bluuebunny/crossref_metadata_2025 huggingface dataset:
Three‐dimensional reconstruction of Prdm family gene expression patterns during mouse development stage by tissue clearing technique
Authors: Woo Jiwon , Kang Haewon , Cho Yong‐Eun
Published: June 2021

The Prdm family has recently spawned considerable interest as it has been implicated in fundamental aspects of cellular differentiation and exhibits expanding ties to human diseases. Several Prdm family members are deregulated in human diseases, most prominently in solid cancers, where they can act as both tumor suppressors or drivers of oncogenic processes. Some Prdm genes might play an essential role in gene expression were confined to the mammalian central nervous system. We hypothesized that the Prdm gene family may also significantly participate in the development of the mammalian nervous system is required to explore how the gene by the advanced pathological analysis. To assess Prdm family gene expressions patterns at the formation of central nervous system during mouse development stage. The study of the genetic regulation of embryonic development requires the three‐dimensional mapping of gene expression at the high‐resolution microscopic level. We generated transparent mouse embryos by new modified passive tissue clearing technique called mPACT method. Mouse embryos of sequential stage were obtained from three distinct developmental stages, intact embryos was optical transparency to mPACT methods in few days. We reconstructed three‐dimensional expression patterns of Prdm family and blood vessel using transparent mouse embryos. Partial Prdm gene expression was concentrated in the brain and spinal cord regions of each embryo. In this study, we developed embryo‐specific simple clearing method for creating see‐through mouse embryo complete with transparent organ. In addition, our study reconfirmed that some Prdm gene might be a transcriptional regulator for normal neural tissue differentiation during mouse embryonic development using three‐dimensional analysis and transparent embryos.

Support or Funding Information

This work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University. In addition, this work was supported by a grant from the National Research Foundation of Korea (NRF‐2017R1D1A1B03030315).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Large Scale Analysis of the Utilization of Programmed ‐1 Ribosomal Frameshifting as a Recoding Mechanism for the Expression of Human Genes
Authors: Du Zhihua , Zhou Xia , Huang Xiaolan
Published: June 2021

Programmed ‐1 ribosomal frameshifting (PRF) is a translational recoding mechanism widely used in viruses. Efficient PRF events are typically induced by two cis‐acting mRNA signals: a heptanucleotide slippery sequence and a downstream RNA pseudoknot. In human, only three cases of PRF have been reported, all involving retroviral‐like genes and protein products. To evaluate the extent of PRF utilization in the human genome, we have developed a fast and reliable computational scheme for identifying putative pseudoknot‐dependent PRF events and applied the method to a collection of 43,191 human mRNAs in the NCBI RefSeq database. In addition to the three reported cases, our study identified more than two dozen putative PRF cases for further experimental validation. The genes involved in these cases are genuine cellular genes without a viral origin. Moreover, in more than half of these cases, the frameshift site locates far upstream (>250 nt) from the stop codon of the 0 reading frame, which is nonviral‐like. We also performed bioinformatic sequence analysis on the frameshift protein products for more than a dozen of the putative cases. The results provide various degrees of support to the credibility of the cases, indicating that some of these cases may represent real PRF events in human. The outcomes of these events are the production of novel protein isoforms that may have functional difference from the normal proteins.

Support or Funding Information

1. SIUC Seed Grant

2. NIH Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of 2’‐5’‐oligoadenylate synthetase 1 (OAS1) by small double‐stranded RNAs
Authors: Schwartz Samantha L. , Conn Graeme L.
Published: June 2021

The innate immune system comprises a broad collection of critical intra‐ and extracellular processes that limit the infectivity of diverse pathogens. The 2’‐5’‐oligoadenylate synthetase (OAS) family of enzymes are important sensors of cytosolic double‐stranded RNA (dsRNA), and play a critical role in limiting viral infection by initiating signaling to halt viral replication and establish an antiviral state in the cell. Further attesting to the importance of the OAS/RNase L pathway, diverse viruses have developed numerous distinct strategies to evade the effects of OAS activation. Like other innate immune proteins, OAS must be able to accurately distinguish “self” from foreign molecules. Misregulation of the innate immune system can cause increased persistence and susceptibility to viral infection and human diseases, such as interferonopathies. Several recent studies have provided important new insights into the molecular mechanisms of OAS activation by dsRNA as well as unanticipated findings of specific RNA sequences and structural motifs that strongly enhance activation of at least one form of OAS. Although X‐ray crystal structures have given some insight into how OAS1 is activated by dsRNA, we still do not completely understand how specific features of the dsRNA contribute to the level of OAS1 activation. To address this question, we designed dsRNAs to test the impact of specific sequence changes within a strongly activating short (18 base pair) dsRNA. Remarkably, while a single point mutation on one strand resulted in complete loss of OAS1 activity, the equivalent mutation on the opposite strand led to increased OAS1 activity. Despite these stark differences in ability to activate OAS1, both variants appear to bind OAS1 with similar affinity. Given these preliminary findings, I hypothesize that dsRNAs may contain competing OAS1 binding sites with remarkably different capacities to activate the protein in a context‐dependent manner. However, the molecular signatures defining these sites as activating and non‐activating are unknown. On‐going studies to define these features will enhance our understanding of host‐pathogen interactions, such as how viruses might circumvent the OAS1/RNase L pathway by masking activating RNA motifs to evade detection.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Light sensitivity of the photoreceptor cryptochrome of the Drosophila circadian clock and its interaction with other clock components
Authors: Lin Changfan
Published: June 2021

Flavoprotein cryptochromes (CRYs) are blue light sensors that regulate the circadian clock in plants and animals. They consist of a highly conserved photolyase homology region (PHR) and a C‐terminal tail extension (CTE) of various sizes. In Drosophila CRY, photoreduction of the PHR bound cofactor FAD triggers conformational changes within CRY, which include undocking of C‐terminal Tail (CTT) helix. The mechanism of FAD photoreduction remains a key question in understanding CRY activation.

Four tryptophan residues (W420, W397, W342, W394) mediate electron transfer for flavin photoreduction. We have substituted these residues to both redox‐inert phenylalanine residues (F) and redox‐active tyrosine (Y) residues to tune the light sensitivity of CRY. Light‐sensitivities of the variants correlate well with the extent of conformational change at the CTT. Interestingly, the surface tryptophan W394 is the most indispensable residue because it gates electron transfer from exogenous reductant to the photoreduction chain.

In flies, CRY interacts with other signaling proteins of the circadian clock in a light depend‐manner to entrain the 24 hour‐cycle to environmental light. For example, in light CRY binds the protein timeless (TIM) and recruits the E3 ligase jetlag to facilitate TIM degradation. However, the molecular interactions between CRY and TIM are not well understood due to the low stability of this protein complex. We have utilized a chemical cross‐linking strategy to map the interfacing domain in this assembly and stabilize it for future structure study by structural methods

Support or Funding Information

This work was supported by grants from the NIH: R01GM054339 (to M.W.Y.) and R35GM122535 (to B.R.C.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinetic Basis for Selective Inhibition of Hepatitis C Virus RNA‐Dependent RNA Polymerase by Nucleoside Analogs
Authors: Villalba Brian , Li Jiawen , Johnson Kenneth
Published: June 2021

The Hepatitis C Virus (HCV) infects an estimated 71 million people worldwide. Currently, there is no vaccine available to treat or prevent HCV infections. Nucleoside analogs (NA) with a modification on the 2′‐carbon are a group of drugs that target the HCV RNA‐dependent RNA polymerase, NS5B. Among these nucleoside analogs are the prodrug Mericitabine and Sofosbuvir which get metabolized to 2′‐fluoro‐2′‐C‐methylcytidine‐5′‐triphosphate and 2′fluoro‐2′‐C‐methyluridine‐5′‐triphosphate, respectively. These compounds work by mimicking natural nucleotides and competing for binding and incorporation during the replication of the HCV genome. Once incorporated they act as chain terminators by preventing further elongation of the nascent RNA strand. Recently, it has been shown that NA can be excised by a nucleotide‐mediated excision reaction which rescues RNA synthesis. In this study we use transient‐state kinetics to understand the mechanism of inhibition for two CTP analogs and a UTP analog. The results demonstrate that while CTP analogs are readily incorporated and excised, UTP and the UTP analogs efficiently incorporated but are resistant to excision.

Support or Funding Information

This work was supported by funding from the NIH

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Enzyme Kinetics and Small Angle X‐ray Scattering to Understand the Allosteric Regulation of SIRT1
Authors: Wang Ningkun
Published: June 2021

SIRT1 is an NAD+ dependent deacetylase located in the nucleus and cytoplasm of the cell. Research has shown that SIRT1 has been implicated to be involved in many important cellular functions such as neurodegenerative diseases and aging. SIRT1 has also been studied with resveratrol, a polyphenol, which was shown to modulate SIRT's enzyme activity. While the mechanism of SIRT1 has been studied extensively, some exact details are still not known, specifically regarding the allosteric regulation by the N‐terminal domain. We hypothesize that there is a correlation between the activity of SIRT1 and the dynamics of the N‐terminal domain relative to the catalytic core. We are testing this hypothesis by enzyme kinetics and small angle X‐ray scattering (SAXS) methods, using resveratrol as a tool to vary SIRT1 activity. A recent study examined the activity of SIRT1 on peptides of different acetylation sites and found that resveratrol could either increase, decrease or not change SIRT1 activity. We chose peptides from each of these categories as substrates to produce a range of SIRT1 activity change upon resveratrol addition: increase, no change and decrease. First we examined the kinetic parameters (kcat and KM) of SIRT1 with these peptide substrates with and without resveratrol. We are using a continuous enzyme‐coupled assay, rather than an endpoint assay used in the previous study, to see if whether kcat or KM contributes more to the activity change. We also studied the conformational dynamics of SIRT1 in the presence of these peptides and resveratrol using SAXS. The SAXS data informed us about the folding state and overall conformation of the protein, where most changes in conformation would be due to large motions of the more dynamic N‐terminal domain. Combining the data from Michaelis‐Menten enzyme assays and SAXS, we hope to identify a correlation between the conformational dynamics of the N‐terminal domain and the enzyme activity of SIRT1. Learning more about the allosteric mechanism of SIRT1 could aid in the design of therapeutic targets in modulating SIRT1 activity in the cell.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EU‐OPENSCREEN ‐ Innovative collaboration models for molecular biologists in early drug discovery
Authors: Stechmann Bahne
Published: June 2021

As of now, the majority of molecular biologists do not have ready access to suitable drug screening platforms and compound collections, which are generally expensive to purchase, operate and maintain. This represents a major limitation in the field to advance innovative drug discovery projects in academia.

With the aim to address this need, researchers from >20 institutions have joined forces to launch ‘EU‐OPENSCREEN’ (www.eu‐openscreen.eu), which offers international scientists access to a wide range of state‐of‐the‐art technology platforms, resources and expertise. This publically funded initiative integrates high‐capacity screening platforms and medicinal chemistry groups throughout Europe on a not‐for‐profit basis.

The aim of EU‐OPENSCREEN is to collaboratively develop novel molecular tool compounds with external researchers from various disciplines of the life sciences and to support researchers seeking a better understanding on how fundamental molecular processes act to govern biological function at the organismal, tissue, cellular and pathway levels.

Molecular biologists and biochemists who have a robust and suitable assay, and are interested in developing novel chemical tool compounds (inhibitors or activators) for their target of interest, will be able to access access the EU‐OPENSCREEN's screening platforms on a collaborative basis and to screen the rationally selected EU‐OPENSCREEN compound collection, comprising up to 140.000 commercial and proprietary compounds collected from European chemists. After the primary screening, EU‐OPENSCREEN's medicinal chemists optimise the identified ‘hit’ compounds during the hit‐to‐probe program.

I will explain how international pharmacologists and molecular biologists can access and collaborate with EU‐OPENSCREEN to advance their research projects.

Support or Funding Information

This project has received fundng from the European Union's Horizon 2020 research and innovation programme under grant agreement No 654248.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chromosomal Instability Promotes cGAS‐Mediated Cytosolic DNA Response in Metastatic Cancer
Authors: D'Ausilio Morgan E. , Agrawal Anushka , Karande Mira , Kolarov Teodora , Kushen Jemma , Lin Jessica , Mandel Matthew , Patel Sahdev , Pollard Sophie , Bakhoum Samuel F.
Published: June 2021

Recent implication of chromosomal instability (CIN) in metastatic cancer cells has pointed to the frequency of chromosome missegregation as a key difference between primary and metastatic tumors. Understanding the cellular response therefore aids development of treatments to prevent metastasis. Chromosome missegregation creates micronuclei of double stranded DNA (dsDNA) fragments that are prone to rupture in the cytoplasm. The cGAS‐STING pathway responds to cytosolic dsDNA by activating inflammatory response pathways, a reaction useful to protect against viruses. Dimerization of cGAS, induced by dsDNA binding, catalyzes synthesis of cyclic GAMP (cGAMP) from ATP and GTP. Activation of cGAS results in upregulation of IFN‐β and NF‐κB stimulated immune response pathways. Cytosolic dsDNA associated with CIN has been proposed to induce cGAS‐STING mediated immune mimicry in cancer cells, allowing tumors to flourish in otherwise lethal environments. The Pingry School SMART Team, in conjunction with MSOE Center for BioMolecular Modeling, is using 3‐D modeling and printing technology to further examine the binding of cytosolic dsDNA to cGAS and the resultant production of the signaling molecule, cGAMP, by the enzyme. Stimulation of a cGAS‐STING inflammatory response due to CIN in cancer may contribute to the metastatic transition of primary tumor cells. The Pingry School SMART Team aims to utilize the cGAS structural model to better understand the role of this protein in responding to CIN and its link to metastasis through cytosolic dsDNA induced immune response.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Photobiomodulation on Apoptosis Process After Induced Myocardial Infarction in Rats: Analysis of Gene Expression
Authors: Maretti Joao Victor , Manchini Martha Trindade , Silva Jose Antonio , Serra Andrey Jorge , Feliciano Regiane Santos
Published: June 2021

Cardiovascular Diseases (CVD) are the main cause of death in Brazil and in the rest of the world. Ischemic heart diseases represent 30% of deaths by CVD in occidental countries. It is well known that Myocardial Infarction (MI) harms contraction and is responsible of a hypertrophy process of the cardiomyocytes, besides the reduction of ventricle wall thickness, modifying extracellular matrix and promoting metabolic changes. Those metabolic processes stimulate a compensatory rearrangement by targeting three different morphologic regions: infarcted area, borderline area and the remote area to the MI. New treatments are aimed at the modulation of various cardiac processes, such as cardiac muscle contraction, metabolism, apoptosis, fibrosis formation and control of hypertrophy, with the objective of revert or prevent damage on those heart areas. Nowadays, Low‐Level Laser Therapy (LLLT) has become an alternative treatment by its capacity of modulation various biological processes and, depending on dose and wavelength parameters, anti‐inflammatory proprieties and faster cell proliferation. However, the action of the LLLT has not yet been elucidated and little is known about the remaining myocardium behaviour on this treatment, specially when related to apoptosis. Thus, the present study aims the evaluation of LLLT effects on genes related to apoptosis, by analysing TP53, MAP3K2, Bax, MAPK14 and Fas mRNA. The study, approved by the local ethics committee, was designed by the promotion of anterior descending coronary artery occlusion and posterior laser irradiation in the remote region to the myocardial infarction in rats, which were submitted to an echocardiography to evaluate myocardial function after therapeutics. Heart samples underwent an analysis by Real‐Time Polymerase Chain Reaction Array. Data showed that LLLT was capable of modulating apoptosis process, by downregulating TP53 gene, when compared to rats which underwent only anterior descending coronary artery occlusion. The study concludes that LLLT may stimulate cellular survival after MI, by the inhibition of apoptosis of remote region cells.

Support or Funding Information

CNPq, CAPES, UNINOVE

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Photobiomodulation in mRNA Expression of Extracellular Matrix Components of Cardiac Fibrosis in Experimental Post‐infarcted Myocardial in Rats
Authors: Costa Thiago Oliveira D'Alcantara , Manchini Martha T , Serra Andrey J , Atum Allan B , Feliciano Regiane S , Silva Jose A
Published: June 2021

Introduction

Myocardial infarction (MI) is due to irreversible cell death (necrosis) of the heart muscle, secondary to a prolonged inhibition of oxygen supply (ischemia). Fibrosis is the major histologic marker, resulting in heart failure from myocardial infarction. Recently, low intensity laser (LBI) has become an alternative therapy to modulate various biological processes and depending on the wavelength, dosage and condition of the irradiated tissue, has anti‐inflammatory effect, reduces pain and speeds up cell proliferation.

Objective

To evaluate the effect of photobiomodulation on genes involved in the formation of post‐MI cardiac fibrosis by analyzing the mRNA of Col1a1, Col3a1, MMP‐1, MMP‐2, MMP‐9 and TIMP‐1 and TIMP‐2.

Materials and Methods

Fifteen female Wistar rats were divided into three groups: Sham (5), IM (5) and IM + Laser (5) (Ethics Committee: 0015–2012). The infarction induction was made by occlusion of the descending left coronary artery, the laser being applied for 60 seconds at the occlusion site immediately after the infarction. Left ventricle samples from the experimental animals were collected 72h after infarction. Modifications in gene expression of genes: Edn1, Ace, Agtr1a and Ace2 were analyzed by Real‐Time PCR.

Results

There was a significant increase in the expression of Col1a1 and Col3a1 in 1270.0% and 605.0%, respectively, in the IM group compared to the SHAM group. In contrast, the expression of Col1a1 and Col3a1 was decreased by 51.68 %% and 42.70%, respectively, in the IM + Laser group, when compared to the IM group. There was a significant increase in the expression of MMP‐1, MMP‐2 and MMP‐9 by 240%, 166% and 295%, respectively, in the IM group compared to the SHAM group. Photobiomodulation was able to reduce the expression of all genes mentioned above in 64.12%, 62.78 and 76.71% respectively. We also observed a reduction in TIMP‐1 and TIMP‐2 mRNA expression in 32% and 35%, respectively, in the IM group compared to the SHAM group.

Conclusion

Photobiomodulation was able to reduce the expression of COL1A1 and COL3A1 in the IM + Laser group when compared to the IM group, and also related to the reduction of MMPs expression and increase of TIMP‐1 and TIMP‐2 inhibitors, demonstrating the effects of laser therapy in reducing collagen deposition.

Support or Funding Information

FAPESP, CNPq, CAPES, UNINOVE

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative Microbiome Analysis of House Dust Mites, the Most Common Cause of Allergens
Authors: Yong Tai‐Soon , Lee Jinyoung , Kim Ju Yeong , Yi Myung‐hee , Lee In‐Yong , Nam Sung‐Hyun , Yong Dongeun
Published: June 2021

Three species of mites, Dermatophagoides farinae, D. pteronyssinus, and Tyrophagus putrescentiae which are most popular in Korean homes, grown under the same condition would show different patterns of microbiome, and induce different immune reactions. Three species of mites (D. farinae, D. pteronyssinus, and T. putrescentiae), known to cause allergies, are cultured in s same condition (autoclaved media, 80%RH, 25_) and, analyzed for microbiome of each species. Using the next generation sequencing, the difference of microbiome between the dust mite species, and the level of endotoxin were investigated. The extracts of three species of mites were treated to human bronchial epithelial cells (BEAS 2B cells) to measure the expression level of proinflammatory cytokines. Three species of mites, D. farinae, D. pteronyssinus, and T. putrescentiae, grown under the same condition showed significantly different patterns of microbiome. In D. pteronyssinus grown on the autoclaved medium, only a few bacteria were seen in contrast to the other two species. The concentration of endotoxin derived from the extract of mites was proportional to the distribution of the microbiome. The expressed cytokine levels from BEAS 2B cell were comparable among three species of mites. The microbiome pattern of laboratory cultured dust mites differ not on the culture conditions but on the species. The extent to induce Th2 responses in vitro which may closely be related in allergic responses in humans also showed difference.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolomic analysis of a mouse model of NASH and correlation with cytokine expression
Authors: McDermott Caitlin , Eudy Brandon , da Silva Robin
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is a prevalent condition that begins with excess lipid accumulation, termed steatosis. NAFLD is one of the hallmarks of metabolic syndrome with a complex etiology, and over time can progress to nonalcoholic steatohepatitis (NASH), characterized by inflammation and fibrosis. There is no biomarker that can be used to detect fibrosis and therefore the condition can go undetected for a long time. In addition, there are no accepted medical treatments for NASH and extreme cases may progress to cirrhosis and hepatocellular carcinoma (HCC). Past work has identified associations between Glycine N‐methyltransferase (GNMT) and both fibrosis and HCC. The GNMT knockout mouse phenotype ties one‐carbon metabolism to hepatic inflammation and immune function. In humans NASH typically occurs in the obese type 2 diabetic population, but GNMT mice are neither overweight, nor insulin resistant. Therefore, we used a high‐fat diet to investigate the impact of diet on fibrosis. We used a metabolomic approach to identify candidate molecules that may be used to identify hepatic fibrosis. We also use Luminex cytokine analysis to identify altered pathways and immune response that correlate to metabolic changes. Intriguingly we find that GNMT mice receiving a high‐fat diet do not gain weight compared to the wild‐type mice. Our metabolomic analysis has identified several important pathways that contribute to the GNMT phenotype that warrant further investigation. We find interesting correlations with several cytokines including VEGF, M‐CSF, GM‐CSF, IFN gamma, IL‐2, IL‐6 and IL‐10.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Regulation of Lipid Metabolism by a Phosphatidylinositol Transfer Protein and a Phosphatidylinositol 4‐Kinase
Authors: Wang Yaxi , Yuan Peihua , Rodriguez Martin , Tripathi Ashutosh , Lönnfors Max , Schuldiner Maya , Moye‐Rowley W. Scott , Bankaitis Vytas
Published: June 2021

Membrane contact sites (MCSs) are regions in cells where membranes of two organelles are in close apposition. MCSs are proposed to facilitate non‐vesicular trafficking of lipids. Lipid transfer proteins (LTPs) are one of the most commonly‐found components in the MCS system and are speculated to mediate lipid transfer between membranes. However, both the biological functions of MCSs and the functional role of LTP in MCSs remain largely elusive. Herein, we examine in detail a previously‐proposed MCSs model, the ER‐Golgi/endosome MCS facilitating the phosphatidylserine (PtdSer) decarboxylation 2 (Psd2) pathway, which converts PtdSer into phosphatidylethanolamine (PtdEtn) in Saccharomyces cerevisiae. Particularly, we investigate the biological role of a yeast LTP, one of the yeast Phosphatidylinositol (PtdIns)‐Transfer Proteins (PITPs), Sfh4, and a PtdIns 4‐OH kinase Stt4, in this Psd2‐MCS system.

We found that Psd2, Sfh4, and Stt4 are the only essential components facilitating Psd2‐dependent PtdEtn synthesis while other previously proposed elements constituting the Psd2‐MCS (Scs2, Scs22, and Pbi1) are dispensable. Surprisingly, neither the PtdIns‐transfer activity of Sfh4 nor its capacity to activate Stt4 is required to stimulate the Psd2 pathway. Instead, Sfh4 activates the Psd2 pathway via an Sfh4‐Psd2 physical interaction involving the F175 residue on the surface of Sfh4. Stt4 also displays physical interaction with Psd2 in an Sfh4‐independent manner. Instead of directly activating Psd2, Stt4 regulates the substrate pool accessible to Psd2, thereby indirectly controlling the Psd2‐mediated synthesis of PtdEtn. These results demonstrate that the ER‐endosomal MCS model is an inaccurate description of the Psd2 system in yeast, and provide an outstanding example where PITP biological function is uncoupled from its ‘canonical’ activity as a PtdIns transfer protein. Additionally, our findings reveal a novel mechanism in which a PITP and a PtdIns 4‐OH kinase regulate cellular phospholipid homeostasis.

Support or Funding Information

This work was supported by grants from the National Institutes of Health (GM44530) and the Robert A. Welch Foundation (BE‐0117) to VAB. The authors declare no financial conflicts.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sulforaphane Induces Colorectal Cancer Cell Proliferation through Nrf2 Activation in a p53‐Dependent Manner
Authors: Oh Jisun , Kim Jong‐Sang
Published: June 2021

Sulforaphane is a well‐known phytochemical that stimulates nuclear factor erythroid 2‐related factor 2 (Nrf2)‐mediated expression of antioxidant and detoxifying enzymes. In this study, we found that sulforaphane promotes cell proliferation in HCT116 human colon cancer cells harboring a normal p53 gene in a dose‐dependent but biphasic manner. Since p53 has been reported to contribute to cell survival by regulating various metabolic pathways to adapt to mild stress, we further examined cellular responses in both p53‐wild‐type (WT) and p53‐knockout (KO) HCT116 cells exposed to sulforaphane in vitro and in vivo. Results demonstrated that sulforaphane treatment (i) activated Nrf2‐mediated antioxidant enzymes in both p53‐WT and p53‐KO cells, (ii) decreased apoptotic protein expression in WT cells but increased in KO cells in a dose‐dependent manner, (iii) marginally changed mitochondrial DNA abundance in WT cells but decreased in KO cells, and (iv) increased the expression of a mitochondrial biogenesis marker PGC1α in WT cells but decreased in KO cells. Moreover, a low dose of sulforaphane promoted tumor growth, upregulated the Nrf2 signaling pathway, and decreased apoptotic cell death in p53‐WT HCT116 xenografts compared to that in p53‐KO HCT116 xenografts in BALB/c nude mice. These findings suggest that sulforaphane can influence colon cancer cell proliferation and mitochondrial function through a crosstalk between the Nrf2 signaling pathway and p53 axis.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT), Republic of Korea (Grant No. 2017R1A2B4005087).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Delineating the Link Between Dilated Cardiomyopathy and Arrhythmogenic Symptoms
Authors: Muser Tyler , Feyen Dries , Bruyneel Arne , Mercola Mark
Published: June 2021

Dilated cardiomyopathy (DCM) is a common cause of heart failure and frequently involves life‐threatening ventricular arrhythmia and sudden cardiac death. In addition, DCM patients often show a heightened susceptibility to drug‐induced arrhythmia. The mechanisms that link DCM to ventricular arrhythmia are not well understood but are thought to involve structural alterations in the heart wall and in the ion channel makeup of individual cardiomyocytes. About 50 genetic mutations are known to cause congenital forms of DCM. Induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) generated from patient tissue recapitulate many pathophysiological phenotypes characteristic of congenital heart disease and offer an unprecedented tool to study disease mechanisms. To model DCM, we used iPSC‐CMs harboring a DCM‐causing mutation in phospholamban (PLN), a protein involved in calcium regulation. Using the PLN mutant cells, we tested whether the disease would increase arrhythmic susceptibility to drugs that target certain ion channels. Initial experiments from my internship found that both Sodium and human Ether‐à‐go‐go Related channel agonists elicited an exacerbated proarrhythmic response from mutant cells. Action potential durations were disproportionately increased in the DCM‐afflicted mutants. If these data are sustained throughout the experiment, they support the model that the PLN mutant DCM involves a channelopathy that increases proarrhythmic sensitivity to drugs.

Support or Funding Information

TM acknowledges support from the American Heart Association and the Stanford Cardiovascular Institute.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A comparative analysis between PrPc and PrPsc to determine the structure‐function relationship of protein misfolding in Scrapie prion disease
Authors: Peterson Alison , Borisova Catherine , Bunday Priscilla , Gong Meili , Merlin Daniela Hernandez , Kachkovsky Nicholas , Quist Eli , Yeoh Ming Ying
Published: June 2021

The Minnetonka MSOE Center for BioMolecular Modeling SMART Team used 3D modeling and printing technology to examine structure‐function relationships of PrPsc. Scrapies is caused by prions, misfolded proteins in animals and humans, resulting in the development of fatal neurodegenerative encephalitis. Scrapies was one of the first prion diseases to be isolated and studied, and was found to be caused by the misfolding of the PrP protein into the PrPsc prion. We will do this by creating, annotating, and comparing two models, one of the original PrP protein, and one of the PrPsc prion. Scrapies disease is caused when the alpha‐helix rich prion protein (PrP) is converted into into a beta‐structure‐rich insoluble conformer (PrPsc) and is thought to be highly infectious. These structural changes in PrPsc contribute to its build up in the brain of sheep and goats PrP, leading to a deadly swelling of the brain. The buildup is caused by the inability of proteinase K (PK) to fully degrade PrPsc. PK has the ability to fully degrade PrP, but, interacting with PrPsc, can only create N terminally truncated PrPsc, resulting in a PK resistant core. There is currently no cure for Scrapies; however, further research is being done to discover how to reverse the functional effects of prion diseases. Our goal is to present which structural changes occur in the original protein PrP that cause the defective protein PrPsc, as well as determine how this misfolding occurs and what effect it has on the protein's function.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Role of an Atypical Anti‐Termination Two‐Component System in Mycobacterial Gene Regulation and Pathogenesis
Authors: Davenport Haley , Rohde Kyle
Published: June 2021

M. tuberculosis, the etiologic agent of tuberculosis, caused 1.3 million deaths and infected 10 million people globally in 2017. During infection, many bacteria respond to their environments using two‐component systems (TCS) consisting of a membrane‐bound histidine kinase (HK) and a response regulator (RR) of transcriptional initiation. Rv3220c (HK) and Rv1626 (RR) are an atypical TCS with a cytosolic HK and an RR acting as a transcriptional anti‐terminator via its ANTAR domain. This domain causes formation of an anti‐terminator structure to allow transcription to proceed past its normal termination point. Rv1626 was previously identified by a transposon screen as involved in cholesterol metabolism, but the role of this TCS in pathogenesis is unknown.

To determine the function of this TCS, knock‐out (KO) constructs were created using PCR, TOPO cloning, and recombineering. Due to the safety, faster growth, and genetic similarity of M. smegmatis to M. tuberculosis, we targeted the orthologs of the HK and RR in this surrogate organism, MSMEG_1918 and MSMEG_3246 respectively, for inactivation. After generating KO mutants, a phenotypic study was done to determine if cholesterol metabolism was inhibited by the gene deletion. Secondly, mycobacterial protein fragment complementation was used to confirm protein‐protein interactions between the putative TCS proteins. Findings from M. smegmatis will be validated in future work by conducting similar studies in pathogenic mycobacteria.

Support or Funding Information

The project was supported by startup funds to Dr. Kyle Rohde from University of Central Florida College of Medicine.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Prognostic Value of Ephrin Type‐A2 Receptor (EPHA2) and Ki67 in Renal Cell Carcinoma Patients: An Immunohistochemical and Bioinformatical Approach
Authors: Talaat Iman Mamdouh , Okap Israa Sobhy , El Sheikh Samar Mohamed , Youssif Tamer Mohammed Abou , Hachim Ibrahim Yaseen , Hachim Mahmood Yaseen
Published: June 2021

Background

The incidence of renal cell carcinoma (RCC), the most common malignant renal epithelial tumour, is increasing worldwide. Patients usually present with advanced disease and mostly have unpredicted clinical behaviour. A variety of prognostic factors have been proposed as useful parameters, however many of them showed limited clinical value. Thus, the discovery of novel prognostic markers, which might help in predicting patients' outcome and additional new targets to treat this disease are still in great demand. EphA2, a receptor tyrosine kinase, was found to be overexpressed in several malignancies and its expression in different tumours was found to be associated with poor prognostic features. In addition, a group of emerging strategies was recently introduced to target this receptor and found to show anti‐tumorigenic activity in various pre‐clinical cancer models.

Aim

To investigate the prognostic value of EphA2 expression in RCC patients and its association with other clinicopathological parameters as well as Ki67 expression, which is a well‐known proliferative and prognostic marker.

Materials and Methods

The expression of EphA2 and Ki67 was investigated immunohistochemically, and the results were correlated with the different clinicopathological parameters in 50 primary tumour blocks obtained from RCC cases surgically managed in the Urology Department, Alexandria University Main Hospital, during the period (2012–2015). In addition, the association between the EphA2 mRNA expression and the tumour stage as well as the patient outcome was also evaluated using two large publicly available databases.

Results

Our results showed a significant association between EphA2 expression and the tumour size, the nuclear grade, the tumour stage, the patient's outcome and the Ki‐67 expression (P < 0.05 for all). The same trend was also observed with EphA2 mRNA expression using larger patients' cohorts in two publicly available databases. Notably, EphA2 protein expression showed higher levels of co‐expression with the proliferative marker Ki67.

Conclusion

We concluded that higher expression of EphA2 and Ki67 in tumour tissues predicts a locally aggressive behaviour and poor outcome of RCC patients. Moreover, our results give a rationale for the potential benefits of using novel therapeutic strategies with the aim of targeting EphA2 receptor in RCC that might help in improving their outcome.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Dry Eye Syndrome Induced by Urban Particulate Matter Using Aerosol Exposure System
Authors: Lee Tae Gu , Hyun Soo‐Wang , Park Bongkyun , Jo Kyuhyung , Lee Ik Soo , Kim Jin Sook , Kim Chan‐Sik
Published: June 2021

Dry eye syndrome (DES) is caused by multi‐factors and it is featured as tear deficiency and eye irritation. Air pollutions, represented as particulate matter (PM), are emerging threat of human health which induce various diseases including DES. However, the pathogenic mechanisms of DES via PM exposure are not completely identified. Though, several in vivo models of PM induced DES have been suggested, these were not properly reflect the phenomena of human who exposed to PM. In this study, we utilized inhalation device to mimic the atmospheric environment of PM exposure. Rats were exposed to urban PM (UPM) for 5 hours a day during 5 days. After exposure, tear volume was reduced in UPM exposure group. In addition, corneal irregularity and corneal epithelial cell apoptosis were significantly increased by UPM exposure. UPM also affected the tear film disruption via mucin‐4 reduction. Our purpose in this study was to demonstrate the application of new in vivo exposure model for DES induced by PM. We suggest that aerosol exposure system of PM can be used for risk assessment of DES.

Support or Funding Information

This study was carried out with the support of ‘R&D Program for Forest Science Technology (Project No. 2017035A00‐1919‐BA01)’ provided by Korea Forest Service (Korea Forestry Promotion Institute). This work was also supported by Korea Institute of Oriental Medicine (Project No. KSN1812080).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimizing Student Use of Quiz Questions as a Learning Tool
Authors: Peffley Dennis , Di Rito Meghan , Bickett Skye , Lee Jiehyun M
Published: June 2021

OBJECTIVE

Students often utilize practice questions to improve understanding and retention of session content and to prepare for exams. However, a major challenge is aligning questions with session learning objectives. The goal of this study is to change student behavior to use questions as a learning tool, create intuitive question searching at the correct level, and align commercial and professor‐written question banks.

METHOD

A developmental model was created combining faculty‐generated questions with commercial question bank questions. Three surveys were sent out to assess perceptions of question bank utilization. The first two surveys were sent out simultaneously, one to faculty and one to students, to assess how practice questions were used for study purposes. Following the developmental model, a pilot study was created using two practice quizzes with faculty‐generated practice questions for a pharmacy anatomy course. After the completion of the pharmacy anatomy course, the third survey was sent out to pharmacy students to assess their perception of the utility of practice quizzes and to assess whether student utilization of quizzes had changed.

RESULTS

Twenty‐three percent of faculty responded to the survey, indicating that most faculty create their own questions, but do not direct students to use commercial question banks. Seventy‐two students (a 12.04% response rate) responded to the first survey, indicating that 53% of students use faculty provided questions while 40% use commercial question banks and 34% use both. Most students reported using question banks to study the week before a test, three days before, or the day before the test. The first survey indicates that roughly 15% and 5.6% of students quiz themselves the same week of the lecture and the day of the lecture, respectively. In comparison, the post‐survey after the pilot study with a 15% response rate demonstrates that students began studying earlier using quiz questions. The post‐survey indicates 28.6% and 7.1% of students in the pilot study test themselves the week of the lecture and the day of the lecture, respectively. The percentage of students who quizzed themselves a week before the test also jumped from 44.4% to 57.1%. Additionally, in the original survey, only 37.5% of students said that they always incorporate practice questions as part of their studying. In the follow up survey, this jumped to 50%.

CONCLUSION

The initial survey showed students inhibit their study strategies by not quizzing themselves early enough. However, the curated set of progressive questions helped students incorporate question banks into their studying. Additionally, they used the question banks to start studying more effectively by quizzing themselves earlier.

Support or Funding Information

Philadelphia College of Osteopathic Medicine Georgia

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EXAMINING THE PRE‐POST MATRICULATION ACADEMIC METRICS AS CORRELATES OF OSTEOPATHIC MEDICAL STUDENT CLINICAL PERFORMANCE
Authors: Eldeeb Khalil , Dogbey Godwin
Published: June 2021

PURPOSE

The clinical years of medical school are critical in the training of medical students. It is a period that informs and shapes their professional decisions. Results from studies have been perverse about students' academic metrics that predict their clinical success. This study seeks to examine the academic performance metrics that are correlates of osteopathic medical students' clinical performance.

METHODS

This is an IRB approved study involving a retrospective review of admission and academic performance records of the first‐two graduated student cohorts from the Campbell University Jerry M. Wallace School of Osteopathic Medicine in North Carolina. For categorical variables, summary descriptive measures—frequencies/percentages will be generated while means, standard deviations/ranges computed for continuous variables. Correlational analyses will be conducted between the academic and clinical performance variables. Secondary exploratory analyses will be conducted inferentially as necessary. All statistical inferences will be considered significant whenever, P ≤0.05.

RESULTS (Preliminary)

Entering science, non‐science and total GPA are positively correlated with third and fourth‐year medical school GPAs. While entering science and total GPA are positively correlated with COMLEX‐USA Level 2‐Cognitive Evaluation (CE) total score they are not positively correlated with any of the Clinical Sciences Disciplines scores or COMLEX‐USA Level 3 total score or any of the Clinical Sciences Disciplines scores. Entering non‐science GPA is positively correlated with COMLEX‐USA Level 3 ‐ Family Medicine only.

While the Physical component of the MCAT is not correlated with enrolled third and fourth‐year medical school GPAs or board exams level 2 or 3, the verbal component is positively correlated with fourth‐year medical school GPAs only. The biology component and the Total MCAT score of the MCAT is positively correlated with both COMLEX‐USA Level 2‐Cognitive Evaluation (CE) total score and COMLEX‐USA Level 3 total score. The biology component also positively correlate with level 2 family and internal medicine and level 3 emergency and internal medicine scores. The third and fourth‐year medical school GPAs are positively correlated with both COMLEX‐USA Level 2‐Cognitive Evaluation (CE) total score and COMLEX‐USA Level 3 total score and all Clinical Sciences Disciplines scores.

CONCLUSION

Preadmission academic variables are associated with matriculation academic and second and third‐level national board examinations performance of osteopathic medical students. Admission processes should consider the importance of these metrics in student recruitment to maximize their clinical success post‐matriculation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.