bluuebunny/crossref_metadata_2025_split huggingface dataset:
Peritoneal mesothelial cells give rise to myofibroblasts via mesothelial‐mesenchymal transition in peritoneal fibrosis (60.10)
Authors: Lua Ingrid , Asahina Kinji 
Published: June 2021

Mesothelial cells (MCs) cover the surface of the internal organs, and the peritoneal cavities as it have a phenotype intermediate between epithelial and mesenchymal cells, expressing markers indicative of both cell types. Peritoneal mesothelium is used as a semipermeable barrier for the exchange of toxic substance in peritoneal dialysis, a renal failure therapy. When peritoneal MCs are exposed to non‐physiological dialysis fluids in long term, tissue fibrosis and failure of treatment occurs. Myofibroblasts (MFs) synthesize excessive extracellular matrix proteins and participate in peritoneal fibrosis. However, the origin of MFs remains obscure. Epithelial‐mesenchymal transition (EMT), typically defined by the loss of E‐cadherin and up regulation of mesenchymal markers, been suggested to involved in this process. However, as MCs do not express E‐cadherin, this mechanism needs to be clarified as we propose mesothelial‐mesenchymal transition (MMT) instead of EMT. In the present study, we examined whether peritoneal MCs undergo MMT and give rise to MFs in mouse peritoneal fibrosis induced by injections of chlorhexidine gluconate (CG) solution. Peritoneal MCs expressed glycoprotein M6A (GPM6A), vimentin, cytokeratin 19, and Wilms tumor 1 (WT1). We isolated peritoneal MCs from mice using antibodies against GPM6A and magnetic beads. In culture, MCs formed epithelial colonies and changed shape to myofibroblastic cells in the presence of TGF‐β. To trace lineages of peritoneal MCs, we used Wt1‐CreERT2 and Rosa26mTmGflox mice. By tamoxifen injection, we specifically labeled peritoneal MCs as GFP+ cells. After injections of CG, most of the single MC layer was eliminated and GFP+ MFs accumulated in the fibrotic area of the peritoneum. Interestingly, when we stopped injections of CG, the mesothelial layer re‐formed and some of MCs expressed GFP in the regenerating mesothelium. These results demonstrate that mesothelial cells are the source of myofibroblasts in peritoneal fibrosis.Grant Funding Source: Supported by NICHD T32 Training Program
Evidence for a role of p130Cas in JAM‐A‐dependent signaling events (60.3)
Authors: Matthews Jason , Weight Caroline , Williams Holly , Leoni Giovanna , Neumann Philipp , Farkas Attila , Nava Porfirio , Nusrat Asma , Parkos Charles 
Published: June 2021

Junctional adhesion molecule‐A (JAM‐A) is a tight junction protein that plays a key role in regulation of intestinal mucosal homeostasis. Previous studies have shown that loss of JAM‐A decreases beta‐1 integrin‐mediated cell migration and increases Akt‐dependent proliferation however signaling pathways downstream of JAM‐A remain incompletely defined. Levels of known actin‐dependent regulators of cell migration and proliferation in epithelial cells derived from JAM‐A deficient mice and ex vivo cultures of small intestinal enteroids were thus examined. We observed dramatic changes in p130Cas, a critical signaling component of focal adhesions that is regulated by ABL kinase. Western blots of JAM‐A deficient intestinal epithelial cells (IEC) and enteroids revealed significantly decreased levels of phosphorylated p130Cas. In addition, JAM‐A deficient colonic IEC showed decreased levels of the migration‐regulating integrin beta‐1 in parallel with decreased E‐cadherin, a negative regulator of proliferation. Treatment of cultured IEC with the ABL kinase inhibitor Imatinib resulted in delayed epithelial wound closure, inhibited cell spreading and decreased p130Cas phosphorylation at Y410 similar to that observed with JAM‐A deficiency. These findings suggest that p130Cas may act to transduce JAM‐A‐dependent signals to beta‐1 integrin to regulate migration, and E‐cadherin to inhibit proliferation.Grant Funding Source: Supported by NIH T32 DK007771‐12, Pathobiology of Mucosal/Epithelial Disease
The mechanism of serine protease‐induced increases in epithelial barrier function in intestinal epithelial cells (60.7)
Authors: Lahey Kelcie , MacNaughton Wallace 
Published: June 2021

Apical serine proteases increase barrier function in intestinal epithelial cells (IECs). We investigated the requirement for proteolytic, protein kinase C (PKC)ζ, and epidermal growth factor receptor (EGFR) activities in the prolonged transepithelial resistance (TER) response to trypsin in IECs.Methods. 1. SCBN epithelial cell monolayers grown on Transwells were treated with trypsin (45BAU/ml) and collected for measurement of phospho‐PKCζ at 2, 5, 10, 15, 30, and 60 min using Western blot. 2. TER across SCBN monolayers was measured in Ussing chambers. Trypsin activity was inhibited by soybean trypsin inhibitor (SBTI) treatment at 10 min (start of plateau phase of TER response) or 30 min (after plateau phase had been established). 3. To investigate the role of EGFR, cells were pretreated with an EGFR inhibitor (PD15305) before addition of trypsin.Results. 1. Upregulation of phospho‐PKCζ was observed in trypsin‐treated SCBNs for up to 60 min. 2. Apical trypsin caused an increase in TER that reached a sustained plateau by 10 min. SBTI at 30 or 60 μg/ml decreased the trypsin‐induced increase in TER by 65% and 80% (respectively) at 10 min and by 70% and 80% at 30 min. 3. Inhibition of EGFR with PD153035 reduced the trypsin‐induced TER by 55%.Our data show the requirement for ongoing proteolytic activity, sustained PKCζ activation and EGFR activity in the trypsin‐induced increase in intestinal epithelial barrier function.Grant Funding Source: Supported by the Canadian Institutes of Health Research
Regulation of ALIX ubiquitination and function in GPCR lysosomal sorting by the alpha‐arrestin ARRDC3 (604.3)
Authors: Lin Huilan , Dores Michael , Trejo JoAnn 
Published: June 2021

We recently discovered that protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, is sorted to lysosomes through an ALIX‐mediated pathway independent of receptor ubiquitination. The mechanisms responsible for regulation of ALIX‐mediated ubiquitin‐independent sorting of GPCRs are not known. Here, we show that the arrestin‐domain containing protein‐3 (ARRDC3) is a key regulator of ALIX‐dependent lysosomal sorting of PAR1. We found that activated PAR1 internalized and colocalized with ALIX and ARRDC3 on late endosomes/lysosomes in HeLa cells. ARRDCs, also known as α‐arrestins, contain PPXY motifs within the C‐terminus that mediate interaction with E3 ubiquitin ligases. Co‐expression of an ARRDC3 C‐terminal truncation mutant lacking the PPXY motifs or mutant in which the PPXY motif was mutated to AAXA resulted in mislocalization of ARRDC3 and disruption of agonist‐induced PAR1 lysosomal sorting. Depletion of ARRDC3 by siRNA also inhibited activated PAR1‐induced ALIX ubiquitination. Thus, ARRDC3 may regulate ALIX activity by controlling the ubiquitination status of ALIX through the recruitment of E3 ubiquitin ligases and consequently regulate activated PAR1 sorting into intraluminal vesicles of late endosomes/multivesicular bodiesGrant Funding Source: Supported by GM090689
Manipulation of phospholipid composition alters protein binding to lipid droplets (606.3)
Authors: Hains Anastasia , Listenberger Laura 
Published: June 2021

Lipid droplets are cytoplasmic organelles that store excess lipid. Each lipid droplet consists of a core of neutral lipid surrounded by a phospholipid monolayer. Proteins interact with the phospholipid surface and regulate lipid droplet synthesis and degradation. We have shown that the phospholipid composition of synthetic lipid droplets can influence protein binding in an in vitro assay. We aimed to determine if protein binding to lipid droplets is influenced by the lipid droplet phospholipid composition in cells. We altered the phospholipid composition of lipid droplets in NIH3T3 cells with hexadecylphosphocholine (HePC), an inhibitor of phosphatidylcholine (PC) synthesis. Isolation of lipid droplets from inhibitor treated and control cells and separation of phospholipids by thin layer chromatography, demonstrated a decrease in PC relative to phosphatidylethanolamine in HePC‐treated cells. Furthermore, HePC treatment prevented the lipid droplet localization of CTP:phosphocholine cytidylyltransferase, the enzyme that catalyzes the rate‐limiting step in PC synthesis. Other changes in protein localization to lipid droplets were observed by SDS‐PAGE and silver staining of lipid droplet proteins in HePC‐treated and control cells. Current and future experiments continue to examine how changes in phospholipid composition alter the protein composition of this important storage compartment.Grant Funding Source: Supported by an award from Research Corporation for Science Advancement
3‐O‐sulfation provides high affinity binding to neuropilin‐1 and modulates endothelial cell sprouting and neuronal growth cone collapse (607.7)
Authors: Thacker Bryan , Lawrence Roger , Seamen Emylie , Liu JIan , Esko Jeffrey 
Published: June 2021

Heparan sulfate structural heterogeneity is driven, in part, by the placement of sulfate groups at various positions in the polysaccharide. While many ligands bind to heparan sulfate without strict requirements for the positions of sulfate groups, binding of a small number of known ligands is influenced by the presence of a sulfate at the C3 position of a glucosamine residue. In mammals, seven enzymes can catalyze the addition of 3‐O‐sulfate groups, suggesting the possibility of other, previously unidentified, ligands whose binding is influenced by 3‐O‐sulfated sequences. To identify these ligands, affinity matrices were created using CHO‐S heparan sulfate, with and without modification by recombinant 3‐O‐sulfotransferases. Serum was fractionated on the matrices and several proteins binding specifically to the 3‐O‐sulfated resins were identified by mass spectrometry. Neuropilin‐1, a modulator of angiogenesis and axonal guidance, bound specifically to affinity matrices with 3‐O‐sulfation. In addition, high affinity binding of neuropilin‐1 to the cell surface required 3‐O‐sulfation. In cell culture, 3‐O‐sulfation modulated two neuropilin‐1 dependent processes. (1) In VEGF‐induced endothelial cell sprouting, 3‐O‐sulfation endowed CHO‐S heparan sulfate with greater inhibitory potential than native heparan sulfate. (2) In semaphorin‐3a‐induced neuronal growth cone collapse, a 3‐O‐sulfated heparan sulfate dodecamer was a potent inhibitor of collapse. An identical dodecamer lacking 3‐O‐sulfation inhibited with much less efficiency. Furthermore, genetic removal of a 3‐O‐sulfotransferase (Hs3st‐2) attenuated the response of the growth cone to semaphorin‐3a. In conclusion, these results demonstrate that 3‐O‐sulfation endows heparan sulfate with enhanced affinity to neuropilin‐1 and modulates two neuropilin‐1 dependent processes, angiogenesis and axonal guidance.Grant Funding Source: Supported by GM93131 and T32CA067754
Stress‐activated signaling in endothelial cells (609.21)
Authors: Rowan Aislinn , Farwell Sara , Lowe‐Krentz Linda 
Published: June 2021

Endothelial cells (ECs) form the interior layer of blood vessels, creating a selectively permeable barrier. Damage to this monolayer triggers inflammatory responses which may eventually lead to vascular disease, making it relevant to characterize EC reactions to inflammatory stress. Pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) activates several pathways in target cells, which interact to determine cell fate. Binding of TNF‐α to its receptor causes activation of certain caspases, advancing apoptosis. c‐jun N‐terminal kinase (JNK) is activated as well, regulating transcription factor activity and possibly promoting an apoptotic response. Also activated is nuclear factor κ‐light‐chain‐enhancer of activated B‐cells (NFκB), a transcription factor which has pro‐survival effects, partially due to interference with both JNK and caspases. ECs encounter TNF‐α as it travels in the blood, however, while most cells are rarely exposed. Thus, we explored whether EC response to TNF‐α is similar to that of other cell types, using immunofluorescence microscopy and Western blotting to analyze TNF‐α‐induced JNK, NFκB, and caspase‐3 activity in primary bovine aortic ECs (BAOECs). NFκB is usually kept in the cytoplasm by an inhibitor; TNF‐α‐binding leads to inhibitor degradation and allows NFκB to move to the nucleus. Upon TNF‐α treatment (10 ng/mL), this translocation begins by 30 minutes, maximizes from 60 to 120 minutes, and declines thereafter. JNK activation is transient, with p‐JNK levels peaking after 30 minutes before decreasing. Excitation of BAOECs with high TNF‐α levels (50 ng/mL for 20 hours) induces caspase‐3 activation, suggesting significant apoptosis. This reaction profile resembles that of other cell types, though it is possible that there are important differences in pathway cross‐talk and communication.Grant Funding Source: supported in part by NIH grant HL54269
Molecular dynamics analysis of CYP2D6 polymorphisms (615.4)
Authors: DeWaal Parker , Furge Laura 
Published: June 2021

The objective of the current study was to use Molecular Dynamics (MD) computational approaches to understand how the interaction of four different CYP2D6 allelic variants with common substrates (bufuralol and N‐desmethyltamoxifen) and with an inactivator (SCH66712) compare to those of reference CYP2D6 (*1). Comparative models of each allelic variant examined (*17‐2, *17‐3, *34, *53) were constructed using AMBER force fields and the starting PDB structure 3MQ4. Analysis of RMSF revealed that allelic variants displayed areas of both greater flexibility and greater rigidity compared to *1. Greater flexibility was seen in the region of helices B’ and C and helices F and G while a region of greater rigidity was clear near helix F’/G’. Samples of final structures for each allelic variant were used for docking studies using AutoDock Vina and channel analysis using CAVER.Grant Funding Source: Supported by NIH 1R15‐GM086767‐02
A mechanism for sequential replication‐coupled destruction of CRL4‐Cdt2 substrates (616.1)
Authors: Coleman Kate , Varma Dileep , Brantley Kristen , Shibata Etsuko , Dutta Anindya , Cook Jean 
Published: June 2021

The CRL4Cdt2 ubiquitin E3 ligase targets a cohort of key cell cycle regulators during both S phase and DNA repair synthesis. These substrates include the p21 CDK inhibitor, the Cdt1 origin licensing protein, and the Set8 lysine methyltransferase. The intracellular concentrations of each of these proteins has profound consequences for cell proliferation and genome stability. CRL4‐Cdt2 only targets substrates if they are bound to DNA‐loaded PCNA, coupling substrate destruction to DNA synthesis. PCNA loading does not trigger simultaneous destruction of all CRL4‐Cdt2 substrates however; Cdt1 degradation begins nearly instantaneously with PCNA loading whereas p21 degradation is significantly delayed for example. As a consequence, the substrates of CRL4‐Cdt2 are destroyed in a stereotypical order, and we have observed this order in multiple cell lines during both DNA repair synthesis and in early S phase. To identify the molecular determinants of delayed vs. immediate destruction, we generated a series of fusion proteins between Cdt1 and p21. We demonstrate that fusing just the Cdt1 PCNA‐interacting motif, a.k.a. “PIP degron,” to the p21 N‐terminus is sufficient to confer rapid rather than delayed destruction. It is not the N‐terminal location, the presence of a second PIP box, or E2 utilization, but rather the degron sequence itself that defines the order of destruction. Surprisingly, rapid vs. delayed destruction is not due to degron features previously shown to be critical for either PCNA or CRL4‐Cdt2 interaction. We generated cell lines that destroy p21 according to the Cdt1 pattern (rapid) rather than the normal p21 pattern (delayed), and we will show the effects of this conversion on DNA replication parameters during S phase and on DNA damage checkpoint responses. This study sheds light on how the substrates of a single ubiquitin E3 ligase are targeted sequentially rather than simultaneously.Grant Funding Source: Supported by NIH GM083024 to J.G.C. and NIH F31 CA165891 to K.C.
Spent engine oil pollution and its effects on selected antioxidants of Telfairia occidentalis leaves (617.9)
Authors: Onyegeme‐Okerenta Blessing , Alozie Chukwuemeka 
Published: June 2021

Spent Engine Oil (SEO) pollution and its effects on selected antioxidants [Protein oxidation (protein thiol (PSH), glutathione (GSH), and total thiol (TSH)] of Telfaria occidentalis leaves was studied. Soil sample was collected from the farmland in Ohii, Owerri West Local Government Ares of Imo State, Nigeria, weighed and polluted with different volumes of spent engine oil (SEO) to give the various percentage pollutions. The polluted soil samples were poured in several nursery bags. Three viable seeds of Telfaria occidentalis were planted in each. An unpolluted soil sample served as the control for the study. Harvest of the leaves was done after 50 days of planting and taken to the laboratory for the assessment of protein oxidation parameters (protein thiol, glutathione and total thiol) concentrations in each of the sample. Results obtained from protein thiol concentration show that there was a significant difference (P>0.05) between the control sample and the 6% polluted sample. There was a progressive reduction in the protein thiol concentration as the volume of the SEO increased. That obtained for GSH also showed a similar trend. There was no significant difference (P>0.05) between control sample and 1% sample pollution but a significant difference (P>0.05) was observed between 0% and 6%. There was also a progressive reduction in the GSH concentration as the volume of the pollutant increased. The same trend was observed for total thiol (TSH) concentration as the SEO volume increased. The mean values obtained for all the parameters (PSH, GSH and TSH) were found not to be significantly different (p>0.05) These results show that increased pollution of soil by spent engine oil have an effect on the protein oxidation parameters of edible fluted pumpkin (T. occidentalis) leaves in areas where there is indiscriminate dumping of spent oil.Grant Funding Source: NIL
Role of undergraduate biochemistry education in protein function assignment (618.26)
Authors: Craig Paul , Dodge Greg , Bernstein Herbert 
Published: June 2021

There are over 3000 unannotated structures in the PDB. Students in our labs have proposed functions for >90 of these structures using template‐based alignments with the ProMOL plugin for PyMOL, BLAST, Dali and Pfam. Subsequently the students confirmed the function of four of these structures in work that they completed with minimal supervision. First they reviewed the putative enzyme function and searched the literature for an assay for that function. If the reagents were available and relatively inexpensive, they obtained the plasmid containing the gene encoding that protein, which they expressed and purified, using metal ion chromatography of the his‐tagged protein. The purified enzyme was then characterized in vitro. The process typically requires 10 ‐ 20 weeks for an individual undergraduate student working full time. We believe that this approach can be easily implemented in undergraduate biochemistry teaching labs, where it would benefit the students and the community. The students will learn about protein expression, purification and characterization using both traditional methods (electrophoresis, UV‐Visible spectroscopy) and modern approaches (plasmid expression, single step chromatographic methods). At the same time, a small army of undergraduates could make a significant dent in the number of unannotated structures in the PDB. Supported by NIGMS 2R15GM078077‐02 and 3R15GM078077‐02S1.Grant Funding Source: Supported by NIGMS 2R15GM078077‐02 and 3R15GM078077‐02S1
Stimulating 5th grade science students' interests through engaging hands‐on inquiry‐based lessons (618.37)
Authors: Booth Rachell , Banta Marilyn , Woodard Addie , Brown Susan , Matias Cindy , Maeder Corina 
Published: June 2021

Students in the 5th grade at Hernandez Elementary School in the San Marcos Consolidated Independent School District (SMCISD) engaged in inquiry based hands‐on activities with their teachers led by faculty and students from the College of Science and Engineering at Texas State University. Two distinct events were used to increase the excitement for and conceptual understanding of concepts required for the 5th grade STAAR (State of Texas Assessment of Academic Readiness) test. First, fifty‐seven fifth‐grade students, four teachers, and four parents from Hernandez Elementary School attended a STEM field trip at Texas State University. Students rotated through a series of hands‐on activities, tours and demos spanning across chemistry, biology, physics, and computer science departments. Assessment of the field trip suggested that enthusiasm for science increased. Second, inquiry‐based activities were brought to the elementary school in a two‐week “science camp” in the spring. Junior level biochemistry students led the activities, which covered a range of concepts, including landforms, properties of water, and the food chain. Impact of the hands‐on experiments was assessed by monitoring the 5th grade STAAR test results. The findings indicated that the 5th grade students had performed better in not only science, but also reading and math.Grant Funding Source: Supported by ASBMB HOPES Grant
Process Oriented Guided Inquiry Learning to enhance learning of concepts in a biochemistry lab (618.9)
Authors: Campbell Naomi 
Published: June 2021

The objective of this study was to develop laboratory modules that incorporated Process Oriented Guided Inquiry Learning (POGIL) to enhance minority students' learning of important concepts in the biochemistry laboratory. The biochemistry laboratory was divided into two groups that met on different days. The exact same laboratory techniques were taught in both labs. The control group had the regular laboratory instruction and the test group recieved POGIL modules. The POGIL group scored significantly higher on the mid‐semester assessment exam than the control group.Grant Funding Source: Supported by National Science Foundation DUE Award #0941955
Iron‐biofortified pearl millet improves attentional function in Indian adolescents, a 6‐month randomized controlled trial (619.2)
Authors: Scott Samuel , Murray‐Kolb Laura , Wenger Michael , Udipi Shobha , Ghugre Padmini , Haas Jere 
Published: June 2021

Iron deficiency affects an estimated one in four persons globally and has been shown to impair cognition, though this relation in adolescents has not been substantiated. In a double‐blind efficacy trial, Indian adolescents (n=264) were randomized to groups consuming either high‐iron biofortified (Fe‐PM) or normal‐iron (CN‐PM) pearl millet. Here, we present results from a subset (n=161; 12‐16 y; 59 F) who performed tests of perceptual and cognitive functioning before and after 6 months of daily pearl millet consumption. Three speeded tests were administered: 1) Simple Reaction Time (SRT), a test of perceptual processing speed, 2) Go/No‐Go (GNG), a test of inhibitory control and 3) Attentional Network Task (ANT), a test of three critical functions of attention (low‐level attentional capture, high‐level attentional selection, and volitional control under distraction). In the intention‐to‐treat analyses, improvement was greater, though not statistically significant, in the Fe‐PM vs. the CN‐PM group for all three tests. In subgroup plausibility analyses, ferritin responders exhibited greater improvement (p<0.05) for the SRT and ANT vs. non‐responders. Hemoglobin responders improved more than non‐responders on the GNG (p<0.05) but not the SRT or ANT. These findings offer promise that iron treatment may improve specific aspects of perceptual and cognitive performance in adolescents.Grant Funding Source: Supported by HarvestPlus/IFPRI