bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vitro Reversal of Multidrug Resistance in Cancer
Authors: Holcomb-Webb Kasi L. , Gard Gabrielle E. , Vogel Pia D. , Wise John G. 
Published: April 2020

Multi‐drug resistance (MDR) occurs when cancer cells become resistant to multiple chemotherapeutic drugs that are functionally and structurally different. MDR usually leads to failure of chemotherapy treatment and subsequently to poor patient prognosis. Cancers can become resistant to antineoplastic drugs by over‐expressing one or more ATP‐binding cassette (ABC) transporters that act as drug efflux pumps and expel xenobiotics from cells. One such protein is the Breast Cancer Resistance Protein (BCRP, or ABCG2), which is believed to confer MDR to several types of cancer. Inhibition of BCRP cellular activity is therefore thought as a way to restore sensitivity to chemotherapeutics. In previous work, we identified several potential inhibitors of BCRP using high‐throughput in silico screens. Using cell viability and cellular accumulation assays of BCRP substrates, we assessed these compounds for toxicity to cells and the ability to reverse MDR in the BCRP‐overexpressing breast cancer cell line, MCF7‐M100. Here we expand our studies of the potential BCRP inhibitors to two other BCRP‐overexpressing cell lines, the BCRP overexpressing colon cancer and non‐small cell lung cancer cell lines, S1M1‐80 and MX20, respectively. For each cell line, the level of BCRP overexpression was assessed at RNA and protein level and the MDR phenotype was confirmed using cell viability assays. The effects of our newly discovered inhibitors on reversing MDR in these two cell lines is reported.Support or Funding InformationThis work is supported by the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D., of Naples, FL.
Role of Calcium in Proteolytic Activity of a Type I Metacaspase from Schizophyllum commune
Authors: Fox Kristin M. , Mineo Charlotte R. 
Published: April 2020

Metacaspases are members of the C14 class of cysteine proteases that also includes the caspases. They are found in fungi, plants and protozoa, but not in animals. Unlike the caspases, metacaspases cleave after positively charged residues such as Lys and Arg and are activated by calcium ions. The role of metacaspases in vivo is not yet clearly defined. They have been implicated in cell death and protein quality control, and recent work in A. thaliana suggests that they are activated in response to plant wounding as part of a primitive immune system. Our work centers on understanding how the Type I metacaspases of the fungus Schizophyllum commune are activated by calcium ions. Metacaspase 1 (MC1) activity can be reduced by >90% by mutation of conserved aspartic acid residues implicated in calcium binding. A homology model for MC1 based on the structures of Type I metacaspases from S. cerevisiae and T. brucei was refined. Using the model we identified a loop near the active site that also contains the putative calcium binding site and may undergo conformational changes upon calcium binding. Mutation of a residue in the hinge region of this loop from Gly to Ala significantly reduced enzymatic activity. Kinetic solvent viscosity effects suggest that at low calcium concentrations the enzymatic reaction is limited by substrate binding, while at high calcium levels product release becomes rate‐limiting. We will combine these data to present a model for the role of calcium in Type I metacaspase activation.Support or Funding InformationCRM was supported by a Scholarship from the Arnold and Mabel Beckman Foundation
Interrogating the Mechanism of Eukaryotic Initiation Factor 3 (eIF3) through Molecular Dissection
Authors: Yoo Kyungyoon , Aitken Colin Echeverría 
Published: April 2020

Eukaryotic translation initiation is the rate‐limiting and most regulated step of protein synthesis. It begins with the formation of a pre‐initiation complex (PIC) on the small (40S) ribosomal subunit. The PIC is then recruited to the 5′ end of an mRNA, from which it scans in the 5′ to 3′ direction to identify the start (AUG) codon. This convoluted pathway is mediated by multiple protein factors called eukaryotic initiation factors (eIFs).eIF3, the largest and the most complex of these protein factors, is comprised of five essential subunits (eIF3a/b/c/g/i). Previous genetic and biochemical studies have shown that eIF3 mediates PIC formation, is essential for mRNA recruitment, and participates in scanning for the start codon. eIF3 has also been shown to play a critical role in the stabilization of mRNA in the exit channel and contributes to the rate of recruitment at the entry channel. This latter role of eIF3 may involve its interaction with the 40S latch elements Rps2/3 and h16/h18. In fact, mutations of Rps3 exacerbate 40S dependence on eIF3 for stabilizing mRNA binding by the PIC. Using an in‐vitro reconstituted system, we are interrogating the interaction between eIF3 and the 40S latch by combining Rps2/3 mutations and mutations within elements of eIF3 thought to interact the latch.To further dissect the role of the eIF3 entry and exit‐channel arms, we are reconstituting yeast eIF3 by recombinant expression in bacteria. Previous work has relied on eIF3 purified from yeast cells. This new approach will alleviate the limitations previously imposed by the essential nature of the eIF3 subunits. Our initial work will examine the activity of reconstituted eIF3 in mRNA recruitment and PIC stabilization. Then, we will focus on a set of mutations targeted at interrogating the many functions of eIF3: truncation of a region of eIF3c thought to promote accurate start‐codon selection; isolation of the eIF3a/c heterodimer that stabilizes mRNA at the ribosomal exit channel; isolation of a eIF3b‐g‐i complex found near the mRNA entry channel; and truncation of an RNA binding domain of eIF3b. The effects of these mutations on component events during translation initiation will shine new light on the role of eIF3 and its subunits.
A Novel Data Analysis Pipeline for Bottoms‐Up Mass Spectrometry Proteomic Analysis of Primary Tissues
Authors: Silverwood Tyler , Radosavljevic Adriana , Charntikov Sergios , Chu Feixia 
Published: April 2020

Modern mass spectrometry‐based proteomic studies require reliable, and robust data analysis for a comprehensive and quantitative profile of histone post‐translational modifications (PTM). Studies involving primary tissue samples face additional challenges in comparative sample analysis, due to various protein concentrations, multiple modification sites and modification states. A novel data analysis pipeline was developed in order to streamline the analysis process and normalize samples of varying concentration. This is relevant in the proteomic analysis of addiction, where various modification isoforms can correlate with different addiction phenotypes. A self‐administration model addiction study of methamphetamine (METH) addiction was used to develop this pipeline.Following nuclear protein isolation, proteins underwent tryptic digestion and histone H3 samples were derivatized with propionic anhydride. Identification, validation, and label‐free quantification of histone modifications was performed. Protein loading of different samples was normalized, using high abundance, proteotypic unmodified peptides. To localize changes in specific site and state, peptide isoforms for each site‐ and state‐specific modification were binned. Using this approach, we were able to identify histone modifications that underwent statistically significant changes in METH self‐administrated rat brain samples. In addition, we were able to identify histone modifications that are indicative of METH controllability in tested individual subjects. This data analysis pipeline has led to robust analysis of histone PTMs. Information on changes in site‐ and state‐specific histone modifications can be integrated with RNA‐seq data to elucidate driving chromatin pathways in the formation and maintenance of pathophysiological epigenetic states.Support or Funding InformationThis project was supported in part by the University of New Hampshire Hamel Center for Undergraduate Research.
Role of MAB21L2 in Ocular Disease
Authors: Klestinski Keith , Malloy Joseph , Patel Siddharth , Colella Benedetto , Homberg Logan , Khan Taha , McLaughlin Andrew , Panichikudiyil Abraham , Sajanlal Ahmed Ali , Scaffidi Isaiah , Warner Luke , Kaiser Carl 
Published: April 2020

MAB21L2 is a protein linked to fetal eye development. The MAB21L2 encoding gene is found on the fourth human chromosome and is found in all vertebrate species. Exome sequencing in human cousins affected by coloboma, microcornea, cataracts, and skeletal dysplasia identified an p.Arg51Gly variant in MAB21L2. Sanger sequencing confirmed that five affected family members were heterozygous for the wild‐type p.Arg51Gly variant, while unaffected family members were wild‐type, thus identifying this variant as a cause of coloboma, a disease characterized by missing tissue in one or more structures of the eye in humans. Studies using zebrafish models confirms the role of this highly conserved gene in ocular development. The affected amino acid (Arg 51) is conserved across all family members and vertebrate species.The crystal structure for MAB21L2 has yet to be successfully solved. The MUHS SMART team submitted four amino acid sequences from the MAB21L2 gene to I‐TASSER, a bioinformatics method for predicting three‐dimensional protein structure, in order to study the effect of the p.Arg51Gly variant on protein structure and function. The first submissions contained human and zebrafish wild‐type amino acid sequences, while the second contained the human and zebrafish p.Arg51Gly variant. The predicted protein structures were 3‐D printed in order to visualize any structural differences between the wild‐type and variant proteins. Through JMOL we focused on the residue 51 mutation site. The mutation site was found on the exterior of the protein. Modeling results showed that in the mutant type residue 51 was glycine rather than the arginine which is typical of the wild type. About 48% of the protein is made up of alpha‐helices and 15% of the protein is made up of beta‐sheets. Research conducted by the Semina Lab of the Medical College of Wisconsin through Yeast Two Hybrid assays found that a protein top2b interacted with wild‐type MAB21L2 but the interaction appeared to be weakened in the mutant. Other proteins typically involved with misfolded proteins that could lead to degradation (hspa8 and hspa5) both interacted with mutant but not wild‐type MAB21L2.Future research into the effects of the protein mutation may provide a useful step towards better understanding of ocular development and ultimately finding cures for various ocular diseases. Possible treatment of this protein mutation may involve gene‐editing technology like CRISPR‐Cas9 to replace the mutated amino acid.
Interactions between chemical and environmental factors and bacterial community composition in a Great Lakes watershed
Authors: Cooper Carolyn , DeLoach Idgena , McMorris John , Slater Adam , Brokus Sarah , Wade Randall , Best Aaron , Krueger Brent , Pikaart Michael 
Published: April 2020

Urbanization and agricultural activity have degraded the water quality of the Great Lakes and their tributaries. These impacts include nutrient and fecal coliform loading and high sediment levels. In this presentation, we report on three years of continuous weekly monitoring of the Macatawa watershed in Ottawa and Allegan Counties, Michigan, a drowned river mouth entering Lake Michigan. Five lake locations and seven stream sites have been analyzed for microbiological content by community 16S rRNA sequencing, fecal indicator bacteria presence, and Escherichia coli whole‐genome sequencing. Along with these biological indicators, we profile chemical and physical parameters including dissolved oxygen, nutrients, temperature, and pH. We find continued high phosphorus, total suspended solids, and E. coli levels, exceeding total maximum daily load targets, or other benchmarks, for the watershed. Patterns in microbial communities show variation influenced by season and geographic location; for example, total E. coli increases in the stream tributaries in summer but decreases in the lake fed by those streams relative to winter. Furthermore, overall diversity of microbial communities increases in late fall and winter. These data provide a baseline for monitoring remediation efforts in the Macatawa Watershed. We suggest these will serve as a comparison for hypereutrophic watersheds around the nation and to aid decisions about remediations and public access to these recreational waters.Support or Funding InformationNSF RUI 1616737
PPP6C Regulation of ERK Signaling in Melanoma
Authors: Cho Eunice , Lou Hua Jane , Turk Benjamin E 
Published: April 2020

Melanoma, the deadliest form of skin cancer, is characterized by aberrant hyperactivation of the ERK mitogen‐activated protein kinase signaling pathway. Inhibitors of BRAF and MEK1/2, both members of this pathway, have shown clinical efficacy and are used in combination to treat BRAFV600E melanoma, the most common melanoma genomic subtype. However, not all BRAFV600E melanomas respond to these inhibitors, and those that do respond eventually acquire resistance. To better understand mechanisms of drug susceptibility in BRAFV600E melanoma, we performed a loss‐of‐function screen to identify kinases and phosphatases that modulate sensitivity to two clinical MEK1/2 inhibitors. In this screen, we identified PPP6C, the catalytic subunit of protein phosphatase 6 (PP6), as a factor promoting sensitivity to MEK1/2 inhibition. PPP6C is a potential tumor suppressor mutated in 7–12% of melanomas, and has roles in cell cycle regulation and DNA damage repair. shRNA‐mediated knockdown and CRISPR/Cas9‐mediated knockout of PPP6C in BRAF mutant melanoma cells both lead to hyperactivation of MEK1/2 and ERK1/2, resulting in decreased sensitivity to pathway inhibitors. This finding was confirmed in a panel of BRAFV600E melanoma cell lines. Through in vitro biochemical and cell‐based studies, we have established that PP6 directly dephosphorylates MEK1/2. As with complete loss of PPP6C, cells expressing most recurrent cancer‐associated PPP6C mutants similarly have hyperactive ERK signaling. Our studies support a novel negative regulatory role for PPP6C in ERK signaling that contributes to its activity as a tumor suppressor and promotes pathway inhibitor sensitivity in BRAFV600E melanoma.Support or Funding InformationFunding was provided by the Gruber Foundation, T32 GM007324, and F31 CA220999.
Rapid effects of aldosterone and phosphatase inhibition on water and urea permeabilities
Authors: Wang Yanhua , Ma Fuying , Klein Janet D. , Sands Jeff M. 
Published: April 2020

Aldosterone promotes renal sodium uptake, thereby increasing water reabsorption. But our previous data showed that aldosterone decreases UT‐A1 urea transporter expression, suggesting aldosterone could inhibit water reabsorption. In this study, we investigated the rapid effect of aldosterone on vasopressin‐stimulated osmotic water permeability in rat inner medullary collecting ducts (IMCDs). In IMCDs from male Sprague Dawley rats weighing 50–75g, we saw that aldosterone significantly decreased osmotic water permeability from 152 ± 21 to 122 ± 17 μm/s (45 min, n=4, p<0.01). Given the rapid nature of the response, we further investigated whether the effect of aldosterone might be through phosphatase‐mediated modulation of transporter phosphorylation. Inhibition of PP1/PP2a phosphatases by calyculin increased basal osmotic water permeability from 58 ± 8 to 92 ± 14 μm/s (n=4, p<0.01). In the presence of vasopressin, stimulation by calyculin remained significant in both male and female rats 77 ± 10 to 119 ± 15 μm/s (30 min, n=6, p<0.01). Addition of aldosterone to the bath in the presence of calyculin reversed the increase in osmotic water permeability from 119 ± 15 to 89 ± 10 μm/s (45 min, p<0.01). Inhibition of pp2B with tacrolimus also stimulated water permeability (88 ± 13 to 108 ± 19 μm/s), which was reversed by addition of aldosterone (81 ± 10 μm/s). Urea permeability was also increased by both calyculin (25 ± 7 × 10−5 to 32 ± 7 × 10−5 cm/s; n=3, p<0.05) and tacrolimus (15 ± 1 × 10−5 to 18 ± 1 × 10− 5 cm/s; n=4, p<0.05). Aldosterone decreased pser256‐ and increased pser261‐AQP2. Inhibition of PP2B by tacrolimus increased pser256‐ and decreased pser261‐AQP2. Calyculin increases pser256‐AQP2 and this was reversed by aldosterone. Treatment of inner medullary tissue with 50 nM calyculin or 62 nM tacrolimus resulted in significantly increased UT‐A1 phosphorylation. We conclude that aldosterone is able to rapidly inhibit water and urea reabsorption, possibly by altering phosphatase activity and changing the phosphorylation states of AQP2 and UT‐A1.Support or Funding InformationAHA 18CDA34060053NIH/NIDDKRO1‐DK41707
Investigating Polycystin‐2‐mediated mitophagy as a stress response through its role in NAFLD
Authors: Brill Allison L. , Kuo Ivana Y. , Ehrlich Barbara E. 
Published: April 2020

Non‐alcoholic fatty liver disease (NAFLD), the most common chronic liver disease worldwide, is characterized by aberrant dysregulation of cellular energy mechanisms, including defective autophagy and mitochondrial function. Inhibiting general autophagy in models of NAFLD has produced contradictory results, resulting in both protection against and causation of hepatic steatosis and insulin resistance. However, the specific role of autophagy to selectively degrade mitochondria—the process known as mitophagy—has not been studied in the context of NAFLD development. The main goals of this project are to establish the function of dysregulated mitophagy in the development of NAFLD, and to determine how Polycystin‐2 (PC2), a Ca2+‐modulating protein on the endoplasmic reticulum (ER) membrane, works to promote hyper‐activation of mitophagy through its interaction with Bcl‐2 associated athanogene 2 (BAG2). My interest in PC2 and BAG2 is based on published and preliminary data demonstrating 1) increased PC2 and BAG2 expression in human and murine livers with NAFLD, 2) BAG2 as an interacting partner of PC2 that promotes PTEN‐induced putative kinase 1 (PINK1)‐mediated mitophagy following PC2 up‐regulation, and 3) protection against insulin resistance and hepatic steatosis in PC2 haploinsufficient mice fed a high fat diet (HFD). In this work, I examine how PC2 is up‐regulated under stress to modulate mitophagy and mitochondrial function, as well as how PC2‐null hepatocytes respond to HFD‐induced NAFLD, and determine how observed mitochondrial morphological and functional alterations that occur in hepatocytes following the onset of NAFLD are altered by PC2 and BAG2. These results will confirm PC2 as a stress response protein capable of regulating mitophagy, and will help resolve the role of dysregulated autophagy in NAFLD development.Support or Funding InformationFunding provided by T32GM007324 and F31DK118836.
Alternately Translated Product of Cx37 Fails to Arrest Growth in Rin Cells but Alters Cx37 Hemichannel Function when Co‐Expressed
Authors: Alamban Andrew , Fye Margret , Pontifex Tasha , Burt Janis 
Published: April 2020

Connexin proteins facilitate transmembrane and intercellular communication via channel formation (hemichannel) and intercellular docking of apposed hemichannels to form gap junction channels, respectively. Additionally, they play a role in determining growth phenotype (e.g., arrest, proliferation, death). Connexin 37 (Cx37), expressed in connexin‐deficient rat insulinoma (Rin) cells induces growth arrest or cell death in a manner dependent on the phosphorylation of specific carboxyl terminus (CT) residues. Notably, phosphorylation at serine 319 (S319) is both necessary and sufficient for growth arrest, and mimicking phosphorylation with aspartate substitution at either S275 or S321 is sufficient to induce cell death. 13 of the 21 members of the connexin gene family have a conserved methionine at position 213; in Cx43 mRNA, this methionine codon serves as an alternate translation start site that produces a CT‐spanning translation product. This 20kDa (20k) product interacts with Cx43, chaperoning its trafficking to the membrane. Here we test the hypotheses that 1) this conserved codon in Cx37 mRNA also serves as an alternate translation start site to produce a 13kDa product (13k) that, when expressed alone, regulates growth phenotype of Rin cells and 2) when co‐expressed with Cx37, regulates its channel function. To test these hypotheses, we expressed V5‐tagged 13k or one of two phosphomimetic mutants, 13k‐S321D and 13k‐S275D, in Rin cells or in Rin cells expressing Cx37. Despite a similar phosphorylation profile to Cx37, as determined by mass spectrometry, 13k did not arrest Rin cell growth and neither phosphomimetic mutant induced cell death nor growth arrest. In co‐expressing cells, immunoprecipitation (IP) of 13k failed to co‐IP full‐length Cx37, suggesting no direct interaction between these isoforms; however, 13k and the phosphomimetic mutants altered Cx37 hemichannel conductance and open probability properties, suggesting an indirect interaction of 13k isoforms with Cx37. In conclusion, these data suggest that while 13k and phosphomimetic mutants alone are insufficient to alter the growth phenotype of Rin cells toward growth arrest or death, they nevertheless lead to altered channel properties of full‐length Cx37. The findings herein will inform further research in understanding how connexins are regulated and, in turn, the role they play in regulating cell growth phenotypes.Support or Funding InformationR01 HL131712 | Undergraduate Biology Research Program | University Funds
Obesity in Vascular and Alveolar Morphogenesis after Pneumonectomy
Authors: Mammoto Tadanori , Hendee Kathryn , Mammoto Akiko 
Published: April 2020

Obese population is rapidly growing worldwide. Obesity is associated with chronic lung diseases including chronic obstructive pulmonary disease (COPD). Impairment of lung regeneration and repair contributes to the pathogenesis of COPD. Angiogenesis – the formation of new blood capillaries ‐ plays a key role in organ regeneration. Inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and obesity is accompanied by endothelial cell (EC) dysfunction. However, the effects of obesity on lung vascular and alveolar regeneration remain unclear. Compensatory lung growth and vascular and alveolar morphogenesis after PNX are suppressed in leptin‐deficient ob/ob mice treated with high‐fat diet compared to that in control lean mice. The levels of the major angiogenic factor, vascular endothelial growth factor (VEGF) are higher in the serum and the brown adipose tissue collected from post‐PNX mice compared to those from sham‐operated control mice, while these effects are attenuated in post‐PNX leptin‐deficient ob/ob mice with high‐fat diet. The serum collected from post‐PNX control mice stimulates EC sprouting, while EC sprouting is inhibited when treated with serum collected from leptin‐deficient ob/ob mice after PNX. These results suggest that obesity may inhibit post‐PNX regenerative lung growth through VEGF signaling. Modulation of VEGF signaling may be the efficient strategy to restore lung regeneration and repair in obese population.Support or Funding InformationNIH R01HL142578