bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of mineral analytical method for food composition data‐up construction (1043.5)
Authors: Hwang Jinbong , Oh Mira , Kim Soyoung , Kim Se‐na , Nam Jin‐sik
Published: June 2021

The analytical method for the determination of minerals (sodium, calcium, potassium, phosphorus, magnesium, iron, copper, manganese, and zinc) in foods by inductively coupled plasma optical emission spectrometry (ICP‐OES) was validated in terms of precision, accuracy, and linearity. SRM 1849a was used as a test sample which was purchased from NIST in USA. Regression analysis revealed good correlation coefficient (r2) that was higher than 0.999. Recovery efficiencies of the minerals ranged from 90.36 % to 110.63 %, and the limit of detection (LOD) and the limit of quantification (LOQ) by ICP‐OES were less than 0.1005 mg/kg and less than 0.3351 mg/kg, respectively. The inter‐day of accuracy was between 1.19 and 18.19 % (n=12), and intra‐day of accuracy was from 0.09 to 4.80 % (n=3). In addition, the amounts of minerals were determined that several kinds of cereals and variety vegetables were purchased from Rural Development Administration (RDA) in Korea. The levels of sodium, calcium, potassium, phosphorus, magnesium, iron, copper, manganese, and zinc in cereals and vegetables ranged from 0.33 to 1371.55 mg/kg, 2.70 to 1494.81 mg/kg, 5.07 to 4939.06 mg/kg, 7.95 to 564.47 mg/kg, 3.92 to 606.94 mg/kg, 0.13 to 31.61 mg/kg, 0.01 to 1.58 mg/kg, 0.05 to 16.98 mg/kg and 0.07 to 11.27 mg/kg, respectively. These results suggest that analytical method verified in the present study will be used as a public analytical method to draw up food composition database for national health promotion.
Antioxidant bioaccessibility from wheat aleurone throughout the canine gastrointestinal tract: an in vitro study (1044.14)
Authors: Garcia Campayo Vicenta , Han Sonia , Aimutis William , Bosscher Douwina , Franck Anne
Published: June 2021

Enhancing overall health status through nutrition has growing interest in humans and animals and thus, the market for food and feed formulations with added functional dietary ingredients is on the rise. Health‐promoting properties of wheat are documented in vivo and in vitro, and are partially attributed to antioxidant compounds in the bran fraction. Cargill isolates the aleurone fraction from wheat bran which is used as an ingredient in companion animal diets with the expectation of promoting a healthier diet. However, for bioactive compounds to be efficacious, they must be bioaccessible (available for absorption) during the digestive process. Our studies indicated antioxidants from aleurone are more bioaccessible than those from beet pulp fiber (BP) in an in vitro dynamic digestive system (TNO) validated for the GI‐tract of dogs. Results show low molecular weight antioxidant compounds become bioaccessible in the jejunum compartment of the small intestine but not in the ileum (TIMdog‐1), and antioxidant activity is comparable among the substrates. Furthermore, additional antioxidant compounds are released during fermentation (lower GI, TIMdog‐2) of the non‐digested fraction, with cumulative antioxidant activity being about 25% higher from aleurone than BP. Thus, supplementing pet food with wheat aleurone fractions may be a strategy for improved health benefits in a canine diet.
A nano‐emulsion of lutein is more effective than regular lutein in reducing hepatic inflammation in guinea pigs (1044.15)
Authors: Murillo Gabriela , Aguilar David , Missimer Amanda , Kakumanu Srikanth , Nicolosi Robert , Bolling Bradley , Fernandez Maria Luz
Published: June 2021

The purpose of this study was to compare the effects of consuming regular lutein versus lutein in a nano‐emulsion on hepatic liver enzymes and gene expression inflammatory markers in guinea pigs. For that purpose, we fed guinea pigs a hypercholesterolemic (0.25 g/100g dietary cholesterol) diet for 12 weeks to induce liver injury. Guinea pigs consumed either 0.9 mg/d of regular lutein (control) (n= 8 guinea pigs) or a nano‐emulsion (NANO) (average diameter 422 nm) (n = 8 guinea pigs). Plasma lipids and glucose were not different between treatments. However hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST), markers of liver function were 54 and 50% lower, respectively in guinea pigs fed the NANO emulsion (p< 0.005). Hepatic and plasma lutein were measured by HPLC and gene expression was evaluated by use of RT‐PCR. There were no significant differences between groups in either plasma or hepatic lutein concentrations, although there was a trend for higher plasma lutein in the NANO group (p=0.084). Gene expression of monocyte chemoattractant protein‐1 was not different between groups. In contrast although the expression of tumor necrosis factor (TNF)‐alpha was 70% higher in guinea pigs fed the control group, they were not different between groups due to the high variability. These results indicate that using the same amount of dietary lutein, a nano‐emulsion is more effective in protecting the liver against diet‐induced inflammation.

Grant Funding Source: UConn Experimental Station
Assessment of nutritional status in subjects of MonaVie product users and non‐users ő a cross sectional study (1044.18)
Authors: Quan Danyi , Xiong Wade , Talbott Shawn
Published: June 2021

BACKGROUND: Phytonutrient intake is directly related to the risk of various diseases. Carotenoid levels in both serum and skin are well correlated with fruit and vegetable intake. Various non invasive techniques, including Reflectance Spectroscopy, are able to accurately detect skin carotenoid levels, and MonaVie's View scanner has been tested for a noninvasive method of assessing antioxidant levels. OBJECTIVE: Across‐sectional study by using MonaVie View Scanner to noninvasively measure skin carotenoid levels in subjects of MonaVie product users and non users. METHODS: Total 68 subjects were enrolled. 31 subjects were MonaVie product users and 37 subjects were non MonaVie product users. All subjects had the reflectance score measurements and blood draw. The plasma concentration of carotenoids were extracted and assayed by HPLC method. RESULTS: The carotenoid skin levels measured by View scanner are well correlated with the β‐carotene plasma concentration (r2 = 0.63). A logarithm values of plasma concentration have further confirmed the correlations between skin levels of carotenoids and plasma concentration of carotenoids (r2 = 0.64). CONCLUSION: Skin carotenoid levels are well correlated with plasma carotenoid concentration. Obviously, the users had higher skin carotenoid levels as well as plasma carotenoid concentration than non users. The noninvasive View scanner is feasible to evaluate the carotenoid plasma profiles.
Enhanced bioaccessibility and transport of reduced coenzyme Q10 is dependent on GSH status in Caco‐2 human intestinal epithelial cells (1044.8)
Authors: Faillai Mark , Chitchumroonchokchai Chureeporn
Published: June 2021

Recently, the bioavailability of orally administered reduced CoQ10 (CoQH) was reported to be markedly greater than that of oxidized CoQ10 (CoQ10) in healthy adults. We have investigated the basis for the influence of redox state on absorption using the coupled in vitro digestion/Caco‐2 cell model. Aliquots of CoQH and CoQ10 in hydrogenated castor oil were added to yogurt before simulated gastric and small intestinal digestion. Partitioning of CoQH in aqueous (i.e., bioaccessible) fraction of chyme was significantly greater than that of CoQ10. Similarly, when diluted bioaccessible fraction was added to the apical compartment of monolayers of Caco‐2 cells, uptake and trans‐epithelial transport of CoQH were 2.3 and 3.4 times greater than that of CoQ10, respectively. Reduction of cellular GSH significantly decreased both uptake and transport of CoQH and CoQ10 by Caco‐2 cells, although the extent of inhibition was much greater for CoQH. This suggests that maintaining a reduced intracellular environment is essential for efficient absorption of the CoQ. Auranofin‐mediated inhibition of thioredoxin reductase activity also decreased apical uptake and transport of CoQH, but not CoQ10. However, this differential effect was associated with auranofin‐associated oxidation and degradation of micellar CoQH in apical medium rather than changes in intracellular GSH status. These data suggest that the increased bioavailability of CoQH compared to CoQ10 results from its greater micellarization, more efficient uptake, and enhanced trans‐epithelial transport across the epithelial barrier, and that the cellular processes are dependent on a high ratio of GSH to GSSG for maintaining CoQ in the reduced state.
Capscoside G suppresses adipogenesis by regulating the expression of adipogenic transcription factors and the activation of AMP‐activated protein kinase (1045.12)
Authors: Sung Jeehye , Lim Yongrae , Lee Junsoo
Published: June 2021

Obesity is a health hazard which is closely associated with various complications including insulin resistance and type 2 diabetes. Capsicoside G isolated from pepper seeds is widely known for antioxidant and anti‐inflammatory activities. However, the mechanism for its anti‐adipogenic effect has largely remained unknown. The objective of this study was to elucidate anti‐adipogenic mechanism of capsicoside G. Preadipocyte differentiation was induced in the presence or absence of capsicoside G for 4 days. After differentiation intracellular lipids were stained with Oil Red O and colorimetric intensity was measured. The expression of proteins associated with adipogenesis was measured using western blotting. The mRNA levels of fatty acid oxidation genes were also confirmed by real‐time PCR. Capsicoside G dose‐dependently inhibited intracellular lipid droplet accumulation in adipocyte without cytotoxicity. Treatments with capsicoside G inhibited the expression of the key adipocyte differentiation regulators and their target genes such as fatty acid binding protein (aP2). Moreover, capsicoside G significantly increased activation of 5’‐adenosine monophosphate‐activated protein kinase (AMPK), a major regulator of cellular energy balance, phosphorylation. This study also investigated the involvement of AMPK in the expression of a major transcription factor, PPARγ. Pretreatment with compound C, a cell permeable inhibitor of AMPK, abolished the inhibitory effects of capsicoside G on PPARγ expression. Moreover, capsicoside G suppressed the expression of glycerol synthesis enzyme GPAT‐1 and increased expression of fatty acid oxidative enzyme CPT‐1. These results suggest that capsicoside G exerts anti‐adipogenic effect on adipocyte differentiation through the activation of the AMPK signaling pathway. Capsicoside G may be a promising compound for the treatment of obesity.
Role of polyphenol‐rich blackcurrant and black chokeberry extracts in the stimulation of transintestinal cholesterol excretion in vitro (1045.22)
Authors: Kim Bohkyung , Park Young‐Ki , Koo Sung , Lee Ji‐Young
Published: June 2021

We previously reported that polyphenol‐rich blackcurrant extract (BCE) and black chokeberry extract (CBE) altered the expression of genes involved in transintestinal cholesterol excretion (TICE) of LDL‐derived cholesterol in Caco‐2 cells. BCE and CBE increased the expression of LDL receptor (LDLR) via post‐transcriptional and transcriptional mechanisms, respectively. As mechanistic target of rapamycin complex 1 (mTORC1) is known to increase LDLR protein by inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) that promotes LDLR degradation, we explored this pathway to gain mechanistic insight into the post‐transcriptional regulation of LDLR by BCE. PCSK9 mRNA and protein were drastically increased, while LDLR was markedly induced, by BCE and CBE. However, when Caco‐2 cells were treated with rapamycin, an mTORC1 inhibitor, together with 100 μg/mL of BCE or CBE, rapamycin partly abolished the induction of LDLR protein by BCE, but with no effect on CBE‐induced LDLR protein. Importantly, BCE significantly increased TICE of LDL‐derived cholesterol by ~30% compared to control. The results indicate that BCE induces TICE via the post‐transcriptional induction of LDLR expression partly by mTORC1‐dependent, but PCSK9‐independent, pathway in the intestine.
Clove extract regulates energy metabolism in differentiated C2C12 cells (1045.38)
Authors: Tu Alex , Moss‐Pierce Tijuana , Jiang Alan , Ford Paul
Published: June 2021

An incidence of diabetes is rising dramatically worldwide. Plants and herbs have been used for the treatment of diabetes for centuries in folk medicine. Clove extract (CE) has been shown to respond similar to insulin by repressing genes encoding gluconeogenic enzymes; however, the mechanisms are essentially unknown. We investigated the effects of clove on metabolism in differentiated C2C12 myotubes and demonstrated that CE significantly increased glucose consumption as well as phosphorylation of AMP‐activated protein kinase (AMPK) and its substrate, acetyl‐CoA carboxylase (ACC). Additionally, CE transcriptionally regulated genes involved in metabolism were upregulated including SIRT1 (2‐fold) and PPARγ coactivator 1α (PGC1α) 2‐fold. Furthermore, treatment of CE dose‐dependently increased muscle glycolysis up to 23% and mitochondrial spare respiratory capacity up to 45%. Overall, our study suggested that CE potentially increases muscle glycolysis and fatty acid oxidation by activating the AMPK and SIRT1 pathways.
Assessment of NETosis in patients with primary immunodeficiencies: evidence for a ROS‐independent pathway (1046.6)
Authors: Byrd Angel , O'Brien Xian , Johnson Courtney , Zarember Kol , Sampaio Elizabeth , Laforce‐Nesbitt Sonia , Bliss Joseph , Newburger Peter , Holland Steven , Reichner Jonathan
Published: June 2021

Neutrophil‐mediated host defenses against pathogens include the extrusion of a lattice of DNA and microbicidal enzymes known as Neutrophil Extracellular Traps (NETs). Since the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with extracellular matrices, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of the purified fungal‐associated molecular pattern, β‐glucan, by normal human neutrophils causes rapid (蠄 30 mins) homotypic aggregation and NET release by a mechanism that requires Fn. NET formation to Candida albicans hyphae depends on β‐glucan recognition by CR3 (CD11b/CD18), requires Fn and ERK but not production of ROS, and results in hyphal destruction. To better understand the genotypic and phenotypic correlations of NET disgorgement in the host defense we assayed responses to Fn + β‐glucan by neutrophils from various patient populations including: GATA2 deficiency, Chronic Granulomatous Disease (CGD), Leukocyte Adhesion Deficiency (LAD), STAT3 loss‐of‐function mutation, STAT1 gain‐of‐function mutation, IL‐12R β2 chain mutation, IFNγ receptor mutation, IRAK 4 deficiency, and neonatal PMNs. Surprisingly, and in contrast with previous reports, CGD and neonatal patient neutrophils formed aggregates and NETs even when treated with DPI to block residual ROS production. These data support our previous findings that the respiratory burst is dispensable for PMN aggregation and NET extrusion. Mechanisms involving STAT1, STAT3 and GATA2 may potentially mediate Fn + β‐glucan‐induced NETosis or aggregation, whereas IRAK4, IL‐12 and IFNγ do not appear to play a major role. Further studies will challenge patient cells with C. albicans to better understand mediators of phagocyte responses and help elucidate the mechanisms in rapid release of NETs to fungal pathogens.

Grant Funding Source: Supported by NIH Grant GM066194; A.S. Byrd is supported by the UNCF/Merck Graduate Fellowship
The effects of extracorporeal shockwave therapy on matrix metalloprotease activity in tendinopathy (1046.8)
Authors: Waugh Charlie , Jones Eleanor , Riley Graham , Langberg Henning , Morrissey Dylan , Screen Hazel
Published: June 2021

INTRODUCTION Extracorporeal shockwave therapy (ESWT) is an effective, non‐invasive treatment for degenerative tendinopathies, however, the biological mechanisms underpinning its therapeutic effects are largely unknown.

METHODS The local tissue response of matrix metalloproteinase (MMP) ‐2 and ‐9 (gelatinases involved in extracellular matrix remodelling) to ESWT was examined in 18 healthy (18 Achilles) and 10 degenerative (4 Achilles, 6 patellar) tendons using microdialysis. Dialysate samples were collected from the peritendinous space before and after a single ESWT session. Analysis of MMP‐2 and ‐9 activities were completed using gelatin zymography.

RESULTS Active MMP‐9 increased ~350% after ESWT, whereas pro‐MMP‐9 was not detected in our samples. There was a ~200% increase in a heavy MMP‐2 complex (~135 kDa; likely an MMP‐2 dimer) after ESWT when compared to baseline, however, levels of pro‐ and active MMP‐2 monomers did not change significantly. There were no response differences between healthy and degenerative tendons.

CONCLUSION These findings provide evidence of the biological mechanisms underpinning ESWT in humans in vivo. Our findings are consistent with studies examining MMP activity after exercise and suggest that the mechanical stimulus provided by ESWT might initiate tendon remodeling in tendinopathy by promoting the catabolic processes required to remove damaged matrix constituents.

Grant Funding Source: Engineering and Physical Sciences Reserach Council Knowledge Transfer Partnership
The template tissue arrayer ő a simplified, low cost tissue microarray instrument (1048.15)
Authors: Hewitt Stephen , Morris Jennifer , Ylaya Kris
Published: June 2021

The use of tissue microarrays (TMAs) has enabled high‐throughput analysis of tissue at the molecular level. Widely used for immunohistochemistry, as well as other in situ detection based methodologies; TMAs have become vital for discovery and validation of new biomarkers. Manual TMA instruments remain expensive, and require a level of technical expertise that prohibits many researchers from adequately accessing the technology. Automated instruments have been developed, however complexity and cost have substantially prevented widespread adoption. A template‐based instrument has been designed and developed that allows the arraying of moderate‐density (96 sample) TMAs with 1.0 mm needles. Test of the instrument demonstrate that all cores are aligned perpendicular to the surface of the array. The template and needle design ensures that cores are aligned flat at the surface of the array. The instrument is smaller than 8 x 10 x 3 inches, and weighs less than 2 pounds. Additional templates are currently in development that allows the use of 1.5 and 2.0 mm needles as well as a specialized TMA template for the production of TMAs for controls in a clinical immunohistochemistry setting.

Grant Funding Source: Supported by the Intramural Research Program of the NIH, NCI, CCR
Immunoblotting analysis of formalin‐fixed and paraffin‐embedded malignant tumor samples (1048.16)
Authors: Celes Mara , Soave Danilo , Freitas Ana Caroline , Ramos Simone , Ribeiro‐Silva Alfredo
Published: June 2021

Introduction: Formaldehyde‐fixed, paraffin‐embedded (FFPE) tissue remains the gold standard technique used for preservation and storage of tissue specimens for pathologic analysis. FFPE‐tissues represent invaluable resources for conducting retrospective disease investigations. Several studies have been undertaken to obtain usable quantities of proteins from FFPE‐samples.

Objective: This study was performed to describe and analyze the potential usefulness of protein extraction protocol from paraffined samples of tumor lesions (salivary gland and lung).

Methods: Paraffin was removed from tissue samples 15 micrometers thick sections with an organic solvent, followed by protein extraction by a modified RIPA lysis buffer and solubilization in 5% SDS. The resultant protein extract was resolved by SDS‐PAGE and submitted to immunoblot analyses using the antibodies to CD31 and GAPDH.

Results: SDS‐PAGE and immunoblot analysis presented usable quantities of protein. However, FFPE‐tissues preserved for 10 years presented less usable proteins than FFPE‐samples preserved for 2 years. In addition, protein extraction from salivary glands tissue provided more usable protein than the lung tumor tissues.

Conclusion: In summary, our investigation presented evidence that the evaluation of protein extraction in FFPE‐samples can be used to provide improved information about tumors protein expression. We also believe that some factors such as tissue density, kind and time preservation of FFPE‐samples may influence the quality and quantity of usable proteins.

Grant Funding Source: Supported by FAPESP and FAPEG
New en bloc staining technique improve tissue contrast and electrical conductivity (1050.2)
Authors: Negron‐Morales Celimar , Torres‐Vazquez Irma , Serrano‐Velez Jose , Powell Richard , Rosa‐Molinar Eduardo
Published: June 2021

Here we describe results of our work to develop a conductive resin and en bloc staining technique that together overcome the challenges of “charging” and “electron beam damage” posed by three‐dimensional (3D) electron‐based imaging methods such as serial‐section electron tomography. A conductive epoxy resin optimized for infiltration, immunocytochemistry, and uniform serial‐sectioning did not infiltrate evenly throughout the tissue and did not section well; however, it provided tissue section stability and eliminated tissue section charging, beam damage, and shrinkage. This led to our development of a new en bloc staining technique to use with the epoxy resin. Prior to embedding tissue in a non‐conductive resin, we made the tissue‐electron‐conductive and non‐charging. Together, the resin and the staining technique increase tissue contrast and electrical conductivity and overcome challenges posed by charging, radiation, and ion beam‐and electron‐induced surface contamination/artifacts. New tools such as the ones described here are needed to overcome the challenges of new technologies and take advantage of the increased resolution they provide. Supported by NS‐080687; GM‐108470; MH‐086994; NSF‐1062963, and NSF‐0964114.
Birc6 drives drug resistance and cell survival in chronic myeloid leukemia (1055.6)
Authors: Okumu Denis , Levine Merlin , Jones Lauren , Zhang Raymond , Graves Lee
Published: June 2021

BIRC6 is a member of the inhibitors of apoptosis proteins (IAPs), a family of functionally and structurally related endogenous proteins known to inhibit apoptosis. High levels of BIRC6 have been reported in various cancer cells. However, little is known about the mechanism of its regulation. We examined BIRC6 expression and how it correlates with drug resistance in Imatinib‐resistant MYL‐R, MYL, and K562 chronic myeloid leukemia (CML) cells. These cell lines are characterized by the increased tyrosine‐kinase activity of Lyn and BCR‐ABL, respectively. Inhibiting Lyn using Dasatinib significantly reduced BIRC6 mRNA and protein expression. Because STAT5 phosphorylation was elevated in MYL‐R cells, we tested the effects of STAT5 knockdown in these cells. STAT5 knockdown reduced the expression of BIRC6 in MYL‐R cells. Additionally, treating K562 or MYL‐R cells with the STAT5 inhibitor pimozide produced similar results, suggesting that STAT5 is an intermediary in the signaling pathway initiated by Lyn or BCR‐ABL. Transfecting K562 and MYL‐R cells with BIRC6 shRNA significantly decreased BIRC6 protein expression. Importantly, treating BIRC6 knockdown cells with imatinib resulted in a large increase in caspase‐3 activation and cell death. Our data suggest that BIRC6 plays an important role in mediating drug resistance and may be a novel and promising STAT5‐ regulated target in CML and other cancers.

Grant Funding Source: NIH
The β3 subunit: a key regulator of vascular calcium channel expression (1057.10)
Authors: Srivastava Anup , Song Li , Kharade Sujay , Fletcher Terry , Rhee Sung , Rusch Nancy
Published: June 2021

The pore‐forming α1C subunit of the vascular L‐type Ca2+ (CaL) channels upregulates during hypertension, which may contribute to increased arterial tone. Recently we reported that a small subunit of the CaL channel, β3, is required for the upregulation of vascular CaL channels and the development of hypertension. Here, we begin to explore the hypothesis that the CaL channel α1C subunit is subjected to rapid proteasomal degradation in VSMCs, but during hypertension, β3 rescues α1C from degradation to increase CaL channel abundance. Western blots (WB) of cultured A7r5 aortic VSMCs treated with β3 morpholino antisense (0.5 and 1 µg/ml) for 48 hr revealed a loss of the CaL channel α1C pore. Subsequently α1C band was detected in A7r5 cells on an anti‐ubiquitin WB and blockade of proteasomal degradation by MG132 (1‐10 µg/ml) for 18 hr increased CaL channel expression by 2‐fold. Since chloroquine (100‐200 μM), an inhibitor of lysosomal degradation, did not alter CaL channel expression after 24hr suggesting that CaL channel half‐life (t1/2) is primarily determined by proteosomal degradation in A7r5 VSMCs. We also designed a new method to study the t1/2 of CaL channels in vivo, which indicated α1C is rapidly degraded with a t1/2 of ~3 hr in mesenteric arteries of normotensive rats. Future studies are designed to define the degradation pathway of vascular CaL channels in vivo and identify possible abnormalities in hypertension.

Grant Funding Source: Funded by NIH R01 HL064806 (NJR)
Vascular endothelial growth factor A evokes distinct calcium entry by promoting surface accumulation of Orai3 (1057.5)
Authors: Bruns Alexander , Li Jing , Hou Bing , Turnover Sarka , Moss Nicholas , McKeown Lynn , Foster Richard , Beech David
Published: June 2021

Vascular endothelial growth factor A (VEGF‐A) regulates endothelial cell function and angiogenesis via activation of vascular endothelial growth factor receptor 2 (VEGFR2). One consequence of VEGFR2‐activation is calcium entry into endothelial cells. The responsible pathways are, however, not fully understood. Here we show that the VEGF‐A‐evoked calcium response in human umbilical vein endothelial cells (HUVEC) is dependent on Orai3, a membrane protein reported to form calcium entry channels. VEGFR2 activation and subsequent arachidonic acid release from the plasma membrane caused forward trafficking of Orai3 to the cell surface and association with STIM1. Knockdown of Orai3 via siRNA caused inhibition of endothelial tube formation in vitro and in vivo, indicating a potential role in angiogenesis. Our findings provide evidence for a previously unrecognised Orai3 mechanism that contributes importantly to VEGF‐A signalling.

Grant Funding Source: Supported by the Medical Research Council.
Sweet Patterns:Decoding glycan control mechanisms using systems‐based approaches. (106.2)
Authors: Mahal Lara
Published: June 2021

Carbohydrates play crucial roles in a diverse array of biological processes from viral pathogenesis to tumor cell metastasis. Glycosylation is an inherently complex system whose regulation is poorly understood. Using our glycomic analysis platform, lectin microarrays, on the NCI‐60, a set of human tumor lines, my laboratory has integrated glycomic and genomic information to identify new glycan control mechanisms. Specifically, we have shown that miRNA are a major regulator of the human glycome. By integrating our glycomic dataset with miRNA expression data using a multidimensional analysis, we discovered unique associations of miRNA and glycan structures that map onto the miRNA regulation of genes involved in glycan biosynthetic pathways. We have validated these pathways and begun to interrogate further the relationship between miRNA and the glycome. This has led to a series of discoveries of miRNA controlled glycosylation networks impacting cell fate. Overall our work argues that glycans are both outcomes and inputs into these regulatory networks, providing new insight into the glycome.
Elucidating the role of inhibitory G‐protein, Gz, in β‐cell preservation and regeneration (1062.3)
Authors: Brill Allison , Pasker Renee , Thompson Mary , Cadena Mark , Brar Harpreet , Neuman Joshua , Kimple Michelle
Published: June 2021

Type 1 diabetes (T1D) occurs when β‐cell death causes insufficient β‐cell mass to maintain normoglycemia. Although select T1D patients are candidates for transplantation, no pharmaceutical cure for T1D exists. Such cures would augment the residual β‐cell mass had by most, if not all, T1D patients. We showed the α subunit of the heterotrimeric Gz protein, Gαz, inhibits production of cAMP, a second messenger proposed to potentiate β‐cell function and mass. In an obesity‐linked T2D model, we demonstrated islets from Gαz‐null mice have constitutively increased β‐cell cAMP production, insulin secretion, and replicative capacity. We hypothesized Gαz‐null mice subjected to chemical induction of T1D would have improved β‐cell replication, survival, and, ultimately, mass, especially when treated with drugs known to stimulate cAMP production. To test our hypothesis, we induced diabetes in mice with streptozotocin. The Gαz‐null mutation partially protected against hyperglycemia, as did treatment with the known cAMP potentiator, exendin‐4. Combining the Gαz‐null mutation with exendin‐4 treatment was completely protective. These pancreas sections had both increased β‐cell replication and decreased apoptosis. In vitro assays with novel Gαz variants are pinpointing critical downstream signaling pathways. Overall, our results support targeting Gαz signaling to preserve and regenerate functional β‐cell mass.

Grant Funding Source: Supported by the Juvenile Diabetes Research Foundation
Modulation of the epithelial barrier by a fragment of Clostridium perfringens iota‐toxin (1062.5)
Authors: Iguchi Daisuke , Hayaishi Tomohiro , Krug Susanne , Watari Akihiro , Fromm Michael , Yagi Kiyohito , Kondoh Masuo
Published: June 2021

Epithelial cell sheets have two types of intercellular tight junctions (TJs): bicellular tight junctions (bTJs) and tricellular tight junctions (tTJs). Modulation of bTJs has been investigated in numerous studies; however, modulation of tTJs has not been attempted. Amongst other components, tTJs contain the tetraspan protein tricellulin and a singlespan protein, the lipolysis‐stimulated lipoprotein receptor (LSR). LSR was recently identified as a receptor for Clostridium perfringens iota‐toxin b (Ib) which suggests that a receptor binding domain of Ib might modulate the epithelial barrier by interacting with tTJs. Here, we used the putative receptor binding domain corresponding to amino acids 421‐664 of Ib (Ib421‐664) to investigate whether inhibition of LSR modulates the epithelial barrier. Ib421‐664 bound to LSR‐expressing L‐cells, but not to mock cells. Immunofluorescence analysis revealed that Ib421‐664 colocalized with LSR in LSR‐expressing cells and was also bound in CaCo‐2 cells. Treatment of CaCo‐2 cell monolayers with Ib421‐664 decreased the transepithelial resistance, a marker of epithelial barrier integrity. Removal of Ib421‐664 recovered the barrier integrity. Additionally, permeability for paracellular flux markers was also increased by treatment with Ib421‐664. These findings indicate that Ib421‐664 could be a first modulator of tTJs.

Grant Funding Source: supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan
Lanthanide resonance energy transfer‐based distance measurements in the mammalian glutamate transporter excitatory amino acid transporter 3 (1064.12)
Authors: Saha Kusumika , Venkatesan Santhosh , Sohail Azmat , Stockner Thomas , Sandtner Walter , Ecker Gerhard , Sitte Harald
Published: June 2021

EAAT3 (Excitatory amino acid transporter 3) mediates the regulation of synaptic transmission by reuptake of glutamate in the synaptic cleft. The motivation of the study is to gain insight into the structure function relation of EAAT3. The project utilizes the high resolution crystal structure of GltPh, the bacterial orthologue of Pyrococcus horykoshi to mammalian glutamate transporters. The structural rearrangement of the protein is caused by the helical movements which will be assessed by distance measurements using the technique of lanthanide resonance energy transfer (LRET). LRET relies on Förster's theory of resonance energy transfer where there is distance dependent transfer of energy between the donor fluorochrome to an acceptor fluorochrome. The protein is expressed in Xenopus laevis oocytes. Lanthanide binding tags (LBT) has been inserted into the protein to chelate the lanthanide terbium which serves as the donor element and aminoacid residues in the protein are mutated to cysteine which then reacts to an acceptor dye serving as acceptor. The measured distances allow us to understand the structure function relationship of the glutamate transporters and can be further investigated using different substrates and inhibitors. The results obtained in this project will allow us to better understand pathophysiological conditions associated with loss of function in EAAT3, for instance in ischemia.

Grant Funding Source: F3506 and W1232 MolTag
NADPH oxidase mediates altered vascular responses in allergic mice (1065.10)
Authors: Ponnoth Dovenia , Mustafa S Jamal
Published: June 2021

Asthma is associated with adverse cardiovascular outcomes, though the cause is unclear. We have shown vascular inflammation and altered vascular responses in allergic mice (Ponnoth et al., AJP 2008, 2010). In this study, we investigated the contribution of NADPH oxidase to angiotensin II (Ang II)‐mediated aortic responses in a mouse model of allergic asthma developed in this laboratory. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups. Mice were sensitized (i.p.) on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11‐13. Ang II type 1 receptor (AT1) antagonist losartan was given i.p. as a single bolus dose (20mg/kg) on day 14 to SEN mice. Organ bath studies were done on day 14 and Western Blot analyses were done to determine aortic expression of AT1, NOX 1, 2 and 4 as well as p22phox and p67phox, the subunits of NOX. In the SEN group, Ang II (10μM) produced higher aortic contraction (33.48±3.26% vs. 18.06±3.07% in CON; p<0.05) that was lowered with the use of apocynin (10μM; 33.48±3.26% vs.13.11±6.89), while no effect was observed in CON. SEN mice had lower endothelial response to acetylcholine (30.95±5.73% vs. 57.6±4.3% in CON; p<0.05), that was improved with i.p. losartan (56.1±4.39%). AT1 expression was unchanged in SEN mice. However, the expression of NOX4 (1.27±0.06 vs. 0.99±0.09) and p22phox (3.45±0.35 vs. 1.35±0.62) were significantly higher in SEN mice compared to CON. Treatment with losartan significantly attenuated the increases in NOX4 (from1.27±0.06 to 0.67±0.11) and p22phox (from3.45±0.35 to 0.26±0.11) in SEN mice. These data suggest that most likely NOX4, through p22phox association, may contribute to altered Ang ll and endothelial responses in allergic mice, and that losartan can improve these outcomes.

Grant Funding Source: Supported by HL027339, HL094447
J20619 inhibits PDGF‐BB‐stimulated vascular smooth muscle cell proliferation and migration (1065.2)
Authors: Fang Lian‐hua , Guo Jing , Li Li , Wu Yu‐jie , Yan Yu , Xu Xiao‐na , Wang Shou‐bao , Yuan Tian‐yi , Du Guan‐hua
Published: June 2021

J20619 is a natural pigment isolated from the heartwood of Traditional Chinese Medicine. J20619 has been reported to exhibit various biological activities including anti‐platelet aggregation, anti‐inflammation, and vasorelaxation, etc. However, the effects of J20619 on vascular smooth muscle cell (VSMC) remain unexplored. The present study investigated the effects of J20619 on platelet‐derived growth factor (PDGF)‐BB induced VSMC proliferation and migration and its possible mechanisms. VSMC proliferation and migration were measured by Crystal Violet Staining, wound‐healing and Boyden chamber assays, respectively. Cell cycle was analyzed by flow cytometry. Enzymatic action of matrix metalloproteinase‐9 (MMP‐9) was carried out by gelatin zymography. These study demonstrated that pretreatment with J20619 dose‐dependently inhibited PDGF‐BB stimulated VSMC proliferation and migration, which were associated with cell‐cycle arrest at G0/G1 phase, reduction in the adhesion molecule expression and MMP‐9 activation in VSMCs. Furthermore, the increase in PDGF‐Rβ, Src, ERK1/2 and Akt phosphorylation induced by PDGF‐BB were suppressed by J20619. These findings indicate that J20619 inhibits PDGF‐BB induced VSMC proliferation and migration, and the inhibitory effects may be associated with the blockade of PDGF‐Rβ‐ERK1/2 and Akt signaling pathways. In conclusion, the present study suggests that J20619 may be useful as an anti‐proliferative agent for the treatment of vascular diseases.
Thrombin‐bound dabigatran modulates protease‐activated receptor‐1 expression (1066.14)
Authors: Chen Buxin , Soto Antonio , Goss Ashley , VanRyn Joanne , Trejo JoAnn
Published: June 2021

Thrombin is the key effector protease of the coagulation cascade and major drug target for anticoagulation therapy. Dabigatran is a synthetic oral direct thrombin inhibitor and is currently being used for multiple thromboembolic disease indications. Dabigatran binds with high affinity to free and clot‐bound thrombin, suggesting that dabigatran‐bound thrombin may modulate expression and activity of protease‐activated receptor‐1 (PAR1), the major effector of thrombin signaling. The N‐terminal LDPR‐S sequence of PAR1 is recognized by thrombin’s active site, which is inhibited by dabigatran. However, a second interaction occurs between thrombin’s exosite‐I, which is spatially separated from the active site, and the “hirudin”‐like sequence present in the N‐terminus of PAR1. In this study, we examined whether thrombin‐bound dabigatran regulates PAR1 expression and function using culture human endothelial cells and other cell models. As expected, we found that dabigatran inhibited thrombin cleavage of PAR1, blocked activated PAR1 internalization and G protein signaling. Intriguingly, however, prolonged incubation of cells with dabigatran‐bound thrombin resulted in elevated expression of PAR1 at the cell surface. These findings suggest that thrombin‐bound dabigratan modulates PAR1 expression and future studies are directed towards understanding the biological consequences of this effect.

Grant Funding Source: Supported by Boehringer Ingelheim International Collaborative Research Agreement and NIH/NIGMS R01
Two approaches to the pressure‐volume relationship after traumatic brain injury using respiratory stimuli (1068.17)
Authors: Haubrich Christina
Published: June 2021

How to determine the individual pressure‐volume relationship after traumatic brain injury? We have tested two different approaches based on respiratory stimuli.Analysis of 51 TBI patients of which 29 were tested with 30min of moderate hypocapnia (4.4±3.0kPa) and 22 patients were monitored. The analysis was based on intracranial pressure (ICP), Doppler flow velocity (FV) in the middle cerebral arteries, and arterial blood pressure (ABP). Parameters included transfer function Gain for respiratory blood pressure waves (R waves; 9‐20 cpm), vasomotor response (VMR) to moderate short‐term hypocapnia, as well as indices of the pressure ‐ volume reserve (RAP).The four day monitoring of spontaneous ICP changes has shown that rising ICP dampened the transfer of respiratory waves to cerebral vasculature. Moderate short‐term hypocapnia on the other hand, has decreased FV and ICP. Both testing conditions have shown that the largest impact of ICP increase on R wave gains (dGain /dICP right: 0.14±0.06; left: 0.18±0.08) as well as the the largest impact of the hypocapnic VMR on the ICP levels (left:1.8±0.6, right:2.2±0.9 cm/s/kPa/mmHg) were associated with an exhausted p/v reserve (RAP>0.8). The p/v compliance can be assessed by the interaction between intracranial compartments ‐ as between brain parenchyma and cerebral vasculature.
The cytokines TNF, MCP‐1 and CINC‐1 mediate diminished dilation of middle cerebral artery after ischemic stroke in rats (1068.2)
Authors: Broskova Zuzana , Anthony Daniel , Bagi Zsolt
Published: June 2021

The level of inflammatory cytokines TNF, Monocyte Chemoattractant Protein (MCP‐1) and Cytokine‐Induced Neutrophil Chemoattractant (CINC‐1) is elevated after ischemic stroke (IS). The vasomotor effects of TNF, MCP‐1 and CINC‐1 on cerebral arteries have not yet been fully elucidated after IS. We aimed to evaluate the direct effect of these cytokines on MCA diameter under normal conditions and also after IS. In isolated, pressurized middle cerebral arteries (MCA) of normal Wistar rats, TNF, CINC‐1 (10‐14 ‐ 10‐11 M) and MCP‐1 (10‐16 ‐ 10‐13 M) elicited vasodilation (max: 26±3%, 21±4% and 28±7%, respectively), which was enhanced after endothelial denudation. In rats with transient MCA occlusion, a well‐known model of IS (90 min occlusion and 48 reperfusion) we observed a significantly reduced dilation to TNF, MCP‐1 and CINC‐1 in the ipsilateral MCA, whereas constriction of contralateral MCA was seen in response to TNF and CINC‐1. We also found reduced dilations of both ipsilateral and contralateral MCA in response to bradykinin and serotonin, but not to the NO‐donor, sodium nitroprusside in IS rats. Thus, TNF, MCP‐1 and CINC‐1 elicit endothelium‐independent vasodilation in MCA, which is significantly reduced after IS. Our data suggest reduced dilator or even enhanced constrictor signaling from damaged endothelium and indicate important role for these cytokines in regulating cerebral perfusion after IS.

Grant Funding Source: Supported by NIH HL104126
The impact of aging on cerebral vasomotor reactivity to carbon dioxide (1069.5)
Authors: Riley Jonathan , Tarumi Takashi , Parker Rosemary , Armstrong Kyle , Tinajero Cynthia , Zhang Rong
Published: June 2021

The objective of this study was to determine the age‐related differences in cerebral vasomotor reactivity (CVMR) to carbon dioxide. Methods: 92 healthy adults aged 23‐77 underwent measurements of cerebral blood flow velocity (CBFV) using transcranial Doppler, mean arterial pressure, and end‐tidal CO2 (EtCO2) during baseline, hyperventilation (HVT), and modified rebreathing (RB). CVMR was determined by the maximal gain between changes in normalized CBFV (CBFV%) and EtCO2 using a 3rd order polynomial model and its derivative. Results: Lower CBFV and EtCO2 were related to increasing age at baseline. HVT decreased EtCO2 by 21±4 mmHg while RB increased by 19±3 mmHg. Increasing age was related to smaller hypocapnic but larger hypercapnic changes in CBFV% at the end of HVT and RB, respectively. The maximal gain between CBFV% and EtCO2 did not differ by age. Advanced age was associated with greater differences in EtCO2 between the baseline and the level at which the maximum gain in CBFV% occurred. Conclusions: These findings suggest that age‐related reductions in baseline CBFV and EtCO2 may lead to a decrease in hypocapnic but an increase in hypercapnic CBFV response. The larger differences in EtCO2 between baseline and the maximal gain of CBFV% with aging may lead to blunted response of CBF to given changes in arterial CO2 at levels close to baseline.

Grant Funding Source: Supported by National Institute on Aging
Enhanced endothelial nitric oxide production impairs cerebrovascular tone after brain trauma (1070.1)
Authors: Villalba Nuria , Longden Thomas , Nelson Mark , Wellman George , Freeman Kalev
Published: June 2021

Enhancend endothelial nitric oxide production impairs cerebrovascular tone after brain trauma

Nuria Villalba, Thomas Longden, Mark T. Nelson, George C. Wellman and Kalev Freeman

Department of Pharmacology, University of Vermont, Burlington, VT Cerebral autoregulation is altered after traumatic brain injury (TBI) reflecting an impairment in myogenic tone and that may contributeing to patient morbidity in patients with TBI. We hypothesized that TBI alters nitric oxide (NO)‐dependent signaling mechanisms leading to decreased cerebral vascular tone. We studied isolated cerebral arteries from adult rats after moderate fluid percussion TBI or sham surgery. We found that both endothelial and smooth muscle (SM) NO levels—indexed by 4,5‐diaminofluorescein (DAF‐2) fluorescence—were increased in cerebral arteries from TBI animals. Arteries from TBI animals exhibited decreased cytosolic SM Ca2+ and reduced myogenic tone compared to controls. Endothelial removal restored myogenic response constriction in TBI animals. Further, inhibition of NO synthase with Nω‐L‐arginine (L‐NNA) restored both myogenic tone and SM Ca2+, and reduced DAF‐2 fluorescence in arteries from TBI animals. The guanylyl cyclase inhibitor (ODQ) and protein kinase G (PKG) inhibitor (RP‐8‐Br‐cGMPS) both elicited robust enhanced constrictions in TBI animals. Further, constriction caused by inhibition of SM large‐conductance Ca2+‐activated potassium (BK) channels with paxilline was augmented in TBI arteries. Addition of 30 nM clamped NO to control arteries provided vasodilation equivalent to that observed in TBI and increased the DAF‐2 signal to a comparable level observed in untreated TBI preparations. These data demonstrate that trauma causes persistent changes in endothelial NO production underlying profound cerebral artery dilation, and provides a quantitative measure of the degree of NO elevation, which is on the order of 30 nM.
Rhythmic variations in BKCa channel activity, [Ca2+]i level and PKC‐delta expression in rat cerebral arterial muscle cells (1070.6)
Authors: Gebremedhin Debebe , Rarick Kevin , Harder David
Published: June 2021

Objectives: The objective of the present study is to investigate preence of rhythmic variation in KCa single‐channel activity, intracellular Ca2+ ([Ca2+]i ) level and PKC‐δ protein expression in rat cerebral arterial muscle cells.

Methods. Although the underlying signaling mechanisms have not been elucidated yet, a circadian variation in the occurrence of cerebrovascular events and higher stroke incidence during morning hours has been reported. In the present study we investigated and compared changes in activities of a 238 pS Ca2+‐activated K+ channel (KCa) current, levels of intracellular Ca2+ ([Ca2+]i ) and PKC‐δ expression in freshly isolated rat cerebral arterial muscle cells during the early afternoon (2:00 PM) and in the early morning (2:00 AM) using the cell‐attached mode of the Patch Clamp technique, the ratiometric fluorescent dye Fura‐2AM to measure [Ca2+]i. and western blotting, respectively.

Results. We found that the isolated cerebral arterial muscle cells exhibited altered NPo of a 238 pS KCa channel current (0.0038 ± 0.0004 at 2:00 PM and 0.0086 ± 0.0003 at 2:00 AM (n=5. P < 0.05) when recorded at a patch potential of +40 mV using symmetrical KCl solution, changed [Ca2+]i levels (172 ± 21 nM at 2:00 AM and 343 ± 76 nM at 2:00 PM, n= 3 trials, p < 0.05) and reduced expression of PKC‐δ protein at 2:00 AM compared to that determined at 2:00 PM of the day.

Conclusions. Given that activities of KCa channel currents, [Ca2+]i level and PKC‐δ activity are requisite for the initiation and development of cerebral arterial myogenic tone, the observed rhythmic changes in these endogenous signaling events may be regarded as possible contributing factors for altered vascular reactivity and increased incidence of cerebral stroke at different times of the day.