bluuebunny/crossref_metadata_2025_split huggingface dataset:
AraL from B. subtilis is a sugar phosphatase and member of the nitrophenyl phosphatase family of the HAD superfamily (585.6)
Authors: Armeli Jordan , Madaio Michael , Hill Jacqueline , O'Handley Suzanne 
Published: June 2021

The HAD (Haloacid Dehalogenase) superfamily is a diverse superfamily with a majority of the enzymes being phosphatases. One family of the HAD superfamily is the nitrophenyl phosphatase family, so named because the first family members were yeast enzymes found only to hydrolyze p‐nitrophenyl phosphate. Since that time, a number of enzymes with a variety of activities have been identified and characterized, most recently, a sugar phosphatase from B. subtilis. We have cloned the araL gene, expressed and purified the AraL protein, and partially determined its activity. Like the other family members, AraL is a phosphatase, but unlike any other member, it is the first sugar phosphatase to be reported for this family. Currently, we have found activity on ribose 5‐phosphate, but this may not be the best substrate, as araL is within the arabinose operon. If AraL degrades sugar phosphates found in the arabinose pathway, its cellular role may be to help regulate this important sugar metabolism pathway.Grant Funding Source: Supported by an NIH AREA grant.
Investigating the mechanisms of esophageal squamous cell carcinoma invasion (59.9)
Authors: Lehman Heather , Welsh Patricia , Massé Julie , Salzberg Ann , Berg Arthur , Stairs Douglas 
Published: June 2021

Esophageal squamous cell carcinoma (ESCC) is a highly invasive disease and understanding the mechanisms involved is necessary to develop effective therapeutics to more successfully treat ESCC. p120‐catenin (p120ctn) is down‐regulated in 60% of ESCC tumors while the epidermal growth factor receptor (EGFR) is the most commonly overexpressed oncogene in ESCC. We hypothesized that p120ctn and EGFR both play integral roles in modulating ESCC invasion. Down‐regulation of p120ctn and overexpression of EGFR together in esophageal keratinocytes resulted in increased migration and invasion of these cells. Further, these cells invade into the extracellular matrix of our 3D organotypic culture system only when p120ctn and EGFR are manipulated. Ingenuity Pathway Analysis of gene expression microarray data generated from our 3D cultures identified cancer‐associated and metastatic genes as the most overrepresented in our datasets. We have identified a putative signaling pathway that involves PKD3 signaling through NFκB and PLAUR, resulting in increased MMP activity and invasion. We have identified increases in protein expression and activity of the components of this pathway. Knockdown of these genes will allow us to assess their role in ESCC invasion. In summary, we have identified a novel synergistic relationship between p120ctn and EGFR that involves a pathway including NFκB and PLAUR that induces ESCC invasion.Grant Funding Source: Supported by R00CA138498
A computational evaluation of the steric and electronic contributions to the stability of the structures of α− and β−D‐glucopyanose Part 4: energy versus geometry for α− and β−D‐glucopyanose in aqueous solution (593.5)
Authors: Balilli Rahih , Abdulsalam Maryam , Baello Rofe , Brutofsky Christian , Kasner Marc 
Published: June 2021

Previous work has established the stable structures and energetics of α− and β−D‐glucopyranose in the gas phase but some examination of the behavior of glucose in aqueous solution is necessary to provide a more realistic picture. By starting with the gas phase optimized geometries of α− and β−D‐glucose, thermochemistry calculations were done and gas phase conformational free energies (ΔG°) were calculated. The geometry optimizations were done using 6‐31G+(d,p), 6‐31G++(d,p), 6‐311G+(d,p) and 6‐311++(d,p) basis sets with HF, DFT (B3LYP) and MP2 methods. The B3LYP/6‐311G++(d,p) gas phase optimized geometries were reoptimized with several solvent models: IEF‐PCM (Integral Equation Formalism of the Polarized Continuum Model) , CPCM (the Conductor‐like Polarized Continuum Model, and SMD (the Solvation Model based on electron Density). CPCM gave results most consistent with experimental values and was used for examining the relationships between energy and geometry.Two‐dimensional scans of energy versus the H‐O‐C1‐O dihedral, the H‐O‐C6‐C5 dihedral and the O‐C6‐C5‐O dihedrals show results consistent with the gas phase results: Only the orientation of hydroxymethyl substituent at C5 ring makes any significant contribution to the energy. The stable rotamers seen in the gas phase (gg, gt, and tg relating the orientation of the hydroxymethyl group at C5) are also seen the solvated molecules but with different relative energies.Three‐dimensional scans of energy versus the two dihedrals in in the hydroxymethyl group (HOCC and OCCO) were done for both α− and β−D‐glucopyranose to develop a potential energy surface to confirm the geometries of the low energy structures and establish consistency with the gas phase results. These scans were also relaxed scans in which the geometry was optimized at each 5° step using the B3LYP method with the 6‐311G++(d,p) basis set and the CPCM solvation model with water as the solvent.
Vitamin E protects against PUFA‐induced behavioral and motor deficits (596.8)
Authors: Ulatowski Lynn , Thakur Varsha , Manor Danny , Parker Robert 
Published: June 2021

Vitamin E is a major lipid soluble antioxidant that scavenges free radicals generated by lipid peroxidation of polyunsaturated fatty acids (PUFAs). Deficiency of this essential nutrient manifests primarily as neurological degeneration. Mutations in the alpha‐tocopherol transfer protein (TTP), the key regulator of whole‐body vitamin E levels, present with extremely low vitamin E levels, oxidative stress, and progressive cerebellar ataxia. Western diets trend towards increased and imbalanced consumption of n‐6 PUFA, thought to be associated with cardiovascular disease, asthma and Multiple Sclerosis and other diseases. We aimed to understand the relationship between PUFA intake, vitamin E status, and neurological function. To this end, we utilized the Ttpa‐null mouse model that faithfully recapitulates vitamin E deficiency in humans. We aimed to exacerbate oxidative stress and neurological deficits by maintaining the mice on a diet high in n‐6 PUFA (corn oil). The wild type mice, which exhibited ample vitamin E levels showed no signs of ataxia, regardless of the PUFA intakes. Vitamin E deficient Ttpa‐null mice fed high‐PUFA diet displayed the initial symptoms of ataxia at ~ 6 months of age, significantly earlier than animals that that were not supplemented with an n‐6 diet. Behavioral tests that assess anxiety and reactivity indicated a correlation between vitamin E status and the levels of PUFA in the diet. Most notable was a spontaneous jumping phenotype displayed when animals were removed from their cage. Future biochemical and histological assessments will explain the relationship between anxiety, oxidative stress and anatomic injuries. These findings indicate that adequate vitamin E status is required to combat deleterious neurological effects of PUFA‐rich diets.Grant Funding Source: Supported by T32DK07319 and RO1DK067494
Structural basis for forming the prion‐like MAVS filament on the mitochondrial membrane (599.1)
Authors: Jiang Qiu‐Xing , Xu Hui , Zheng Hui , Huang Lily , He Xiaojing , Zhang Xuewu , Chen Zhijian 
Published: June 2021

Mitochondrial anti‐viral signaling (MAVS) protein is a critical adaptor required for innate immune responses against RNA viruses. In virus‐infected cells MAVS forms prion‐like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo‐electron microscopic structures of the helical filaments formed by both the N‐terminal caspase activation and recruitment domain of MAVS and a truncated MAVS lacking its C‐terminal transmembrane domain. Both structures display a left‐handed three‐stranded helical filament, revealing specific interfaces between individual subunits that are dictated by electrostatic interactions between neighboring strands and conserved hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super‐resolution imaging of virus‐infected cells revealed the spatial features of rod‐shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α‐helical domain uses distinct chemical interactions to form self‐perpetuating filaments.Grant Funding Source: NIH, Welch Foundation, AHA and HHMI
A role for hypoxia inducible factor in the junctional integrity and barrier function of intestinal epithelial cells (60.1)
Authors: Saeedi Bejan , Kendrick Agnieszka , Schwisow Kayla , Bayless Amanda , Colgan Sean , Glover Louise 
Published: June 2021

The gastrointestinal mucosa provides the primary physical barrier against gut luminal contents. In mucosal diseases such as inflammatory bowel disease, hypoxia and inflammation occur coincidentally at the level of the epithelium. Adaptive transcriptional responses to oxygen deprivation are mediated primarily through the hypoxia‐inducible factor complex (HIF), comprised of an oxygen‐labile ‘ Α’ subunit and constitutively expressed ‘Β’ subunit. A role for HIF has been implicated in orchestrating a signaling program that promotes intestinal epithelial function and homeostasis. Using lentiviral shRNA‐mediated knockdown of HIF1Β in T84 epithelial cells we sought to define the contribution of HIF signaling to epithelial barrier function. HIF1β knockdown resulted in significantly increased permeability, as measured by transepithelial resistance and FITC‐dextran flux assays. Immunolocalization studies of tight junction and adherens junction markers ZO1 and E‐cadherin, respectively, revealed non‐uniform, undulating junctions in HIF1Β depleted T84 monolayers. RT‐PCR and western blot analysis of HIF1Β‐depleted T84 monolayers revealed significantly reduced levels of claudin‐1, a transmembrane tight junction protein that is important for junctional integrity. To date, little is known concerning the transcriptional regulation of claudin‐1, and this work provides an important mechanistic link between the observed influences of HIF signaling on epithelial barrier function, the formation and maintenance of the apical junction complex and ultimately intestinal tissue integrity.Grant Funding Source: Supported by NIH and CCFA
Identification, classification and differential expression of oleosin genes in tung tree (605.3)
Authors: Cao Heping , Zhang Lin , Tan Xiaofeng , Long Hongxu , Shockey Jay 
Published: June 2021

Triacylglycerols (TAG) are the major molecules of energy storage in eukaryotes. TAG are packed in subcellular structures called oil bodies or lipid droplets. Oleosins (OLE) are the major proteins in plant oil bodies. Multiple isoforms of OLE are present in plants such as tung tree (Vernicia fordii), whose seeds are rich in novel TAG with many industrial applications. We identified five tung tree OLE genes coding for small hydrophobic proteins. Genome‐wide phylogenetic analysis and multiple sequence alignment demonstrated that the five OLE genes represent the five OLE subfamilies and all contained the “proline knot” motif (PX5SPX3P) shared among 65 OLE from 19 tree species, including the sequenced genomes of cacao, castor bean, grapevine, peach and poplar. TaqMan and SYBR Green qPCR methods studied the differential expression of OLE genes in tung tree. Expression results demonstrated that 1) All five OLE genes were expressed in developing tung seeds, leaves and flowers; 2) OLE mRNA levels were much higher in the seeds than leaves or flowers; 3) OLE1‐3 genes were expressed in tung seeds at much higher levels than OLE4‐5 genes; 4) the amounts of OLE mRNA rapidly increased during seed development; and 5) the expression levels of OLE genes were well‐coordinated with tung oil accumulation in the seeds. These results suggest that tung OLE genes 1‐3 probably play major roles in tung oil accumulation and/or oil body development. Grant Funding Source: USDA‐Agricultural Research Service Quality and Utilization of Agricultural Products Research Program
Phosphatidylethanolamine increases perilipin 2 binding to synthetic lipid droplets (606.5)
Authors: Rickertsen Cassandra , Townsend Elizabeth , Listenberger Laura , Kharbanda Kusum 
Published: June 2021

Excess lipid is stored in organelles known as lipid droplets. Each lipid droplet is composed of a neutral lipid core surrounded by a phospholipid monolayer. The surface of each lipid droplet is decorated with proteins that facilitate fat storage and utilization. Our lab studies perilipin 2, a protein that binds to the surface of lipid droplets and slows the degradation of stored fat. We aim to understand how the phospholipid composition of the lipid droplet influences perilipin 2 binding. Here, we show that perilipin 2 binding to synthetic lipid droplets is influenced by the phospholipid composition of the surface. Specifically, perilipin 2 binding decreases as phosphatidylcholine (PC) levels decrease relative to phosphatidylethanolamine (PE). This result may influence the progression of alcohol‐induced fatty liver disease. Our experiments show that alcohol consumption in rats not only increases hepatic triacylglyceride storage and lipid droplet accumulation but also alters the phospholipid composition of hepatic lipid droplets. We observe a decrease in the PC:PE ratio of hepatic lipid droplets following alcohol consumption. Future experiments will continue to explore the link between the lipid droplet phospholipid composition, perilipin 2 binding, and alcohol‐induced fatty liver disease.Grant Funding Source: Supported by a grant to St. Olaf College from the Howard Hughes Medical Institute through the Precol
A congenital disorder of deglycosylation: biochemical characterization of N‐glycanase 1 deficiency in patient fibroblasts (607.3)
Authors: He Ping , Ng Bobby , Cresswell Peter , Grotzke Jeff , Gunel Murat , Jafar‐Nejad Hamed , Kodali Vamsi , Kaufman Randal , Freeze Hudson 
Published: June 2021

N‐glycanase 1, encoded by NGLY1, catalyzes the deglycosylation of misfolded N‐linked glycoproteins. Using whole‐genome and ‐exome sequencing, we identified 6 cases with mutations in NGLY1. The patients show developmental delay, seizures, peripheral neuropathy, abnormal liver function, and absent tears. The mutations in NGLY1 resulted in drastic reduction of N‐glycanase 1 protein in patient‐derived fibroblasts. We probed N‐glycanase 1 enzymatic activity firstly by measuring the enzyme released free oligosaccharides (fOS). The patient fibroblasts produced 2‐3 folds less fOS and showed an altered size distribution. Applying a recently established cellular deglycosylation dependent Venus fluorescence assay, we found that patient fibroblasts had dramatically reduced fluorescence indicating pronounced enzymatic activity reduction in N‐glycanase 1. Since deglycosylation of misfolded glycoproteins precedes proteasomal degradation, we speculated that N‐glycanase 1 deficiency might also accumulate misfolded glycoproteins, leading to ER stress. We observed 5‐10 fold increase of transfected ERAD substrates accumulation in the cytoplasm. However, under physiological conditions, we did not observe abnormalities of the three branches of UPR signal transduction (IRE1, PERK, and ATF6) in patient fibroblasts, indicating that the N‐glycanase 1 deficiency itself is not sufficient to cause ER stress.Grant Funding Source: Supported by the Bertrand Might Research Fund
Canonical and non‐canonical pathway of unfolded protein response signaling (607.5)
Authors: Banerjee Dipak , Banerjee Aditi , Santiago Jesus , Longas Maria , Baksi Krishna 
Published: June 2021

The endoplasmic reticulum (ER) is a principal site for folding and maturation of transmembrane, secretory and ER‐resident proteins. Perturbations that alter ER homeostasis can lead to accumulation of unfolded proteins, and a threat to all living cells. To cope with such stress, cells activate an intracellular signaling pathway ‐ the unfolded protein response (upr). It is an integrated intracellular signaling pathway that transmits information about the protein folding status in the ER lumen to the cytoplasm and the nucleus. If the protein‐folding defect is not corrected, cells undergo apoptosis. We have used Tunicamycin, a chemical mediator, a potent asparagine‐linked protein glycosylation inhibitor to induce upr and study the capillary endothelial cell fate, i.e., angiogenesis, a hallmark for tumor progression. Our results indicate ER stress developed due to protein denaturation, causes upr‐mediated cell cycle arrest in G1 and induction of apoptosis. Most importantly, Tunicamycin prevented double‐ and triple‐negative breast cancer progression ~55‐65%. To enhance the efficacy, we have examined nano‐formulated Tunicamycin. The results indicated gold anoparticles (20nm‐50nm) of Tunicamycin (1μg/ml) were three times more potent in inhibiting angiogenesis. There was upr‐mediated cell cycle arrest but no activation of apoptosis. This raised the possibility of a canonical and non‐canonical pathway.Grant Funding Source: Supported in part by grants from Susan G. Komen for the Cure BCTR0600582 (DKB) and NIH/NIMHD 2G12MD0
To cleave or not to cleave: the proteolytic and non‐proteolytic functions of ADAMs in neural crest induction (609.11)
Authors: Li Jie Jing , Perfetto Mark , Wei Shuo 
Published: June 2021

The induction and subsequent development of the neural crest require highly coordinated interactions among genes and pathways. The disintegrin metalloproteinase (ADAM) family proteases have emerged as key regulators of gene and pathway interactions, mainly through their functions as proteolytic “molecular scissors”. Here we show that both the proteolytic and non‐proteolytic activities of ADAMs are important for neural crest induction. Knockdown of either ADAM13 or ADAM19 inhibits the induction of cranial neural crest (CNC) in Xenopus tropicalis. ADAM13 functions as a major regulator of canonical Wnt signaling, a signaling pathway that is essential for CNC induction. This is achieved through ADAM13‐mediated cleavage of class B ephrins, which we identified as new Wnt antagonists. By contrast, the proteolytic activity of ADAM19 seems to be dispensable for its function in CNC induction. Surprisingly, we found that ADAM19 directs CNC induction by binding and stabilizing ADAM13. These results highlight the importance of protease‐protease interaction as part of the gene interaction network in neural crest development.Grant Funding Source: Supported by the NIH
Upregulation of PRKD1 gene expression by activated K‐ras in pancreatic cancer cells (609.20)
Authors: Storz Peter , Doeppler Heike 
Published: June 2021

Summary Activating mutations of the K‐ras proto‐oncogene are detected in over 95% of pancreatic adenocarcinoma (PDA) and its precursor lesions. One mechanism by which activated K‐ras can drive the development of PDA is activation of the transcription factor nuclear factor κ‐B (NF‐κB), which has been shown to regulate genes involved in all aspects of tumor formation and progression. We here describe the PRKD1 gene promoter as a target for K‐ras‐NF‐κB signaling. We identify the binding sites for NF‐κB in the PRKD1 promoter and show that K‐ras‐driven upregulation of expression of PRKD1 and its gene product protein kinase D1 (PKD1) is linked to increased cell survival and oncogenic signaling in PDA.Conclusion Our data provide a functional link between oncogenic K‐ras, NF‐κB and PKD1, a targetable kinase in this cancer.Grant Funding Source: Supported by the NCI R01 grant CA140182
Investigating the cytotoxic effects of mycobacteriophage Vix gene 80 (611.3)
Authors: Krumm Drew , Neevel Andrew , Goodman Danielle , Ludwig Megan , Stukey Joseph , McDonough Virginia 
Published: June 2021

A bacteriophage is a virus that infects and reproduces in bacteria. During productive infections ‐ those that result in construction and release of infectious phage particles ‐ key host cell metabolic processes are modified by the infecting phage and redirected toward making new phage particles. Protein‐protein interactions are likely involved in this process. In this work, gene 80 of mycobacteriophage Vix, a gene cytotoxic to host strain Mycobacterium smegmatis, was studied. Our hypothesis was that an interaction between the Vix80 gene product and a host cell protein caused growth inhibition. The Vix80 protein shares 68% amino acid identity with the product of gene 77 of mycobacteriophage L5. The L5_77 protein has been previously shown to exhibit cytotoxic properties, and interacts with MSMEG_3532, a L‐serine dehydratase. The Vix80 and MSMEG_3532 proteins were expressed in Escherichia coli and purified, but attempts to show a physical interaction in vitro have not succeeded. In addition, no interaction between of Vix80 and MSMEG_3532 was observed through two‐hybrid analysis. HHMI‐Pred analysis found that Vix80 contains a conserved domain of unknown function (DUF) near the N‐terminus. The Vix80 gene was dissected, and the N‐terminal 66 residues, encompassing the entire DUF, was found to be cytotoxic to M. smegmatis. DUF was found to be homologous to a region of three M.smegmatis ORFs, two of which are related by alternate initiation points of the same sequence. Efforts to test the interaction of Vix80 and the VIX80 DUF with themselves and with the three host proteins are described. Identifying the relevant phage and host gene products and understanding how phage exploit their host’s weaknesses could lead to new therapeutic options for many bacterial illnesses.Grant Funding Source: Supported by a Richard Decker Biology Summer Research Award
Investigation of PKCδII and BAD protein complex formation (613.7)
Authors: Morris Amanda , Anselme Ann , Roki Antonio , Patel Niketa , Shor Audrey 
Published: June 2021

Protein kinase C δ (PKCδ) has been shown to have dual functions both as a regulator of apoptosis and cellular survival due to alternative splicing. One splice variant, PKCδII, is integral in promoting cell survivability. The pro‐apoptotic protein, BCL‐2 antagonist of cell death (BAD), will complex with and inhibit the function of pro‐survival proteins. Upon phosphorylation at Ser‐136, BAD disassociates from these complexes, conferring survival. It has been found that the overexpression of PKCδII coincides with increased BAD phosphorylation. Although the exact mechanism is unknown, we believe PKCδII enhances this phosphorylation via direct interactions with BAD. We aim to elucidate the amino acid residues of PKCδII that bind BAD. We propose the interactions that complex PKCδII with BAD using information gained from sequence analyses and computational simulations. The amphipathic groove (Lys‐49, Val‐176, Leu‐220) of 14‐3‐3 has previously been demonstrated to interact with BAD. Upon alignment of the 14‐3‐3 and PKCδII amino acid sequences, it was noted that the residues that comprise the amphipathic groove may be conserved and also facilitate binding of PKCδII with BAD. The PKCδII residues Gln‐376, Val‐547, and Val‐586 may enable the formation of this complex. A homology model of PKCδII and BAD has aided in visualizing this interaction. Subsequent site‐directed mutagenesis will further solidify our understanding of this protein complex. These proposed interactions provide evidence to suggest that BAD complexes directly with PKCδII in a conserved amphipathic groove, thereby leading to increased survivability.Grant Funding Source: The CREST Program is supported by NSF‐DUE #1022793 and #1323414.