bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Vacuolar H+‐ATPase Inhibition and the a4 Isoforms on Secretion and Activation of Cathepsin D in MDA‐MB231 Breast Cancer Cells
Authors: Swickard Jamie Nicole , Henry Abhijeet , Hinton Ayana 
Published: April 2020

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are important for acid‐base balance in the blood and sperm maturation. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. V‐ATPases are hypothesized to create a localized low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells are involved in metastasis by cleaving extracellular matrix proteins and activating other proteases, which facilitates tumor invasion. We are interested in a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor showed a trend of reducing the activity of secreted cathepsin B, but not cathepsin L. However, that trend, was not statistically significant. Cathepsin D is another protease that is secreted by cancer cells including metastatic breast cancer. For this study we looked at the activity of cathepsin D following inhibition of V‐ATPases. Our preliminary data indicates that inhibition of V‐ATPases reduces the activity of secreted cathepsin D. To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for the a4 isoform of the V‐ATPase “a” subunit. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. We hypothesize that the a4 isoform knockout cells will have reduced activation of secreted cathepsin D while not effecting activation of cathepsin L. Separate abstracts submitted by our lab are focused on the a1, a2 and a3 isoform knockouts.Support or Funding InformationAnderson Summer Research Assistantship
MMP‐12 knockdown during acute and chronic phases promotes post‐stroke neurological recovery
Authors: Nalamolu Koteswara Rao , Challa Siva Reddy , Fornal Casimir A. , Martin Ryan C. , Olson Elsa A. , Ujjainwala Ammar L. , Klopfenstein Jeffrey D. , Pinson David M. , Wang David Z. , Kalyanasundaram Ramaswamy , Vemuganti Raghu , Veeravalli Krishna Kumar 
Published: April 2020

Matrix metalloproteinases (MMPs) are known modulators of ischemic brain damage. We recently showed that ischemic stroke in adult rodents induces MMP‐12 in brain during both acute and chronic phases of ischemic stroke. We also showed that MMP‐12 is detrimental during acute phase as it promotes BBB disruption leading to brain damage. In the present study, we evaluated the significance of elevated MMP‐12 during the chronic phase after focal ischemia on neurological recovery. Adult male Sprague‐Dawley rats were subjected to a 2h transient middle cerebral artery occlusion (MCAO). These rats were administered with either a single dose (at 10–15 min of reperfusion) or 3 doses (at 10–15 min, 7 days and 14 days of reperfusion) of plasmids expressing either MMP‐12 shRNA or a scrambled control shRNA (1 mg/Kg; i.v.; n = 9–11/group) in nanoparticle formulation. To assess reflexes, balance, sensory and motor functions, rats were subjected to modified neurological severity scoring, adhesive removal test, beam walk test and rotarod test at 1, 7, 14 and 21 days after reperfusion. The cohort of rats treated with a single dose of MMP‐12 shRNA showed significantly better functional recovery as compared to control shRNA cohort. Longer‐term suppression of MMP‐12 also resulted in better functional recovery than control shRNA cohort. Thus, these studies indicate that induction of MMP‐12 during both acute and chronic phases after focal ischemia contributes to neurological deficits and hinders recovery.Support or Funding InformationThis work was supported by the NIH Grant 1R01NS102573‐01A1 to KKV
Impact of Aging and G Protein‐Coupled Estrogen Receptor Deletion in Arterial Stiffening and Cardiac Function in Male and Female Mice
Authors: Ogola Benard Ojwang , Zimmerman Margaret A. , Harris Nicholas R. , Kilanowski-Doroh Isabella , Groban Leanne , Lindsey Sarah 
Published: April 2020

Arterial stiffening assessed by pulse wave velocity (PWV) independently predicts cardiovascular events and is exacerbated in aging men and women. Our previous studies demonstrated that G protein‐coupled estrogen receptor (GPER) plays a protective role in the heart and vasculature. The current study evaluated sex differences and the impact of GPER and aging on arterial stiffening and cardiac function. We hypothesized that genetic deletion of GPER induces sex differences in arterial stiffness and cardiac function during aging. Male and female wildtype (wt) and global GPER knockout (ko) mice were used between 4–6 months (adult) and 12–13 months (middle‐aged; n=6–17 per group). Echocardiography was performed under isoflurane anesthesia in short axis view to assess cardiac function, and local PWV was obtained within the carotid artery. Data was subjected to 3‐way ANOVA and correlation analysis. PWV was significantly higher in adult male ko versus wt mice (1.60 ± 0.07 m/s vs. 1.21 ± 0.05 m/s, P<0.001) but was not altered by GPER deletion in female mice. Aging significantly increased PWV (P<0.0001) in both males and females. PWV did not correlate with ejection fraction (P=0.69) or heart rate (P=0.89) but inversely correlated with early left ventricular filling deceleration time (Edec time; P=0.0005, r= −0.37) and left ventricular ejection time (LVET; P=0.044, r= −0.22) and positively correlated with relative wall thickness (RWT; P=0.008, r= 0.281). In conclusion, GPER deletion impacted PWV in adult male mice, while aging increased PWV in all groups. Future studies will investigate whether other pathological conditions such as hypertension reveal a phenotype of vascular stiffness in female GPER ko mice. The inverse relationship between PWV and Doppler‐derived indices of left ventricular distensibility underscores the important relationship between arterial and cardiac stiffness.Support or Funding InformationNIH Grant # R01HL133619 to SHL
Annotation of Contig53 on Chromosome 3L of Drosophila takahashii
Authors: Garcia Janette , Fung Siaumin 
Published: April 2020

DNA wraps around histones to form chromatin in the nucleus of eukaryotic cells. DNA densely packed histones, heterochromatin, is associated with silencing or transcription inactive genes. While, the loosely packed chromatin, euchromatin, is associated with actively transcribed genes. The F element, dot chromosome in Drosophila melanogaster is composed of mostly heterochromatin, yet has active transcribed genes. The goal of our research is to elucidate the regulatory motif within the dot chromosome by comparing the dot chromosome genes with gene located in the euchromatin, D element. In this study, Contig 53 in D element from Drosophila takahashii is characterized with 7 features. Through the creation of a gene model or outline of where exons and introns begin and end, instances of where regulatory motifs occurs can be observed. The Genome Browser allows for visualization of the predicted genes and isoforms. BLAST allows for the alignment of genes between the two species. FlyBase contains genetic and molecular data for Drosophila melanogaster. By annotating and comparing different contigs or gene segments of the two species and the divergence between two different species D. melanogaster and D. takahashii can be visualized as well as any silencing of genes that has occurred through evolution. Our research shows that although many of the jim isoforms vary slightly, data can be correlated with RNA seq, Gene scan predications, and TopHat predictions. Of 11 total isoforms of D. melanogaster, seven share almost 90% similarity to D. takahashii. This shows that through evolution, contig 53 of D. takahashii and D. melanogaster have only diverged marginally.Support or Funding InformationThis project was supported in part by grants from the Department of Education (Award #P031S150199), from Southern California Edison, and through Vanguard University’s SURP Program generously supported by the Office of the Provost and the Institute for Faculty Development.”Special thanks to the Genomics Education Partnership (GEP)
Elimination of Wnt Secretion from Hepatic Stellate Cells Impairs Hepatocyte Proliferation After Partial Hepatectomy
Authors: Hu Shikai , Ko Sungjin , Kim Min-wook , Preziosi Morgan , Bell Aaron , Monga Satdarshan 
Published: April 2020

Wnt‐dependent β‐catenin activation contributes to hepatocyte proliferation following 2/3 partial hepatectomy (PH) through regulation of Cyclin‐D1 (Ccnd1) expression, as we have previously shown using β‐catenin or Wnt co‐receptors, low‐density lipoprotein receptor‐related protein 5–6, conditional knockouts (KO). However, given the redundancy between the 19 mammalian Wnt proteins and various cell sources for Wnt secretion, the precise mechanisms of Wnt signaling in liver regeneration (LR) are not completely understood. Recently, using endothelial cell (EC)‐specific deletion of Wntless (Wls), a cargo receptor required for Wnt secretion, we have reported that sinusoidal ECs secrete Wnt2 and Wnt9b to activate β‐catenin in hepatocytes and enhance Ccnd1 expression, which is indispensable for normal and optimal proliferation of hepatocytes during LR after PH. Alternatively, conditional deletion of Wls in hepatocytes or cholangiocytes did not impact LR, whereas Wls deletion in macrophages only marginally affected LR post‐PH. Here, to investigate the roles for hepatic stellate cell (HSC)‐secreted Wnts in LR, we characterize the HSC‐Wls‐KO, generated by interbreeding Wls‐floxed and Lecithin Retinol Acyl Transferase (LRAT)‐driven Cre (Lrat‐Cre) transgenic mice. HSC‐Wls‐KO mice showed no effect on liver weight or β‐catenin‐dependent pericentral gene expression at baseline. However, hepatocyte‐specific CCND1 protein normally evident in midzonal hepatocytes, was diminished in baseline KO liver as also evident by WB, whereas Ccnd1 expression was unaltered as seen by RNA‐seq and qPCR, suggesting posttranslational regulation. No difference in CCND1 protein was seen in the non‐parenchymal cells of the KO livers when compared to WT. After PH, HSC‐Wls‐KO showed modest decrease in gene expression of Ccnd1 at only 24 hour (h) post‐PH as compared to WT but comparable levels between the two groups were seen at 40h and 72h. While similiar PCNA+ staining in hepatocytes was observed at 40–72h post‐PH in HSC‐Wls‐KO and WT, HSC‐Wls‐KO displayed significantly diminished BrdU+ or pHH3+ hepatocytes at 40h, which rebounded by 72h, suggesting delayed regeneration due to the impaired hepatocyte replication.ConclusionAt baseline, Wnt ligands secreted by HSCs are essential for CCND1 protein expression in midzonal hepatocytes but not for pericentral zonation. Following PH, HSCs‐derived Wnts also contribute to normal Ccnd1 expression and hepatocyte mitosis.Support or Funding InformationThis work was supported by NIH grants 1R01DK62277, 1R01DK100287, R01CA204586 and Endowed Chair for Experimental Pathology to S.P.M., and by NIH grant 1P30DK120531‐01 to Pittsburgh Liver Research Center (PLRC) and by PLRC Pilot & Feasibility grant PF 2019‐05 to S.K.
Mechanistic Insights into pH‐sensing GPCRs
Authors: Rowe Jacob , Kapolka Nick , Taghon Geoffrey , Morgan William , Isom Daniel 
Published: April 2020

Many aspects of protein function, cell biology, human physiology, and disease are influenced by pH changes. However, our mechanistic understanding of pH effects in most of these areas is limited. This is especially true in the case of cell signaling, for which very few pH‐sensing receptors and signaling cascades have been identified. To address this issue, we are studying how pH regulates G protein‐coupled receptors (GPCRs), the largest and most therapeutically targeted family of cell‐surface receptors in humans. Unlike most cellular proteins, GPCRs are regularly exposed to a wide range of dynamic pH values (from pH 7.4 on the cell surface, to pH 5.0 in endosomes) and can be chronically exposed to acidic microenvironments caused by cancer and processes such as inflammation. While there are more than 800 GPCR family members, only three receptors (i.e. GPR4, GPR65, and GPR68) are known to be activated directly by pH. In this study, we used structural informatics and cell‐based signaling assays to predict and validate residues responsible for pH sensing by these receptors. As part of these efforts, we engineered and interrogated 44 GPR4, GPR65, and GPR68 point mutations in a library of 440 cell strains that covered all possible GPCR‐Gα subunit signaling combinations. This comprehensive strain library included 14 mutations predicted by our structural informatics software, pHinder, and the full set of 30 mutations for these receptors available in the literature. Our results for the known mutants agree with previous reports and expand our understanding of the relationship between Gα‐signaling specificity and pH sensing. More importantly, several of our newly predicted mutations appear to have a greater influence on the pH‐sensing capabilities of these receptors than any of the previously reported GPR4, GPR65, and GPR68 mutations to date. Most of the pH‐sensitive residues predicted by pHinder were found within the protein core, a region that has been largely overlooked when considering pH‐sensing properties of GPCRs. Our cell‐based experiments confirmed that these residues are critical for pH sensing and provide a more thorough knowledge‐base for understanding GPCR pH sensing. We anticipate that the outcome of these studies will enable us to more comprehensively quantify the extent of pH sensing across the GPCRome and rationally engineer new pH‐sensing GPCRs for use in industry, synthetic biology, and medicine.Support or Funding InformationR35GM119518R01NS103906R03TR002908
The influence of high fat, high cholesterol diet on expression of myocardial remodelling biomarkers in the mouse model of atherosclerosis ApoE−/−
Authors: Kowara Michał Krzysztof , Czarzasta Katarzyna , Jędrzejewski Michał , Koperski Łukasz , Segiet Agnieszka , Cudnoch-Jędrzejewska Agnieszka Barbara 
Published: April 2020

IntroductionDespite the significant development of cardiology, little is still known about the molecular basis of myocardial remodelling under conditions of high fat, high cholesterol diet, which is known to be proatherogenic.PurposeThe aim of the project was to investigate whether a diet with high amount of fat and cholesterol contributes to changes in the expression of proteins regulating the structure of extracellular matrix in the heart muscle.MethodsThe study was conducted upon 20 ApoE−/− mice (Charles Rivers Laboratories, Inc.), which from the 10th week of age for the next 14 weeks were fed a standard diet (5% fat, 19.5% casein, Animalab) (n = 10 – control group) or high fat, high cholesterol diet (21% fat, 0,15% cholesterol, 19.5% casein; Animalab) (n = 10 – experimental group). At the end of the experiment, mice were euthanized to collect the heart for immunohistochemical analysis. In myocardium, levels of matrix metalloproteinase 1 (MMP‐1), matrix metalloproteinase 2 (MMP‐2), matrix metalloproteinase 9 (MMP‐9) and pro‐inflammatory cytokines ‐ interleukin 1β (IL‐1β), interleukin 6 (IL‐6) and interleukin 17 (IL‐17) were determined. In addition, the levels of type I collagen, type III collagen and tissue metalloproteinase inhibitor type 1 (TIMP‐1) were also examined in the myocardium. Consent was obtained from The Local Animal Research Ethics Committee (20/2015).ResultsApoE−/− mice in the experimental group had significantly higher body weight compared to the ApoE−/− mice in the control group. ApoE−/− mice in the experimental group showed significantly higher levels of MMP‐9 in myocardium compared to control mice. In contrast, TIMP‐1 expression was significantly higher in the control group compared to the experimental group. The high fat, high cholesterol diet also contributed to significantly higher expression of type I collagen and type III collagen in myocardium in the ApoE−/− mice. We also showed that the mice in the experimental group had significantly lower myocardial IL‐6 levels compared to the mice in the control group.ConclusionHigh fat, high cholesterol diet significantly affected the level of proteins regulating extracellular matrix structure in the ApoE−/− mice. It suggests, that the diet influences myocardial remodeling. This subject is worth of further investigations due to potential clinical implications.Support or Funding InformationThis research was supported by the Medical University of Warsaw, Young Researcher’s Grant (1MA/PM1/17/17).
High‐precision and Scalable Fabrication of Biomimetic Culture Environments for Replicating the Extracellular Matrix In Vitro
Authors: Geisse Nicholas A. , Fisher Elliot C.F. , Gray Kevin , Ghazizadeh Hamed , Smith Alec S.T. , Kim Deok-Ho 
Published: April 2020

In vivo, cell and tissue organization is driven by a complex interplay of cells and their environment, including the extracellular matrix (ECM). The ECM can provide varied cues such as structure, protein content, mechanical stiffness, stretch, and electrical stimulation to influence cell and tissue development and function. Traditional in vitro cell culture environments—typically composed of non‐dynamic hard and unstructured glass or plastic—fail to fulfill the roles of the ECM in development. Consequently, many in vitro cell models fall short in correctly reproducing critical in vivo phenotypes as evidenced when cultured cells lose type‐specific characteristics or express phenotypes indicative of an immature developmental stage. Considerable effort is directed at fabricating biomimetic culture environments that maintain or promote mature phenotypes. However, making biomimetic substrates typically involves costly or hard‐to‐reproduce techniques that are often incompatible with many standard assays. These challenges are further compounded when using high‐throughput assays. In this study, our objective is to develop a host of techniques for recapitulating certain aspects of the ECM in vitro. To replicate the shape and stiffness of the extracellular niche, we first developed novel nanopatterning techniques based on high‐precision photolithography methods that are highly reproducible, scalable, and amenable to integration with most industry‐standard endpoint assays, including high‐NA microscopy. Our data demonstrate that a variety of cell types‐‐including cardiac, skeletal, endothelial, neuronal, and cancer‐‐can cue off of patterns that mimic the fibrous structure of the native ECM. We then extended this biomimetic patterning technique to deformable elastomeres which permitted the addition of mechanical strain to recapitulate mechanical signals present in the ECM of specific tissues. Our data show that mechanical strain can orient skeletal muscle cells and tissues along the axis of stretch, resulting in a polarized orientation that is similar to what is seen in vivo but is not recapitulated in traditional culture environments. Last, we demonstrate that a combination of ECM patterning, mechanical stretch, and electrical pacing can reproduce the extracellular environment of the intact myocardium to facilitate studies of induced pluripotent stem cell‐derived cardiomyocytes. We conclude that our approach is a viable method for recreating specific aspects of the ECM that are critical for driving the development and maturation of cells in culture in a cell, instrument, and assay‐agnostic manner.Support or Funding InformationNational Institutes of Health Small Business Innovation Research Grant.
What influences research medical students?
Authors: Elizondo-Omaña Rodrigo Enrique , Muñoz-Leija Milton Alberto , Quiroga-Garza Alejandro , Salinas-Alvarez Yolanda , Gutierrez-de la O Jorge , Zarate-Garza Pablo Patricio , Martinez-Garza Javier Humberto , Guzman-Lopez Santos 
Published: April 2020

IntroductionThere are several influencing factors that aid in promoting student involvement in scientific research. Due to the steady decrease of medical researchers over time, identifying these factors would provide valuable data regarding research productivity and attitudes among medical students. Our objective is to determine the influencing factors in undergraduate students who were already interested in research.Materials and methodsA cross‐sectional study was performed, applying a survey validated by the Delphi method on students involved in the anatomy research group established in our University. Our school has a 6‐year program, and the group was established 17 years ago. Informed consent was obtained from all participants. The survey was voluntary and anonymous,ResultsA total of 85.5% of the 90 surveyed students answered with a mean age of 20.5±2 years. Most students still belonging to their 2nd year, and just starting to get involved, without an active research project. Most had as a goal to contribute new knowledge (45.5%) as well as to the scientific community (42.9%). Their primary limitation is the lack of time. More than half of the students mentioned a professor/doctor was their primary motivation to get involved in research, and half also mentioned interest due to a peer already involved. Only 1.3% have considered a full career in research.ConclusionProfessors are the primary source of motivation for student recruitment, preferably those that lead by example. These same students who get involved in research will also play an important part in leading by example their younger peers. Universities should focus on these two variables to resolve the rapid decrease or physician‐researchers, as well as nourishing the mentor‐mentee relationship.