bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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