealed MMP‐2 in mitochondria of heart sections from mice. However, HaloTagged MMP‐2 expressed in HL‐1 cardiomyocytes showed an ER‐like distribution with greater colocalization with an ER marker (protein disulfide isomerase) relative to a mitochondrial marker, MitoTracker Red. Although MMP‐2 protein and enzymatic activity were present in crude mitochondrial fractions, once these were separated into purified mitochondria and MAM, MMP‐2 was principally associated with the latter. We also found that calreticulin, an ER and MAM‐resident Ca2+ handling protein and chaperone, could be proteolyzed by MMP‐2 in vitro. Thus, although mitochondria may contain minimal levels of MMP‐2, the majority of MMP‐2 previously identified as “mitochondrial” is in fact associated with the MAM. We hypothesize that MAM‐localized MMP‐2 could affect mitochondrial function by affecting ER‐mitochondrial Ca2+ signaling.

Grant Funding Source: Supported by the Canadian Institutes of Health Research
Potential mechanisms of cardiac hypertrophy in mice with a null deletion of MAFbx (1164.7)
Authors: Matern Philip , Gomes Aldrin , Bodine Sue
Published: June 2021

The E3 ubiquitin ligase, MAFbx, has been shown to target specific proteins for degradation through poly‐ubiquitination and the ubiquitin proteasome system. A null deletion of MAFbx reveals no cardiac phenotype in young unchallenged knock out (KO) mice, but leads to the development of congestive heart failure and sudden death (at 18 months) in unchallenged aging mice. The aim of this study was to determine potential mechanisms underlying the development of congestive heart failure in MAFbx KO animals. Echocardiograms demonstrated functional changes (reduced fractional shortening and left ventricular ejection fraction) in KO animals beginning at 11 months and markers for multiple growth pathways were elevated in KO mice at 14 months of age. Calcineurin expression was elevated above wildtype (WT) at 14 months and remained elevated at 18 months. Phosphorylated Akt (S473) and GSK3β (S9) expression were also elevated at 14 months. Markers of the proteasome system demonstrated altered expression and activity relative to WT animals. Expression of 19 and 20S proteasome subunits (RPT1, α3, β5) were elevated in KO mice at 14 months, but only the 26S β1 subunit showed higher activity, with no change in β2 or β5 activity. The 14 month mice also had elevated total ubiquitinated proteins and reduced free ubiquitin. Together, these findings suggest that MAFbx deletion results in a progressive disruption of protein quality control (increased protein synthesis and limited increase in protein degradation) leading to activation of pathological signaling pathways and the development of cardiomyopathy.

Grant Funding Source: Muscular Dystrophy Association
Caffeine does not alter the hypoxic ventilatory response of neonatal rats (1177.3)
Authors: Blegen Halward , Bavis Ryan
Published: June 2021

Rats reared in hyperoxia exhibit a sustained (vs. biphasic) hypoxic ventilatory response (HVR) at an earlier age than untreated, control rats (Bavis et al., J Appl Physiol 109:796‐803, 2010). It has been suggested that adenosine receptors contribute to hypoxic ventilatory depression in neonatal rats, so we investigated the effect of caffeine (a nonselective adenosine receptor antagonist) on the HVR of rats reared in room air (Control) or 60% O2 (Hyperoxia) through 4‐5 days of age (P4‐5). Rats were injected with saline or caffeine (20 mg kg‐1 caffeine citrate, i.p.) approximately 30 min prior to measuring normoxic and hypoxic (12% O2) ventilation via head‐body plethysmography. As expected, Control rats exhibited a biphasic HVR and Hyperoxia rats exhibited a sustained HVR after saline injection. However, caffeine treatment had no effect on the HVR of either group. These data do not support a role for adenosine receptors in the late phase of the biphasic HVR of neonatal rats.
Assessment of student nutrition knowledge and self‐efficacy at Boston University School of Medicine (118.1)
Authors: Ireland Kathy , Robbins Jennifer , Manders Aaron , Lenders Carine
Published: June 2021

Objective: To determine baseline nutrition knowledge and perceived self‐efficacy in BUSM students and identify associated demographic factors.

Methods: We conducted a cross‐sectional survey of 1st to 4th year medical students (n=775) including 6 knowledge and 4 self‐efficacy nutrition questions. Students were encouraged to take the survey via email and Blackboard postings from February‐April 2013. Data included year in medical school, gender, and involvement in a student nutrition interest group (SNAAC). Outcomes included an average composite knowledge score (mean±sd) and self‐efficacy ratings related to confidence to provide dietary counseling, refer to Registered Dietitians (RDs), and refer to food programs (Likert scale ranging from “strongly disagree” to “strongly agree”). Descriptive and multivariate analyses were performed.

Results: 166 (23%) students responded. Average knowledge score was 52±19%. Confidence to provide dietary counseling, refer to Registered Dietitians (RDs), and refer to food programs was 55%, 37% and 24%, respectively. After adjusting for all covariates, knowledge score increased with each education year (3.1 ±1.4%, p<0.05) and was lower in males (‐9.3±3.2%, p<0.05). Students were more likely to feel confident to counsel with each education year (AOR=1.41, 95% CI 1.03‐1.96) and with SNAAC involvement (AOR=2.74, 95% 1.39‐5.40). Confidence to refer to RDs was more likely with involvement in SNAAC (AOR=2.47, 95% CI 1.26‐4.83).

Conclusions: Medical student knowledge and self‐efficacy in nutrition is low. More nutrition education and prospective evaluations are needed. Funded by the New Balance Foundation.
Investigating py‐STAT6 protein levels and mRNA expression of STAT6 regulated genes in renal epithelial cells (1180.8)
Authors: Boslett Brianna , Resnick Andrew
Published: June 2021

The current study will investigate the mechanosensory‐signaling pathway leading to STAT6 gene transcription in mouse cortical collecting duct epithelial cells. Many stimulants are capable of activating STAT6 in the kidney, but it is still unknown how STAT6 is directed to specific gene targets. In several cases, transcription co‐factors, such as a fragment of polycystin 1, have been found to enter the nucleus along with STAT6 dimers. Additionally, the mechanism by which STAT6 becomes activated by mechanosensation is currently unknown. A primary goal of this study is to determine the kinase involved in STAT6 phosphorylation (activation) in changing flow conditions.

The STAT family has cell‐type specific promoter binding sites. Common STAT controlled cis‐regulatory modules (cSCCs) are cis‐regulatory modules (CRMs) that can be recognized by all STAT family members across cell types. 116 cSCCs have been identified and correspond to JAK/STAT signaling genes. I hypothesize that different stimulants (cytokines vs. fluid flow) will lead STAT6 to transcribe different genes. Therefore, STAT6 binding sites that do not correspond to cSCCs will be investigated for cell specific, or stimuli‐specific STAT6 gene transcription.

These results will have implications for ADPKD, which is caused by genetic mutations leading to defects in polycystin 1 (PC1) and polycystin 2 (PC2) proteins.

Grant Funding Source: Supported by the National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716
Proteolytic activation of the human epithelial sodium channel by trypsin and trypsin IV involves distinct cleavage sites (1181.2)
Authors: Haerteis Silke , Krappitz Annabel , Krappitz Matteus , Murphy Jane , Knecht Wolfgang , Bunnett Nigel , Korbmacher Christoph
Published: June 2021

Proteolytic channel activation is a unique feature of the epithelial sodium channel (ENaC) but is not yet fully understood. The serine protease trypsin is known to activate ENaC in vitro but may not be relevant in vivo. In this study, we wanted to investigate whether the trypsin‐like serine protease trypsin IV, known to be expressed in several epithelial tissues, can activate human ENaC. Moreover, using site‐directed mutagenesis we wanted to identify functionally relevant cleavage sites in the γ‐subunit of the channel. In the Xenopus laevis oocyte expression system, we monitored the proteolytic activation of ENaC currents and the appearance of γ‐ENaC cleavage products at the cell surface. We demonstrated that trypsin IV can stimulate ENaC and that this activation involves a critical cleavage site (K189) in the extracellular domain of the γ‐subunit. Moreover, we showed that channel activation by trypsin was prevented by mutating three putative trypsin cleavage sites (K168; K170; R172) in addition to mutating previously described prostasin (RKRK178), plasmin (K189), and neutrophil elastase (V182;V193) sites. In conclusion, we demonstrated for the first time that trypsin IV can activate ENaC and that trypsin and trypsin IV use specific cleavage sites to achieve channel activation. Preferential cleavage sites may provide a mechanism for differential ENaC regulation by tissue‐specific proteases.

Grant Funding Source: Supported by grants of the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) (S.H., M.K.)
Differential role of epithelial Na+ channel lysines in Nedd4‐2 regulation (1181.5)
Authors: Tomkovicz Vivian , Snyder Peter
Published: June 2021

The epithelial sodium channel (ENaC) plays a critical role in blood pressure control. Defects in ENaC regulation by the E3 ubiquitin ligase Nedd4‐2 cause Liddle’s syndrome, an inherited form of hypertension. Nedd4‐2 catalyzes ubiquitination of ENaC lysines, which enhances ENaC endocytosis and degradation. To identify the lysines required for Nedd4‐2 regulation, we mutated them to arginine and tested the effect of Nedd4‐2 on ENaC cell surface expression. Mutation of all cytoplasmic lysines in ENaC reduced the ability of Nedd4‐2 to decrease ENaC surface expression (detected by biotinylation) and ENaC current. In contrast, mutation of all cytoplasmic lysines in ‐ and ENaC had no effect. By mutating ENaC lysines individually and in combination, we identified differential roles for each. K8, K10, and K26 were partially required for Nedd4‐2 regulation, whereas K6, K12, and K13 were not. Mutation of lysines in ‐ and ENaC also revealed differential roles. In ENaC, mutation of K22 and K25 to arginine reduced ENaC inhibition by Nedd4‐2. In contrast, mutation of K83 enhanced Nedd4‐2 inhibition. In ENaC, mutation of K16 and K23 produced a small decrease in Nedd4‐2 regulation, but mutation of other lysines did not. Combination of mutations in ‐, , and ENaC produced an additive reduction in Nedd4‐2 regulation, but did not abolish regulation. Thus, Nedd4‐2 regulates ENaC in part through a subset of ENaC lysines, as well as through a second mechanism that is independent of ENaC lysines.

Grant Funding Source: supported by NIH HL072256 (PMS)
Inducible vascular progenitor cells grown on the biodegradable polymer bundles in the cardiovascular regeneration (216.2)
Authors: Yin Liya , Ohanyan Vahagn , Enrick Molly , Stevanov Kelly , Song Hokyung , Kolz Christopher , Logan Suzanna , Newby Bi‐min Zhang , Chilian William
Published: June 2021

Coronary heart diseases (CHD) caused about one in six death. The interest of cell‐based therapy for CHD is growing and different cell types have been studied in animal models and clinical trials for cardiovascular regeneration. But so far, the benefit is often modest and disputable. The best cell type and optimal approach to deliver the cells have not been determined. Our approach is to deliver the induced vascular progenitor cells (iVPCs) reprogrammed from the vascular endothelial cells by an injectable biodegradable polymer bundles. iVPCs showed enormous potential of angiogenesis in vitro and differentiated into both endothelial cells and smooth muscle cells. Differentiated iVPCs formed tubular structure on regular 2‐D culture dish. iVPCs grown on the polymer bundles formed 3D multi‐ layer cell strands and increased cell survival of the shear stress during injection. In a rat model, PBS, iVPCs or iVPCs grown on the polymer bundles were injected after the LAD was ligated. At 4 weeks, iVPCs increased the cardiac function (by echo cardiograph) and decreased the fibrosis (by Trichrome staining). Moreover, the iVPCs/polymer bundles further augmented the function of iVPCs by aligning and retaining iVPCs, increasing cell survival and vessels formation (by Fluorescence microscopy and staining). In conclusion, the iVPC/polymer bundles promoted angiogenesis, improved the cardiac function and decreased fibrosis after myocardium infarction, thus showed the therapeutic potential in cardiovascular regeneration.
Modulation of O‐GlcNAc levels as a disease modifying approach for Alzheimer disease (223.3)
Authors: Yuzwa Scott , Shan Xiaoyang , Heinonen Julia , Cekic Nevena , Jones Bryan , Gong Cheng‐Xin , Watson Neil , Vocadlo David
Published: June 2021

Protein O‐glycosylation with N‐acetylglucosamine (O‐GlcNAc) is found in the nucleus and cytoplasm of multicellular eukaryotes. O‐GlcNAc is particularly abundant in brain tissue and is a glucose responsive post‐translational modification, which is interesting given that impaired glucose utilization is an early pathological feature occurring in the brains of Alzheimer patients. In this presentation I will discuss the generation of inhibitors that slow removal of O‐GlcNAc as well as the neuroprotective effects of such inhibitors in tissue culture as well as transgenic mouse models of Alzheimer disease.
Structure and function of the beta‐barrel assembly machine and its chaperones (224.2)
Authors: Sousa Marcelo
Published: June 2021

Transmembrane proteins in the bacterial outer membrane display a characteristic beta‐barrel architecture. Folding and insertion of these Outer Membrane Proteins (OMPs) is essential for Gram‐Negative bacteria and requires the action of both periplasmic chaperones and the outer membrane complex known as beta‐Barrel Assembly Machine (BAM). The BAM complex is composed of BamA, a bετα‐barrel OMP itself; and four associated lipoproteins: BamB, C, D and E. BamA and BamD are essential for viability and appear to be present in all bacteria with an outer membrane. Whereas the phylogenetic distribution of BamB, C and E is not well defined, these lipoproteins are also important for OMP biogenesis and their deletion in E. coli results in outer membrane permeability defects. I present the crystal and NMR structures of BAM subunits as well as a complex between BamB and a periplasmic fragment of BamA, which begin to illuminate the architecture of this essential molecular machine. We show that chaperones prevent nascent OMP aggregation in the periplasm and maintain them in an unfolded state. The central BAM component, BamA, undergoes conformational transitions that are essential for its role in OMP folding and insertion, whereas BamB is proposed to modulate these conformational changes.
Who is willing to pay for micronutrient powders for children 2‐5 years of age in Nepal? (250.2)
Authors: Gunnala Rajni , Perrine Cria , Subedi Giri Raj , Mebrahtu Saba , Dahal Pradiumna , Jefferds Maria
Published: June 2021

Background: Nepali children have a high burden of micronutrient deficiencies. A 2011 pilot project gave micronutrient powders (MNP) for free to children 6‐23 months. This project is under review to inform future policy decisions. We describe willingness to pay (WTP) for MNP and pricing attitudes to explore potential for purchase of MNP for children 2‐5 years. Methods: Representative household surveys were conducted with mothers of children 6‐23 months in two districts 3 months after the pilot started. Questions explored MNP WTP for children 2‐5 years and attitude about a suggested price (R150/6 mo supply, ~US$2.10). Chi square tests assessed WTP and price attitudes by demographics; and WTP by MNP knowledge and intervention exposure. Results: Overall, 78% of mothers expressed WTP for MNP and 66% had a positive attitude toward the suggested price. WTP and positive price attitude were associated with higher wealth quintiles and higher maternal education. Urban residence was associated with positive pricing attitude. Receipt of MNP education and printed materials were associated with WTP, but use of MNP among younger children was not. Those with knowledge of barriers to MNP use were less likely to show WTP than those with none (66.3% vs. 92.4%, p<0.01). Conclusions: WTP and positive price attitude indicate potential for mothers to purchase MNP for children 2‐5 years in Nepal. Further studies are needed to determine if stated WTP translates into actual purchase.
Association of parental emotional feeding with preschool child intake of sugar‐sweetened beverages (252.7)
Authors: Lora Karina , Wakefield Dorothy , Ferris Ann
Published: June 2021

The study examined parental feeding practices with preschool children consumption of sugar‐sweetened beverages (SSB). Baseline data from a longitudinal study with low‐income multicultural caregivers of 3‐5 y old children (n=326). Participants provided demographics; two‐consecutive weekend food recalls for the child and responded to the Preschooler Feeding Questionnaire. Children’s height and weight were measured in triplicate. Food recalls were averaged over the two days. General linear models tested association of feeding practices with child intake (oz.) of SSB (juice drinks, soda) among children with SSB intake in their food recalls (consumers). Among consumers, median total child intake of SSB was 8.7 oz/day, while juice drinks and soda were 6.7 oz/day and 5.4 oz/day, respectively. Total SSB and juice drinks intakes were positively associated with caregiver’s “Use of Food to Calm the Child” in Latino and White children (p<.05). Use of Food to Calm the Child was positively associated with BMI percentile in White children only (p<.05). SSB and juice drinks intakes were not associated with child’s BMI percentile. No other feeding practice was associated with child’s intake of SSB. Caregivers’ use of SSB for emotional feeding seems to occur in the context of negative emotions in the child. Ethnic/racial differences exist in the use of foods for child’s emotional regulation.

Grant Funding Source: USDA/NRI 2008‐55215‐19071
Health implications of adopting nutritious, low‐carbon diets in the U.K. (255.3)
Authors: Green Rosemary , Milner James , Dangour Alan , Haines Andy , Chalabi Zaid , Markandya Anil , Spadaro Joseph , Wilkinson Paul
Published: June 2021

Objective: To design diets for the UK adult population that are plausible, conform to nutritional guidelines and reduce greenhouse gas emissions (GHGEs), and to quantify the health impacts associated with these diets.

Methods: We used data from the National Diet and Nutrition Survey and a Life Cycle Analysis of GHGEs to calculate the emissions associated with current average diets in the UK. We then optimised these diets so that they conformed to World Health Organization (WHO) nutritional guidelines while minimising deviation from the current diet and reducing the GHGEs associated with the diet. We assessed the changes in Quality Adjusted Life Years (QALYs) from mortality and morbidity from coronary heart disease, stroke, cancer and type II diabetes if the optimised diets were adopted.

Results: The current average diet among UK adults does not conform to WHO guidelines. If diets were optimised to comply with these guidelines, an incidental reduction of 15% in GHGEs would be achieved. Further reductions in GHGEs could be achieved by modifying diets so that they contain fewer animal products and more fruit, vegetables and cereals, but dietary changes that reduced GHGEs by more than 50% resulted in implausible changes to dietary patterns. Adopting healthy diets 50% lower in GHGEs than the current diet would result in a large saving in QALYs over a 20 year follow‐up period, half of which would be from coronary heart disease.

Conclusions: There are large potential co‐benefits to health from adopting diets with lower associated GHGEs. However, the nutritional value and acceptability of these diets is likely to peak at a reduction in GHGEs of around 50% for a country like the UK, therefore in the absence of major changes in dietary preferences any additional reductions in emissions from food will have to come from other means such as waste reduction and increased production efficiency.