bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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.
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
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
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 traumaNuria Villalba, Thomas Longden, Mark T. Nelson, George C. Wellman and Kalev FreemanDepartment 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.