bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of Rab3C, a secretory Rab GTPase, as a predictive marker of survival outcome and adjuvant chemotherapy response for colorectal cancer patients (59.5)
Authors: Su Chia Yi , Lim Christina , Jan Yi hua , Chang Yu Chan , Chen Chi Long , Hsiao Michael
Published: June 2021

Rab GTPases, a large family of Ras small GTPase, are critical in regulating membrane traffic and cell motility. Rabs has recently been demonstrated to play an important role in carcinogenesis. However, the function role of Rab3C, one of the secretory Rabs, in cancer has not yet been elucidated. To investigate the clinicopathological function of Rab3C, immunohistochemical staining of Rab3C was performed on tissue microarray specimens of 215 colorectal adenocarcinoma. Rab3C immunoexpression was significantly higher in tumors compared to the corresponding normal tissue and high Rab3C expression was correlated with distant metastasis, higher pathological stage, and tumor recurrence. High Rab3C was associated with poor prognosis in either early stage or late stage colon cancer patients. In multivariate analyses, high Rab3C expression retained its independently prognostic significance for both overall survival (P = 0.001) and disease‐free survival (P < 0.001). Further experimental results showed that Rab3C overexpression promoted tumor progression in vitro and vivo. More importantly, our data also showed that stage II and III patients with high Rab3C expression tumors benefit from adjuvant chemotherapy while patients with low Rab3C expression tumors had no benefit from adjuvant chemotherapy. In conclusion, we demonstrated that Rab3C expression, as a predictive marker for prognosis and therapeutic outcome, is critical for cancer progression.
BCR‐ABL residues interacting with Ponatinib are critical to preserve the tumorigenic potential of the oncoprotein (59.6)
Authors: Romano Chiara , Buffa Pietro , Pandini Alessandro , Massimino Michele , Tirrò Elena , Di Raimondo Francesco , Manzella Livia , Fraternali Franca , Vigneri Paolo
Published: June 2021

Chronic Myeloid Leukemia patients that fail Tyrosine Kinase Inhibitors (TKIs) often present mutations in the BCR‐ABL catalytic domain. We noticed a lack of substitutions involving four amino acids (E286, M318, I360 and D381) that form hydrogen bonds with Ponatinib. We therefore introduced mutations in each of these residues either preserving or altering their physico‐chemical properties. We found that E286, M318, I360 and D381 are dispensable for ABL and BCR‐ABL protein stability but are critical to preserve catalytic activity. Indeed, only a “conservative” I360T substitution retained kinase proficiency and transforming potential. Molecular Dynamics simulations of BCR‐ABLI360T revealed differences in both helix‐αC dynamics and protein correlated motions consistent with a modified ATP binding pocket. Nevertheless, this mutant remained sensitive to Ponatinib, Imatinib and Dasatinib. These results suggest that changes in the four BCR‐ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Interestingly, amino acids equivalent to those identified in BCR‐ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.
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
Dynamic regulation of ER‐PM junctions facilitates PI(4,5)P2 replenishment during receptor‐induced Ca2+ signaling (599.3)
Authors: Liou Jen , Chang Chi‐lun
Published: June 2021

ER‐PM junctions are membrane interfaces between the endoplasmic reticulum (ER) and the plasma membrane (PM), permitting direct molecular interactions and exchange of signals between the two subcellular compartments. ER‐PM junctions have been implicated as important subcellular loci for lipid metabolism and calcium signaling. However, the molecular mechanisms underlying the regulation and functions of ER‐PM junctions are not well understood. Using a genetically‐encoded marker and live‐cell imaging, we found that ER‐PM junctions are dynamically regulated during calcium signaling. Elevation of cytosolic calcium induces the translocation of the ER membrane protein extended synaptotagmin‐1 (E‐Syt1) to ER‐PM junctions and enhances ER‐to‐PM connection. E‐Syt1‐mediated enhanced ER‐to‐PM connection facilitates the recruitment of Nir2, a phosphatidylinositol (PI) transfer protein, to ER‐PM junctions during receptor‐induced calcium signaling. Nir2 promotes replenishment of PM PI(4,5)P2 following receptor‐induced hydrolysis. Disruption of the enhanced ER‐to‐PM connection or Nir2 expression resulted in reduced PM PI(4,5)P2 replenishment and defective calcium signaling. These results demonstrate that calcium‐mediated dynamic regulation of ER‐PM junctions is crucial for maintaining PI(4,5)P2 levels at the PM and sustaining calcium signaling.
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
Uncovering regulatory nodes in miRNA‐mediated regulation of glycome (608.7)
Authors: Koppolu Sujeethraj , Kasper Brian , Mahal Lara
Published: June 2021

Glycan biosynthesis is a complex and highly non‐linear process requiring a large number of diverse proteins (glycosyltransferases, glycosidases and sugar nucleotide transporters) to act in accord. An increasing number of recent reports, including previous work from our lab, suggest a possible role of microRNAs (miRNAs), small non‐coding RNA that complement to the 3’ or 5’ untranslated region (UTR) of mRNA to attenuate translation, in the regulation of overall glycan expression (glycome) via post‐transcriptional regulation of glycan related gene expression. In this work, we hoped to identify the key regulatory nodes in glycan biosynthetic pathways that might help in the deconvolution of glycomic signals. Adding a more functional approach, we further generated a condition‐driven model with Ovarian Cancer Metastasis (OCM) to identify any glycosylation patterns. Using miRNA prediction data from microrna.org and 3’ UTR lengths from ENCODE project, a metric, called Hit Density (HD), was calculated for each of the glycogenes that incorporate factors contributing to the miRNA regulation of genes. We identified key regulatory nodes in the glycan biosynthetic pathways at the miRNA regulatory level, and show that the glycogenes are not redundant. We also show that orchestration of several events of the glycome can be regulated by miRNA that are predicted to target multiple glycogenes. Finally, bio‐functional mapping of glycan regulatory networks in OCM revealed specific glycosylation patterns, suggesting that glycan regulatory networks in condition‐driven models could impart valuable information.
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.
Characterization of bacterial chemotaxis receptors sensing and signaling (614.1)
Authors: Payne Molly
Published: June 2021

Motile bacteria navigate in gradients of diverse chemical signals (toward attractants or away from repellents) by chemotaxis. Chemotaxis is essential for the adaptation of motile bacteria to changes in their environment. Bacterial chemotaxis depends on a functioning signal transduction pathway comprising several cytoplasmic proteins that form a phosphorylation cascade. Chemotaxis signal transduction is initiated via sensing cues by dedicated chemoreceptors that are localized in the membrane. The sensing activity of a bacterial chemotaxis receptor, named Tlp1 from Azospirillum brasilense, was recently shown to be regulated by binding to the second messenger molecule, c‐di‐GMP and a novel mechanism of sensory adaptation was proposed. To further characterize this mechanism, a polyclonal antibody against the C‐terminal region of Tlp1 was purified. This antibody was then used to test whether it was c‐di‐GMP bound or c‐di‐GMP free. Tlp1 receptors could be distinguished, using the wild type strain and a mutant derivative over‐expressing c‐di‐GMP. Biophysical methods such as X‐ray crystallography are currently being used to characterize how c‐di‐GMP binding to the C‐terminal region of Tlp1 affects conformational change and signaling activity of this receptor. Although chemotaxis receptors are essential for bacteria to monitor their environments, the sensory specificity of bacterial chemoreceptors is largely unknown (88% unknown function) despite the public databases harboring over 22,000 such chemoreceptors. Tlp2 is a chemoreceptor from A. brasilense with a periplasmic domain of unknown function. Genetic and behavioral evidence indicate that Tlp2 senses nitrogenous compounds (ammonium and nitrate). Further characterization of the sensory specificity of Tlp2 for these compounds is being conducted using Isothermal Titration Calorimetry to determine the affinity of the isolated and purified recombinant sensory domain of Tlp2.
Protein‐protein interactions involved in chemotaxis of Epulopiscium sp. type b (614.4)
Authors: Faughnan Patrick , Piefer Andrew
Published: June 2021

The purpose of this study was to further understand the protein‐protein interactions that occur during chemotaxis of Epulopiscium sp. type B. Epulopiscium is a very large, unculturable, gram‐positive bacterium that lives symbiotically in the gut of the surgeonfish. Chemotaxis is the process in which cells move in response to different chemical stimuli. The specific proteins being studied were methyl accepting chemotaxis sensory transducers (MCP3 and 6), CheA, a sensory kinase, and CheW, a coupling protein. These proteins were selected to study because of DNA sequence homology with E. coli and B. subtilis. We hypothesized that the Epulo MCP proteins should interact with CheW and / or CheA based on the behavior of the homologous proteins in E. coli. The Epulopiscium proteins were produced recombinantly in E. coli and subsequently purified. CheA and CheW were cloned into E. coli using a vector that attached a 6x histidine tag to the proteins and MCP‐3, MCP‐6, and CheW were cloned and expressed in E. coli using a vector that created a GST fusion with the protein. The recombinant proteins were purified via affinity chromatography and interactions were analyzed using a GST pull down assay, followed by western blotting with an anti His‐tag antibody.
Development of whole cell biosensors mediated by bacteria chemoreceptors (614.5)
Authors: Donaldson Teraya , Dattelbaum Jonathan
Published: June 2021

Whole cell biosensors are part of an emerging strategy to build new tools for diagnostic and therapeutic applications. The goal is to re‐engineer bacterial cells to respond to environmental cues or chemicals. Whole cell biosensors typically express a sensor protein targeting the ligand of interest that is coupled with a genetic regulatory system to produce a measureable signal by the host cell. We have adapted an AraC‐based transcriptional reporter assay to create a new whole cell biosensor that may be used for the creation of new sensor proteins. In our bio‐reporter system, Trg is translationally‐fused to AraC such that dimerization of the Trg‐AraC results in expression of the reporter gene. The presence of analyte will be detected in the periplasm of the cell by inclusion of an engineered periplasmic binding protein (PBP) specific for this molecule. The bound protein then interacts with Trg causing a conformational change. This dimerization of the Trg‐AraC fusion in the cytoplasm will then switch on gene expression of the reporter gene, green fluorescent protein (GFP). This mechanism reports dose‐response fluorescence detection in the presence of the analyte. Ultimately this bio‐reporter system will allow us to design new PBPs with selectivity for specific chemical entities and for construction of a genetic switch inside bacterial cells that precisely reports the presence of the analyte using a fluorescence reporter system.
“Building Your Science Toolkit”: a unique laboratory‐based mentoring program aimed at undergraduate students at risk for exiting the science pipeline (618.33)
Authors: Roecklein‐Canfield Jennifer , Yasi Emily , Bixho Ida , Walker Stephanie
Published: June 2021

The status of women in STEM careers continues to remain a problem, both in the recruitment and retention of women in science research and teaching. It is particularly critical for women from underrepresented minorities. One critical need seems to be focused on reaching students at a younger, more impressionable age and a large component of that has been the lack of substantial mentoring early in their careers. We initiated a laboratory‐based project to develop a mentoring program for Simmons College freshman and sophomore undergraduates, utilizing senior research students as mentors. The advantage of this project is to increase mentoring activities and training opportunities for the senior student as well. Studies have long shown that persistence in the STEM field for women requires strong confidence and leadership skills. Including a laboratory component focusing on skill acquisition, younger students are able to visualize themselves as senior independent study students early in their college career, critical to helping those students at risk to remain in the science pipeline. We will discuss the laboratory and mentoring activities of two pilot cohorts illustrating a unique badging system to document laboratory technique mastery. We will show preliminary assessment data on student gains in aptitudes and attitudes towards science. The expected outcome of this program will be an increase in participation in scientific research and greater numbers of women entering into the science career pipeline
Development and implementation of a workshop on inclusive teaching for undergraduate and graduate teaching assistants by postdoctoral scholars at UCSD (618.35)
Authors: Merkhofer Evan , Pollard Daniel , Lee Suzanne , Kadandale Pavan , Vicente‐Crespo Marta , Otto‐Hitt Stefanie , Clement Sandra , Wienhausen Gabriele
Published: June 2021

The loss of talented students from Science, Technology, Engineering and Math (STEM) fields is a major challenge for U.S. technological advancement and global competitiveness in the 21st century. Although inclusive teaching strategies can help student retention, formal instruction in pedagogy is often completely absent from postdoctoral training. Thus, increased access to training in pedagogy for student teaching assistants (TAs) and postdoctoral scholars has the potential to improve the quality of instruction in the STEM classroom. In order to provide TAs with the tools they need to create a safe learning environment that respects student diversity and encourages learning, a group of postdoctoral scholars in the Division of Biological Sciences at UCSD have developed a TA training workshop on inclusive teaching. The workshop is based on the learning goals of (1) raising TA awareness of the impact of identity and classroom climate on student learning, (2) helping TAs recognize the importance of inclusivity in effective teaching, and (3) empowering TAs with a pedagogical toolkit for creating an inclusive classroom. The workshop uses a combination of self‐reflective and group activities to personalize diversity issues and model best practices for inclusive teaching. Based on participant pre‐ and post‐surveys, this workshop has been successful in promoting awareness of unconscious bias and providing strategies that participants can use in their classrooms to facilitate and encourage student learning. This workshop will continue to provide an opportunity for postdocs and undergraduate and graduate teaching assistants to increase their awareness and understanding of the importance of diversity and inclusiveness in learning.
Interdisciplinary collaborative project‐based instruction: students in two distinct courses teach each other by building software to solve biological problems (618.38)
Authors: Goodman Anya , Dekhtyar Alex
Published: June 2021

We developed and piloted a novel approach to interdisciplinary instruction; this approach relies on well‐defined interdependent roles for biology (BIO) and computer science (CS) students in a project‐based laboratory accompanied by two distinct discipline‐specific lecture courses. Student interactions are structured around software development process: (1) BIO students are presented with a biological question, (2) they figure out what data is required and write program requirements, (3) CS partners write a program, (4) BIO and CS students collaborate on testing the program, (5) BIO students use the program to answer the original question. We applied this workflow to five increasingly complex assignments relevant to the genome analysis project conducted by the Genomics Education Partnership (GEP, www.gep.wustl.edu). We tested this approach during two 10‐week quarters (Spring 2012 and 2013) with 65 CS students and 47 BIO students. We assessed cognitive learning gains (skills and knowledge) by evaluating course artifacts and student gains in the affective domain (attitudes and self‐efficacy) using confidential surveys. Assessment data indicate that students appreciate and mutually benefit from interdisciplinary interactions. Students from both disciplines improve communication skills and reinforce mastery of their own discipline by acting as experts and teachers in their field.

Grant Funding Source: Supported by NSF TUES # 1140828.
The double burden of undernutrition and excess body weight in Mexico (621.5)
Authors: Kroker María , Pedraza Lilia , Pedroza Andrea , Rivera Juan
Published: June 2021

Introduction: Stunting (ST) and anemia (AN) have declined but are still high in some regions and subpopulations and overweight and obesity (OW/OB) have increased at alarming rates; therefore, undernutrition and OW/OB coexist. Objective: Describe the coexistence of ST, AN and OW/OB at national (NAT), household (HH) and individual (IND) levels. Methods: Using the Health and Nutrition National Survey 2012 (ENSANUT 2012), prevalences of ST, AN and OW/OB in 0‐59 mo children (<5mo), school‐aged children (5‐11y) and adult women (20‐49y) were obtained at NAT level. Double burden of malnutrition (DB) at HH level was defined as coexistence of a ST < 5y and an OW/OB mother (ST‐OW/OBm). At IND level, DB was defined as concurrent ST and OW/OB (ST‐OW/OB) in 5‐11y, AN and OW/OB (AN‐OW/OB) in 5‐11y and women. To determine if coexistence of the conditions corresponded to expected values, given independent distributions of each of them, observed and expected prevalence were tested statistically. Results: At HH level, the prevalence of ST‐OW/OBm was 8.6%, at IND level prevalence were 1% for ST‐OW/OB in 5‐11y, and 2.9% for AN‐OW/OB and 7.4% for AN‐OW/OB in women. At HH and IND level in 5‐11y, prevalence of DB were significantly lower than expected, while those in women were significantly higher. Conclusion: Although some prevalences of DB were lower than expected, given independent distributions of the two conditions, the coexistence of ST, OW/OB and AN at NAT, HH and IND levels in Mexico call of policies and programs to prevent simultaneously the three conditions.
Genome‐wide analysis of gene transcription by EGR1 in response to TGF‐β2 stimulation of small intestinal cell differentiation (623.23)
Authors: Zhang Man , Liao Yalin , Lönnerdal Bo
Published: June 2021

Human milk contains growth factors that maintain intestinal mucosal homeostasis. However, the molecular mechanisms behind how these growth factors regulate gene transcription are largely unknown. We used IEC‐6 (intestinal crypt cell) cells as a model and treated them with 10 ng/ml TGF‐β2, a milk growth factor known for its stimulatory effect on intestinal cell differentiation. Immunoblotting and immunofluorescent assays showed that EGR‐1 (early immediate gene, regulating cell survival, growth, differentiation and transformation) expression in the nucleus was stimulated, with maximum expression occurring at 1h and then a decrease with time. EGR1 was predicted to have binding sites in the promoters of Axin1 (‐248bp/258bp, the scaffold protein in Wnt signaling), LRP5 (‐78bp/113bp, interacting with Axin1), Notch1 (‐76bp/84bp, Notch signaling) and Hes1 (‐1158bp/1170bp, affecting differentiation of intestinal cells). Chromatin immunoprecipitation (CHIP) verified a binding site in the Axin1 promoter. To comprehensively explore the functions of EGR1 in intestinal cell growth, next‐generation sequencing (NGS) was used to examine the interaction between EGR1 and other genes under the stimulation of TGF‐β2 at 0h, 1h and 48h, respectively. In conclusion, our results for the first time show the genome‐wide interaction between EGR1, a TGF‐ β2 responsive factor, and active genes involved in intestinal crypt cell growth and differentiation.
Insights into infant nutrition through cross‐species proteomic comparison of primate milks (623.9)
Authors: Beck Kristen , Weber Darren , Phinney Brett , Smilowitz Jennifer , Korf Ian , Lemay Danielle
Published: June 2021

Milk has served as the optimal neonatal nutrition source directing infant growth, neurological development, and immunity for millions of years. Its production is an energetic balance between mother and infant needs. Therefore, molecular components providing a competitive advantage against genetically similar peers will be retained. Milk proteins have alternative end points aside from being an amino acid source. Bioactive peptides have antimicrobial and immunmodulatory functions. Recent studies show milk proteins to vary in abundance with disease status. Further biomedical research relies on non‐human models, specifically the rhesus macaque. Currently, little is known about the rhesus macaque milk proteome due to prohibitive small volume of samples collected. Our group developed a small sample method for quantitative milk proteomics using LC‐MS/MS. We identified over 800 proteins in macaque milk (protein and peptide FDR = 4.4%, 0.9%, respectively). In our initial analysis, proteins associated with assisting the innate immune response (complement proteins C3, C4, factors B, H, and I) are more abundant but lysozyme C is less abundant in macaque compared to human milk. By understanding the rhesus macaque milk proteome, we can better utilize this model organism for investigation of health related diagnostic tools. Cross‐species comparisons will also highlight which milk proteins are unique to human nutrition.

Grant Funding Source: K.B. supported by T32‐GM008799 from NIGMS‐NIH and CNPRC Post‐Doc Pilot to DL NIH/NCRR P51 RR00169
Maternal serum n‐6 polyunsaturated fatty acids are related to blood pressure during pregnancy: results from a prospective cohort from Rio de Janeiro, Brazil (628.4)
Authors: Rebelo Fernanda , Pinto Thatiana , Farias Dayana , Lepsch Jaqueline , Vaz Juliana , Kac Gilberto
Published: June 2021

Objective: To evaluate the association of serum n‐3 and n‐6 polyunsaturated fatty acids (PUFA) with blood pressure (systolic [SBP] and diastolic [DBP]), longitudinally during pregnancy. Methods: Prospective cohort of 212 women evaluated at up to 13th, 22‐26th and 30‐34th gestational weeks. Fasting serum samples were assayed employing a high‐throughput robotic direct methylation coupled with fast gas‐liquid chromatography. SBP and DBP were obtained in duplicate using an automatic sphygmomanometer (Omron HEM‐742 INT). Statistical analysis comprised longitudinal mixed‐effects models, adjusted for socio‐economic, demographic, lifestyle, biochemical and anthropometric variables. Results: Mean SBP was 110.3, 108.9, and 111.2 (mmHg) and DBP was 67.2, 65.2, and 67.2 (mmHg) for the 1st, 2nd and 3rd trimester, respectively. The mean n‐6/n‐3 ratio was 11.49 (range: 5.85 ‐ 20.19). There were no associations of n‐3 fractions with SBP or DBP. Total PUFA n‐6 (β=0.007 [95%CI: 0.002‐0.012, p=0.004]) and linoleic acid (LA, β=0.007 [95%CI: 0.001‐0.012, p<0.004]) showed a positive association with SBP. Arachidonic acid (AA) was associated with both SBP (β=0.019 [95%CI: 0.001‐0.038, p=0.046]) and DBP (β=0.020 [95%CI: 0.004‐0.037, p=0.017]). Conclusion: In a sample with high n‐6/n‐3 ratio, higher serum concentrations of total PUFA n‐6, LA and AA may represent a risk for the increase of blood pressure during pregnancy.

Grant Funding Source: Supported by FAPERJ, CNPq and CAPES
Semantic memory processing is enhanced in preadolescents breastfed compared to those formula‐fed as infants: An ERP N400 study of sentential semantic congruity (629.1)
Authors: Pivik R , Andres A , Snow J , Ou X , Casey P , Cleves M , Badger T
Published: June 2021

Studies comparing child cognitive development and brain activity during cognitive functions between children who were fed breast milk (BF), milk formula (MF), or soy formula (SF) have not been reported. We recorded event‐related scalp potentials reflecting semantic processing (N400 ERP) from 20 homologous hemispheric sites in 8 yr old children [BF (n=21), MF (n=19) or SF (n=17)]. Children listened to sentences in which the final words did or did not make sense within the sentence context. Infant feeding histories and current cognitive status (standardized tests of IQ, language, and memory) were documented. Group responses were similar for congruent words, but semantic processing of incongruent words was greater in BF children [higher amplitude responses over left hemisphere central‐parietal sites in BF than other groups (Analyses of Variance with Tukey‐Kramer post‐hoc tests: central: BF > SF; parietal: BF>MF, all p < .05)]. There were no significant group differences on measures of general cognitive development. These findings are the first to demonstrate a relationship between infant diet and preadolescent brain activity associated with semantic activation ‐ a process fundamental to language comprehension ‐ and show this activity is greatest in children who were breastfed.