bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of SecY protein‐translocation channel in action using a novel in vivo tool (LB198)
Authors: Park Eunyong , Akey Christopher , Rapoport Tom
Published: June 2021

Many proteins are targeted by signal sequences to a conserved protein‐conducting channel, formed by the prokaryotic SecY or eukaryotic Sec61 complex, and are translocated across the membrane. The channel forms an hourglass‐shaped pore with a constriction in the middle across the membrane. The channel also has a lateral gate that can open toward the lipid phase. During initiation of translocation, a ribosome‐nascent chain complex binds to the SecY/Sec61 channel, resulting in insertion of the nascent chain. To study the SecY channel engaged in translocation, we developed a novel method generating defined translocation intermediate complexes in E. coli cells. Using this tool, we addressed how the channel is opened by a ribosome‐bound nascent chain and transports large polypeptides and yet prevents passage of small molecules. We determined structures of inactive and active ribosome‐channel complexes with cryo‐electron microscopy. These structures demonstrate that the nascent chain opens the channel through mostly rigid body movements of the two halves of SecY. The polypeptide inserts as a loop into the channel, with the hydrophobic region of the signal sequence intercalated into the open lateral gate. Biochemical analysis shows that the pore residues of SecY form a gasket‐like seal around the translocating polypeptide chain, preventing the permeation of small molecules. This work was supported by HHMI and NIH.
Human cytokines activate JAK‐STAT signaling pathways in porcine ocular tissue (LB214)
Authors: Fasler‐Kan Elizaveta , Barteneva Natasha , Nurzhanova Asil , Wunderlich Kerstin , Meyer Peter
Published: June 2021

Introduction The pig has been used extensively in experimental studies of human eye diseases. Pig retina shares many morphological features with the human eye. The use of porcine ocular cells (POC) as putative xenotransplants appears theoretically possible. We investigated cytokine‐induced changes of the JAK/STAT signaling pathways in POC in response to human cytokines.

Methods POC were used to study the nuclear translocation of STAT proteins by gel‐shift assay, immunohistochemistry, confocal laser microscopy, flow and imaging cytometry.

Results JAK/STAT signaling pathways could be activated in porcine pigmented epithelial ciliary body cells, pigmented iris epithelial cells and in lens epithelial cells in response to porcine and human cytokines. All POC showed very strong STAT1 activation upon stimulation with porcine interferon‐gamma. POC also respond to human cytokines; IFN‐alpha caused strong activation of STAT1 in all assays whereas activation of STAT3 was less strong in EMSA, but strong in flow cytometry and immunofluorescence. Human recombinant IL‐6 activated STAT3 and human IL‐4 activated STAT6. With the help of immunofluorescence assay and flow cytometry we observed nuclear localization of STAT proteins after activation of POC with cytokines and interferons. Human IFN‐α had an inhibitory effect on POC in proliferation assays.

Conclusion Our data demonstrate the upregulation of the members of the JAK‐STAT signaling pathway in POC. Human cytokines and interferon activate JAK‐STAT signalling pathway in POC. We hypothesize that direct stimulation of the JAK‐STAT pathway in POC in response to human cytokines will lead to complications or failure, if pig‐to‐human ocular tissue xenotransplantation were to be carried out. For successful xenotransplantation among other obstacles there must be new approaches developed to regulate signalling pathways.

Grant Funding Source: University of Basel
The transcriptional regulator PecS from Pectobacterium atrosepticum senses both oxidants and ligands (LB233)
Authors: Deochand Dinesh , Grove Anne
Published: June 2021

Pectobacterium atrosepticum (erstwhile Erwinia carotovora) is one of the top phytopathogens, responsible for soft rot in potato. The pathogenicity of this bacterium is mainly characterized by secretion of cell wall degrading enzymes, for instance pectinases, cellulases and proteases. It has been shown that PecS, a ligand‐responsive MarR family transcription factor, controls expression of the pectinase genes as well as global virulence in the related bacterium E. chrysanthemi (now D. dadantii). PecS specifically binds to the promoter region of PecS and PecM genes leading to repression of both. PecM is responsible for effluxing blue pigment i.e., indigoidine, which is instrumental in neutralizing ROS, produced by the plant as a defense mechanism. By EMSA we show that P. atrosepticum PecS binds to the intergenic region of PecS and PecM with Kd = 1.0 ± 0.1 nM. We speculate that oxidants can attenuate DNA binding by disulfide bond formation between redox‐active cysteines at the dimer interface in PecS, as evidenced by SDS PAGE analysis, which demonstrates that in presence of organic and inorganic peroxides, PecS forms dimers and multimers. Additionally, thermal shift assay corroborates that there are structural changes in the protein since Tm of PecS (~50 °C) is significantly reduced in presence of oxidants or ligands. Hence, we propose that PecS modulates global virulence by sensing oxidants in addition to ligands.
Optimization of Chemical Inhibition Conditions for NMD Studies in p53‐Positive Cancer Cell Lines (LB234)
Authors: Soares Robert , Connelly Zachary , Vazquez Oscar , Carastro L. Michael
Published: June 2021

We report on the treatment of p53‐positive cancer cell lines with chemical inhibitors of transcription, translation and PI‐3 kinase‐like kinases (PIKKs). Our goal was to determine optimal concentrations of, and exposure times for, chemical inhibitor treatments. These experiments were prerequisite for our studies of the role nonsense‐mediated mRNA decay (NMD) plays in p53 expression. Cultured p53‐positive cancer cells were treated with varied concentrations of cycloheximide, caffeine, wortmannin, or actinomycin D to determine optimal concentrations. Cells were also treated with constant inhibitor concentrations in time‐course experiments to determine optimal duration of exposure. Treated cell samples were divided equally for protein and total RNA isolation. Protein samples were subjected to SDS‐PAGE and western analyses for p53 and beta‐actin. RNA samples were used to synthesize cDNA, which was subjected to TaqMan quantitative RT‐PCR (qRT‐PCR) analysis for p53 and beta‐actin. Western and qRT‐PCR data are consistent with optimal chemical inhibitor concentrations being substantially higher for HepG2 cells compared to MCF‐7 and HeLa. However, chemical inhibitor exposure times required to achieve optimal effect was not widely varied between cell lines studied. Through our determination of optimal conditions for chemical inhibition of transcription, translation and PIKK activity in p53‐positive cell lines, we conclude that cell lines studied require widely different concentrations of, but not different exposure times to, chemical inhibitors. Therefore, we determined optimal concentrations and exposure times for chemical inhibition in our studies of the role of NMD in p53 expression.

Grant Funding Source: University of Tampa Chemistry, Biochemistry & Physics Departmental Student Research Support Fund
Neutrophil elastase suppresses cigarette smoke extract‐induced activation of anti‐oxidative pathway by mediating nrf2 degradation in bronchial epithelial cells. (LB237)
Authors: Jeong Jiyeong , Lee Kyoung‐Hee , Lee Chang‐Hoon , Yoo Chul‐Gyu
Published: June 2021

Cigarette smoke (CS) induces lung inflammation and simultaneously activates anti‐oxidative pathway as a defense mechanism. Recently, we found that neutrophil elastase (NE) enhances cigarette smoke extract (CSE)‐induced inflammation. Therefore, this study was undertaken to investigate the roles of NE on CSE‐induced activation of anti‐oxidative pathway and its regulatory mechanism in bronchial epithelial cells. CSE activated Nrf2, a transcription factor, which regulates the expression of several antioxidants. CSE increased anti‐oxidants such as HO‐1 and NQO1. Interestingly, NE suppressed CSE‐induced expression of HO‐1 mRNA and protein, but not NQO1. Therefore, we first determined the effects of NE on Nrf2. Although NE did not affect the basal and inducible level of Nrf2 mRNA, it significantly decreased the expression of Nrf2 protein. Nrf2 protein stability has been reported to be regulated by DJ‐1 and Keap1. However, the expression level of DJ‐1 and Keap1 did not change in NE‐treated cells. Nrf2 down‐regulation by NE was not blocked by treatment with proteasomal & lysosomal inhibitors (MG132, PS‐341 & chloroquine, NH4Cl), pan‐caspase inhibitor (zVAD‐fmk), or ROS blocker (NAC). Therefore, we hypothesized that down‐regulation of Nrf2 might be mediated by direct cleavage after NE internalization into cells. Immunofluorescence staining showed that NE was up‐taken intracellularly. Furthermore, in vitro fragmentation analysis supports that NE directly cleaved Nrf2. Taken together, these data suggest that NE accelerates CSE‐induced damage in lung epithelial cells by blocking anti‐oxidative pathway via Nrf2 proteolysis.
High‐throughput screening for inhibitors of iron transport in E. coli (LB238)
Authors: Hanson Mathew , Newton Salete , Klebba Philip
Published: June 2021

Iron acquisition is a component of the pathogenesis of Gram‐negative bacteria, therefore as a form of 'nutritional immunity' host organisms sequester iron. To obtain iron bacteria secrete siderophores that scavenge iron. The E. coli outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We observe this uptake reaction by fluorescently labeling FepA in live bacteria, monitoring quenching that occurs upon binding of FeEnt, and then fluorescence recovery during transport. We developed methods to screen for iron transport inhibitors using a cell‐based high‐throughput screening platform (HTS). This novel assay measures the fluorescence of bacterial cells before and after addition of FeEnt, and monitors its transport. Using this microtiter assay we differentiate between three possible events: no binding of FeEnt to FepA, binding without transport, and binding with transport into the periplasm. After optimizing the assay parameters (cell density, FeEnt concentration, and measurement timing) we found a Z' factor of ~0.7. This test allows for evaluation of libraries of compounds in 96‐well or 384‐well formats using live bacteria, in less than 1 hour.

Grant Funding Source: Supported by: NIH GM53836
Oxidative stress based response of a transcriptional regulator, OhrR, from Burkholderia thailandensis (LB252)
Authors: Pande Anuja , Grove Anne
Published: June 2021

Bacteria face a burst of oxidative species during host invasion. As a protective mechanism, they possess several proteins involved in inorganic peroxide sensing. However, much has to be discovered about the sensor‐responder proteins involved in organic hydroperoxide (OHP) resistance, which is important since OHPs are more toxic. Burkholderia thailandensis, a soil dwelling bacterium, contains genes ohrR and ohr, that are involved in OHP detoxification, with the former encoding an organic hydroperoxide reductase regulator (OhrR), a MarR family transcriptional regulator, regulating expression of the gene encoding organic hydroperoxide reductase (Ohr). EMSAs revealed specificity of B. thailandensis OhrR for the promoter region of ohr, with half‐maximal saturation of 3.6 nM. SDS‐PAGE demonstrated formation of reversible, oligomeric species of OhrR on treatment with organic and inorganic oxidants. Conformational changes were indicated by reduced thermal stability of oxidized protein compared to its reduced form (Tm 65.3 °C). Attenuated DNA binding in the presence of oxidants further confirmed structural changes in oxidized OhrR by virtue of disulfide bond formation between redox‐active cysteines. B. thailandensis shares 80% genetic homology with its pathogenic homologues B. pseudomallei and B. mallei, making characterizing its protein machinery involved in peroxide resistance even more crucial.
Metalloproteomic approach of mercury in breast milk samples of lactating women in the Amazon region, Brazil. (LB254)
Authors: de Queiroz João , dos Santos Felipe , Braga Camila , Leite Aline , Buzalaf Marília , Zara Luiz , Okoshi Marina , Padilha Cilene do , Padilha Pedro de
Published: June 2021

The aim of this study is to present the metalloproteomic results obtained for the protein fractionation in breast milk samples of lactating women from JIRAU Hydroelectric Power Plant ‐ Madeira River/Amazon region, Brazil. The protein spots obtained after the fractionation by 2D‐PAGE were cut from the gel and analyzed by Thermal Decomposition and Amalgamation Coupled with Atomic Absorption and the protein spots, in which were detected mercury, were characterized by mass spectrometry (ESI‐MS/MS). The mercury determinations indicated that this element is linked in two protein spots with molar mass (Mm) of 10.80 (spot 1) and 15.00 kDa (spot 2) and with concentration of 4.70 and 12.10 mg of mercury per gram of protein in the protein spots, respectively. Based on these concentrations was possible to estimate one mercury atom per four protein molecules in relation to the spot 1 and one mercury atom per protein molecule in relation to the spot 2. The ESI‐MS/MS analysis allowed to characterize the spot 1 as a fragment of beta‐casein (Mm = 26.00 kDa) and the spot 2 of lysozyme (Mm = 17.70 kDa).

Grant Funding Source: Financial Support: ESBR PD‐6631‐0001/2012 and Fapesp
Regulation of Snf1 kinase by a cross talk between ubiquitylation and phosphorylation (LB256)
Authors: Hsu Hsiang‐En , Liu Tzu‐Ning , Lo Yi‐Chen , Kao Cheng‐Fu
Published: June 2021

A search for genes reported to interact with UBP8 and UBP10, coding for two highly‐conserved ubiquitin proteases required for deubiquitylation of histone H2B, revealed the yeast homologue of AMPK, SNF1. Snf1 is required when cellular glucose is limited, as it activates stress‐responsive transcription factors. We used a mutant that lacks Ubp8 and Ubp10 to investigate whether Snf1 is regulated by ubiquitylation and degradation. SNF1 mRNA levels in the mutant were no different to the wild type, but Snf1 protein levels were dramatically decreased. The decreased the level of Snf1 protein in the mutant was mediated ubiquitin proteasome pathway. Interestingly, the remaining Snf1 in the mutant was hyper‐phosphorylate when the glucose is exhausted; furthermore, we observed no change in tolerance to stress and lifespan, suggesting the sustainment of Snf1 activity. We hypothesize that Snf1 activity is enhanced in the mutant to compensate for the defect. When Snf1 activity approaches the adaptive level, the cell can overcome the stress and survive. In conclusion, our results suggest that Snf1 is protected from degradation by both Ubp8 and Ubp10. Adaptive hyper‐phosphorylated Snf1 might trigger stress‐responsive transcription and prevent Snf1 from being degraded further, thereby protecting cells against stress. We demonstrate the regulation of Snf1 by a crosstalk between phosphorylation and ubiquitylation.

Grant Funding Source: Supported by National Science Council, Taiwan and Academia Sinica
The use of a digital site (Vizua) in the teaching of gross‐anatomy by the students of the School of Medicine S3V (University of Versailles‐France) (LB26)
Authors: LE FLOCH‐PRIGENT Patrice , Vaniet Chloé , Bouhelal Omar , Ordureau Sylvain
Published: June 2021

Since four years, the students in gross‐anatomy of the “Sciences de la Santé Simone Veil's” School of Medicine (Versailles‐Saint Quentin University) followed an initiation on a digital site (Vizua) during their dissections’ laboratories. This short course was done in small groups (15 students). The site included numerous demonstrations of digitalized reconstructions of isolated organs, regions (thorax, abdomen, pelvis) and of cerebral vascularization. Osseous reconstructions were available. After registration, all the data were in free access for man and for some other vertebrates as well. The students of the school of medicine were well interest and regularly used the site on the web (www.vizua3d.com) in the following years noticing the new contributions.
Beneficial effects of Gelidium amansii ethanol extracts on the adipogenesis in 3T3‐L1 preadipocytes (LB260)
Authors: Park Hey jin , Lee Myoungsook
Published: June 2021

Abstract We believed that the prevalence of obesity could be driven by changes of lifestyle and environments as well as the individual genetics. Gelidium amansii (GA‐3) contains a flush of plant fiber that plays a role of food digestion and excretion. Even though many reports had been shown the beneficial effect of GA‐3 ethanol extracts on the reduction of cholesterol absorption, their effect on anti‐obesity was not proved so far.

To investigate the anti‐obese effects of GA‐3 ethanol extracts, biomarkers for adipogenesis (PPARλ, C/EBPα, FABP, ACC & SREBP‐1), lipolysis (HSL & AMPK) and inflammation (TNF‐a, IL‐6, IL‐10) were tested by western blotting, real time PCR and ELISA in 3T3‐L1 cells.

Intracellular lipid accumulation was significantly decreased in preadipocytes differentiated in the presence of GA‐3 ethanol extracts compared to control as judged by Oil Red O staining. GA‐3 ethanol extracts reduced the lipid accumulation in corresponding to the reduction of protein and mRNA and protein level of PPARλ, C/EBPα, SREBP‐1, LPL, AQP7, and Insulin in concentration–dependent manner. However, adiponectin mRNA expression was not increased significantly. GA‐3 ethanol extracts also increased the phosphorylation of HSL and AMPK expression, which are active forms of lipolysis function on the intracellular signaling. IL‐6 mRNA expression decreased.

We found that GA‐3 ethanol extracts have a anti‐obesity effects because they significantly reduced inflammation‐induced adipogenesis but activated lipolysis. These results suggested GA‐3 ethanol extracts may develop the functional food to prevent obesity in future.

Grant Funding Source: Korea Inst. of Planning Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (811003‐3) and Ministry of Oceans and Fisheries
Expression of synaptopodin in endothelial cells exposed to laminar shear stress and its role in endothelial wound healing (LB272)
Authors: Park Soo Jin , Boo Yong Chool
Published: June 2021

Laminar shear stress (LSS) due to orderly blood flow contributes to the vascular health by multiple mechanisms including promotion of endothelial wound healing. The purpose of the present study was to identify LSS‐inducible actin‐binding proteins which might play a role in endothelial wound healing processes. Analysis of gene expression profiles of human umbilical vein endothelial cells under static and LSS‐exposed conditions provided a list of actin‐binding proteins whose mRNA levels were significantly increased by LSS. The list includes synaptopodin (SYNPO) whose expression or function in endothelial cells has rarely been reported. Western blot and immunocytochemistry showed that the SYNPO protein level was significantly increased by LSS. In the in vitro scratch wound healing assay, LSS treatments of endothelial cells before and/or after wound creation enhanced wound closure, and such effects were attenuated when SYNPO expression was suppressed using small interfering RNA. In addition, overexpression of exogenous SYNPO enhanced endothelial wound closure in the absence of LSS. This study demonstrated that SYNPO is expressed in endothelial cells in response to LSS and mediates the wound healing process.
Calpain‐2 proteolysis of MARCKS and MLP1 regulates ENaC activity (LB273)
Authors: Montgomery Darrice , Al‐Khalili Otor , Ghant Marcus , Eaton Douglas , Alli Abdel
Published: June 2021

MARCKS is a ubiquitously expressed protein while MARCKS like protein (MLP1) is more abundantly expressed in mouse kidney. The effector domain of MARCKS and MLP1 is similar and serves as the functional domain for the sequesteration of phosphatidylinositol phosphates which is essential for normal activity of the epithelial sodium channel (ENaC). PIP strip overlay binding assays revealed identical binding of PIP2 and PIP3 by recombinant MARCKS and MLP fusion proteins. The effector domain of MARCKS is known to be regulated by PKC‐mediated phosphorylation of serine residues, binding to calcium‐calmodulin, and calpain‐2 proteolytic cleavage. The purpose of this study was to determine if ENaC activity is affected by calpain‐2‐mediated proteolysis of either ENaC subunits, MARCKS, or MLP. Calpain‐2 inhibition in MPKCCD cells resulted in a decrease in amiloride‐sensitive transepithelial current. However, exogenous application of purified recombinant calpain‐2 to the apical side of MPKCCD cells did not result in any appreciable change in transepithelial current. Algorithms predicting calpain‐2 cleavage sites revealed multiple putative cleavage sites within ENaC, MARCKS, and MLP. In vitro proteolytic assays showed both MARCKS and MLP but not ENaC alpha, beta, or gamma subunits are sensitive to calpain‐2 proteolytic cleavage. Taken together, these results suggest ENaC activity is regulated by calpain‐2 proteolytic cleavage of MARCKS/MLP in mouse kidney.

Grant Funding Source: This work was supported by an NIH/NIGMS K12 GM000680 (to DCE) and an NIH/NRSA DK093255‐01 (to AAA).
Identification and validation of P. berghei glutathione s‐transferase small molecule inhibitors by virtual library screening (LB274)
Authors: Colon‐Lorenzo Emilee , Colón‐López Daisy , Bosch Jürgen , Ortiz José , Serrano Adelfa
Published: June 2021

Antimalarial drug development is of outmost importance to achieve malaria eradication, especially by targeting the blood stages critical to parasite development. Identification and validation of potential drug targets is required for the development of novel antimalarials. Glutathione S‐transferase (GST) is a detoxification enzyme that has been associated with drug resistance and proposed as a potential drug target in Plasmodium. This study aims to elucidate the biological role of pbGST using a combination of reverse genetics and bioinformatics approaches. Multiple attempts to disrupt the pbgst gene were unsuccessful indicating that GST is essential for P. berghei erythrocytic stages; validating pbGST as a drug target. A pbGST structural model was generated by comparative modeling and used to perform a virtual library screening of the ChEMBL‐Neglected Tropical Disease library and the ChemBridge Express library using the OpenEye software suite. In silico screening identified several hits with potential as pbGST inhibitors. Docking analyses show favorable binding interactions between the small molecule compounds and the pbGST protein. P. berghei in vitro drug luminescence assay is being used to assess the antimalarial activity and the EC50 values of the identified compounds. This work should enhance our understanding on the role of Plasmodium GST and will be relevant for potential development of novel antimalarials.

Grant Funding Source: GM08224, 2G12‐RR003051, 8G12MD007600, ASM Watkins Fellowship (EECL), R25‐GM061838 (EECL), The Bloomberg Family Foundation (JB) and NSF predoctoral fellowship (DDCL)
JunB contributes to the epithelial‐mesenchymal transition in response to TGF‐β (LB277)
Authors: Gervasi Megan , Bianchi‐Smiraglia Anna , Cummings Michael , Bakin Andrei
Published: June 2021

The process of epithelial‐mesenchymal transition (EMT) is important in tissue fibrosis, wound healing and cancer. Transforming growth factor beta (TGF‐β) induces EMT and fibrotic proteins in a variety of responsive cells via a mechanism that is not fully understood. This study identifies a critical role of JunB in the EMT and fibrotic process in response to TGF‐β. Knock‐down of the immediate‐early response gene JunB by siRNA abrogates TGF‐β‐induced disruption of epithelial cell junctions, formation of actin fibers, focal adhesions and expression of fibrotic proteins. JunB contributes to Smad‐mediated repression of Id2 helix‐loop‐helix protein but does not affect the induction of Hmga2, Snail and Slug. Knock‐down of JunB abolishes up‐regulation of ATF3, which is required for Id2 repression. Importantly, JunB contributes to the TGF‐β‐induced fibrotic response by regulating expression of fibronectin, fibulin‐2, tropomyosin (Tpm1) and β3‐integrin, which play critical roles in matrix deposition, cell‐matrix adhesion and actin stress‐fibers. In summary, JunB provides important input in setting the transcriptional program of the EMT and fibrotic responses to TGF‐β. Thus, JunB represents an important target in diseases associated with EMT, including cancer and fibrosis.

Grant Funding Source: Supported by R01 CA95263
Structure and mechanism of bacterial effectors that hijack trafficking small GTPases (LB278)
Authors: Cherfils Jacqueline , Folly‐Klan Marcia , Roy Craig , Alix Eric , Mukherjee Shaeri , Antonny Bruno , Stalder Danièle , Campanacci Valérie
Published: June 2021

Membrane traffic, which is the “cellular postal service” that shuttles biomolecules around the cell and organizes the structure of organelles, is among the primary targets of effectors injected by intracellular pathogenic bacteria to invade their host and avoid from being destroyed. We will present our recent structural and biochemical studies of effectors from Legionella pneumophila (the bacteria that causes the legionnaire’s disease, a severe pneumonia) which divert membrane traffic to generate a membrane‐bound vacuole where the pathogen hides and replicates. One of these effectors, AnkX, is a FIC domain‐containing toxin that alters the functions of a Rab GTPase involved in vesicular traffic at the endoplasmic reticulum, by covalent attachment of a phosphocholine molecule. The other one, RalF, functions as an illegitimate guanine nucleotide exchange factor to illegitimately activate an Arf GTPase on the vacuole. Our studies showed how AnkX binds and process CDP‐choline to transfer phosphocholine to Rab1 [1], and uncover a novel membrane sensor in RalF that controls its localization and activity [2] [1] Campanacci V, Mukherjee S, Roy CR, Cherfils J. The mechanism of phosphocholine transfer by AnkX, an ankyrin repeats and FIC domain‐containing Legionella effector. EMBO J. (2013) 32:1469‐77.

[2] A Novel Membrane Sensor Controls the Localization and ArfGEF Activity of Bacterial RalF. Folly‐Klan M, Alix E, Stalder D, Ray P, Duarte LV, Delprato A, Zeghouf M, Antonny B, Campanacci V, Roy CR, Cherfils J. PLoS Pathog. (2013) 9(11):e1003747.

Grant Funding Source: Supported by CNRS and ANR
FRETting for a benchmark (LB280)
Authors: Lin Tao , Scott Brandon , Hoppe Adam , Chakravarty Suvobrata
Published: June 2021

Fluorescence resonance energy transfer (FRET) is a powerful tool to study molecular interactions, especially protein‐protein interactions (PPI). Recombinant fluorescent proteins enable the study of such interaction in living cells. However, there is no reference study yet detailing the limits of the strength of molecular interactions that can be detected by live‐cell FRET. Using a test set of 11 pairs of small interacting proteins for whom the respective affinities (Kd, dissociation constant) range between mM ‐ nM, we establish the limits with live‐cell microscopy. This study benchmarks intracellular FRET and is a very useful reference for other intracellular PPI methods such as BiFC (Bimolecular Fluorescence Complementation) and IDPRIME (Interaction‐Dependent Probe Incorporation Mediated by Enzymes).

Grant Funding Source: BCAAP (Biological Control and Analysis through Applied Photonics) and AES (Agricultural Experiment Station) of SDSU
I woRk diFferently 4 ‐ Mechanism of activation of auto‐inhibited IRF4 (LB282)
Authors: Govinda Remesh Soumya , Escalante Carlos
Published: June 2021

I woRk diFferently 4 ‐ Mechanism of activation of auto‐inhibited IRF4

Transcriptional regulation involves the orchestrated binding of transcription factors to regulatory DNA binding sites known as enhancers. Interferon (IFN) regulatory factor family member (IRF4) is a transcription factor that serves specific roles in transcriptional regulation of IFN responsive genes, is limited to the immune system and is critical in B‐ & T‐cell differention. Generally, members of IRF family, like IRF3 and IRF5, have carboxy terminal auto‐inhibitory regions that are phosphorylated to generate a transcriptionally active dimer. IRF4 also has a carboxy terminal auto‐inhibitory region and it gets activated by binding to multiple different partners presumably through a general mechanism of activation with slight variations with each binding partner. In the current study, we are investigating the general mechanism of activation of the auto‐inhibited IRF4 and compare it with other members of the IRF family. We have determined the crystal structure of the C‐terminal activation domain of IRF4 and carried out small angle X‐ray scattering (SAXS) studies to generate ab initio models for the full‐length protein and obtain insights into the autoinhibitory mechanism. Using this approach, we have concluded that the putative linker of IRF4 connecting the N‐ and C‐terminus is most likely a folded well‐structured domain that interacts with the auto‐inhibitory carboxy tail. Information about the mechanism of activation of the auto‐inhibited IRF4 highlighting its uniqueness within the IRF family will help in development of novel therapeutics in disease states that are mediated by IRF4 like multiple myeloma, cardiac hypertrophy and certain autoimmune diseases

Grant Funding Source: NIH R01
The MED12/Cyclin C Interface as a Therapeutic Target in Oncogenic Wnt/Beta‐catenin signaling (LB285)
Authors: Clark Alison , Spaeth Jason , Gao Fangjian , Tomita York , Boyer Thomas
Published: June 2021

CDK8 is a colorectal cancer oncoprotein whose amplification‐dependent overexpression identifies a significant subset of colorectal cancer patients with poor prognosis. Mechanistically, Cyclin C (CycC)‐dependent CDK8 kinase drives tumorigenesis by regulating beta‐catenin transcriptional activity. Accordingly, inhibition of CDK8 kinase function offers a promising therapeutic approach to CDK8‐overexpressing colorectal cancers. CDK8/CycC, along MED12 and MED12, comprise a discrete 4‐subunit “kinase” module within Mediator, a conserved multiprotein interface between gene‐specific transcription factors and RNA Polymerase II. Our studies reveal that beta‐catenin binds directly to MED12, which in turn activates CycC‐dependent CDK8 kinase activity and beta‐catenin target gene induction. Notably, we found that MED12 activates CDK8 through its direct interaction with an uncharacterized CycC surface groove that is phylogenetically conserved among CycC family members, but absent from cell cycle‐type cyclins. We employed a focused structure‐based approach to identify small molecule CDK8 inhibitors through disruption of the MED12/CycC interface. Using a virtual ligand screen of the NCI diverse compound library, we identified 36 potential inhibitory scaffolds, five of which were biochemically validated to specifically inhibit MED12‐dependent CycC‐CDK8 kinase activity in a dose dependent manner. These compounds could thus represent promising lead candidates for pharmacological intervention in CDK8‐overexpressing colorectal and other beta‐catenin‐driven malignancies.

Grant Funding Source: Supported by R01MH085320 (TGB) and T32DE14318 (ADC)
Does pre‐medical anatomy experience affect student performance in gross anatomy courses in medical school? (LB31)
Authors: Jordan Rebecca , Boehm Karl , Fraticelli Nicole , Riccardi Margaret , Kondrashov Peter
Published: June 2021

Gross Anatomy is one of the most challenging courses taught in medical school. We investigated the effect of taking an anatomy course prior to entering medical school on student performance in Gross Anatomy. Surveys administered to students covered the number, type, and level of anatomy courses taken prior to matriculation. Students were also asked to rate the perceived or potential benefit that such courses had or would have had. The responses were matched with each student’s semester, lecture, and lab grades from fall and spring semesters of Gross Anatomy. There was no significant difference in these grades between students with previous anatomy experience compared to those without. There was a statistically significant difference in all three grades between students who took anatomy lecture only and those who took courses with a lab. In the fall semester, the latter group’s grade was higher than the former’s by 4.2 % in lecture, 6.7 % in lab, and 5.7 % in semester. The perceived benefit of previous anatomy courses was rated as 6 out of 10 by students with previous anatomy experience and as 8 out of 10 by those without.

Students felt that there was a benefit to taking anatomy courses prior to matriculation, but it did not affect their performance in the medical Gross Anatomy course. Pre‐medical anatomy courses with a lab component have a greater impact on student success in mastering anatomical concepts in medical school.
Biomimetic porous tetracycline loaded PLGA/Silk Fibroin/Ascorbic acid/n‐HA hybrid scaffolds for adipose derived stem cells differentiation into Osteogenic lineage (LB32)
Authors: Gandhimathi Chinnasamy , Venugopal Jayarama , Ramakrishna Seeram , Tay Samuel , Kumar Srinivasan
Published: June 2021

The objective of this study is to fabricate poly (D,L‐lactide‐co‐glycolide) (PLGA)/Silk fibroin(SF)/Ascorbic acid(AA)/Tetracycline (TC) nanofibrous scaffolds and nanohydroxyapatite (n‐HA) was deposited by calcium phosphate dipping method. These nanofibrous scaffolds were characterized using scanning electron microscopy (SEM), surface wettability, functional groups analysis, porosity and tensile properties. Adipose derived stem cells (ADSCs) were cultured on these nanofibrous scaffolds and were made to undergo osteogenic differentiation in the presence of TC/n‐HA. Osteogenic differentiation of ADSCs was confirmed using alkaline phosphatase activity (ALP), mineralization (ARS) and immunofluorescent staining using both ADSC specific marker protein CD105 and osteoblast specific marker protein osteocalcin. SEM micrographs of PLGA/SF/AA/TC/n‐HA fabricated electrospun fiber diameters measured around 430 ± 26 nm to 220 ± 27 nm and FT‐IR analysis showed the amide I, II and III groups. The obtained PLGA/SF/AA/TC/n‐HA scaffolds were hydrophilic, having water contact angle of 0° compared to PLGA nanofibers (119.2 ± 12.11°). Resulting nanofibrous scaffolds were highly porous (85‐91%) and provided desirable architecture for transport of nutrients and metabolic waste and facilitate neovascularization. The PLGA/SF/AA/TC/n‐HA scaffolds had mechanical properties comparable to that of native bone with tensile break of 28.52% and Young’s modulus of 2.94 MPa, while that of PLGA nanofibers was comparatively lower at 1.96 ± 0.68 MPa. Cell culture studies performed on the bioactive AA/TC/n‐HA molecules were incorporated on the nanofibers to develop specific biological functions like proliferation, mineralization and differentiation of ADSCs into osteogenic lineage. The results proved that the ADSCs differentiated cells showed osteoblast‐like morphology, expression of osteocalcin and mineralization. Overall the data suggest that the abundance and accessibility of biodegradable PLGA/SF/AA/TC/n‐HA nanofibrous scaffolds can be used as a carrier for the sustained release of biomolecules and promote greater osteogenic differentiation of ADSCs, proving them to be potential scaffolds for bone tissue engineering.

Grant Funding Source: This work was supported, in part by the Ministry of Education SUG (SD Kumar), LKCMedicine, NTU
Modest increases in serum calcidiol may improve T2DM‐related health outcomes in non‐white, ethnically diverse, postmenopausal women (LB327)
Authors: Alabdulkader Shahd , Malhotra Sonia , Solomon Jesse , Moreira‐Lucas Tracy , Hamadeh Mazen
Published: June 2021

Mounting evidence suggests a crucial role for vitamin D status in the pathogenesis of type 2 diabetes mellitus (T2DM), since vitamin D promotes the survival and function of pancreatic β‐cells. Our objectives were to examine the correlation between serum calcidiol and diabetes‐related health outcomes, and to determine whether vitamin D3 and calcium supplementation would attenuate the severity of T2DM. In this pilot study, 11 non‐white, ethnically diverse (Caribbean, Black, South Asian), post‐menopausal women with T2DM (age, 61 ± 11 y) were supplemented for 3 y with either placebo or 1800 IU of vitamin D3 + 720 mg of calcium (CaD)/day. Spearman’s rank coefficient was used to examine the correlations between serum calcidiol and the different outcome measures. Per‐protocol and retrospective analyses were adopted to determine the effect of CaD on the outcome measures. Significance was established at P 蠄 0.10. The relative change over 3 y in serum calcidiol significantly correlated with the relative change in body weight (r = ‐0.736, P = 0.005), BMI (r = ‐0.736, P = 0.005), body fat (%) (r = ‐0.445, P = 0.085), hip circumference (r = ‐0.664, P = 0.013), serum TC/HDL‐C (r = ‐0.427, P = 0.095), serum PTH (r = ‐0.655, P = 0.014), and serum calcium (r = 0.500, P = 0.059). Retrospective analysis showed differences between the CaD vs. placebo in serum calcidiol (+41.7% vs. ‐30.3%, respectively, P = 0.004), hip circumference (‐3.25% vs. +0.32%, respectively, P = 0.052), systolic blood pressure (‐1.5% vs. +12.0%, respectively, P = 0.126), and serum PTH (‐30.8% vs. ‐3.1 %, respectively, P = 0.003). We conclude that modest increases in serum calcidiol may improve T2DM‐related health outcomes in non‐white, ethnically diverse, postmenopausal women.
Validation of a screener to measure overall lifestyle habits: The MEDiterranean LIFEstyle index (LB375)
Authors: Sotos Prieto Mercedes
Published: June 2021

The aim of this study was to conduct an external validation of the previous developed MEDiterranean LIFEstyle 28‐item (MEDLIFE) index. A full length validated 142‐item questionnaire (full‐Q) (which includes a food frequency questionnaire) and the MEDLIFE index assessing three blocks: food consumption, traditional Mediterranean dietary habits and physical activity, rest and social interaction patterns) were administered to 196 adults men and women (mean age 41.4±9.2 y) from the Program SI! Study. MEDLIFE index had an intraclass correlation coefficient=0.544, showing acceptable ability to rank participants by MEDLIFE‐derived from full‐Q. Absolute agreement measured by kappa statistics showed strong concordance (k=0.81‐1) for 10.7% of the items; and good (k=0.61‐0.80) to moderate (k=0.41‐0.60) for most of the items evaluated. In the crude classification analysis, 52.1% of the participants were classified in the same tertile by both instruments whereas only 7.14% were in the opposite tertile. Bland Alman’s analysis showed limits of agreement ranged from 3.67 to 6.81(mean 1.58). In conclusion, the MEDLIFE index is a valid instrument to measure overall Mediterranean lifestyle, and may be used as a short screening tool in clinical and epidemiological studies

Grant Funding Source: PI11/01885
Inhibitory effect of kohlrabi juices with antioxidant activity on oxidative stress in human dermal fibroblasts (LB394)
Authors: Kim Dan‐bi , Oh Ji‐Won , Shin Gi‐Hae , Kim Young‐Hyun , Lee Jong Seok , Park In‐Jae , Cho Ju Hyun , Lee Ok‐Hwan
Published: June 2021

Inhibitory effect of kohlrabi juices with antioxidant activity on oxidative stress in human dermal fibroblasts Dan‐Bi Kim1, Ji‐Won Oh1, Gi‐Hae Shin1, Young‐Hyun Kim1, Jong Seok Lee1, In‐Jae Park2 and Ju Hyun Cho2, Ok‐Hwan Lee1,* 1Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 200‐701, Korea 2Hurum Central Research Institute, Ochang 363‐880, Korea This study was to investigate the antioxidant activity of green kohlrabi juice (GKJ) and purple kohlrabi juice (PKJ) using different in vitro methods. GKJ exhibited the higher total phenol content (153.74 GAE mg/mL), and antioxidant activities than that of PKJ. However, PKJ exhibited the higher ORAC value (664.32 μM TE/mL) than that of GKJ. Human dermal fibroblasts were co‐incubated with various concentrations (10~25 μg/mL) of GKJ and PKJ for 24 h, and then exposed to 1 mM hydrogen peroxide for 3 h. Hydrogen peroxide treatment significantly decreased cell viability (p<0.05), whereas, GKJ and PKJ protected against hydrogen peroxide‐induced cell death in a dose‐dependent manner. Also, pre‐treatment with GKJ and PKJ suppressed intracellular reactive oxygen species (ROS) production in hydrogen peroxide‐treated human dermal fibroblasts. These results demonstrate that both kohlrabi juices have strong antioxidative activity, which could be a good candidate for natural antioxidant and anti‐aging agents.
Gc‐ms analysis of characteristic aromatic compounds from jujube wine (LB396)
Authors: Zhang Zhisheng , Shu Ying , Li Guijie , Jiao Xueqin , Zhao Renbang , Chen Feng , Zhao Congzhi , Tang Xia
Published: June 2021

The characteristic aromatic compounds in jujube wine was extracted by liquid‐liquid extraction (LLE) and determined by Agilent 7890A gas chromatography system with a split/splitless injector and Agilent 5975C mass spectrometer. The results showed that a total of 50 aromatic compounds, such as one acid, seven alcohol, eight alkanes, twenty five esters, four ketones, three phenols and two others compounds were detected, and they are characteristic flavor of jujube wine. Esters, the compounds of the highest values in the analyses of volatiles, are consisted mostly of ethyl esters which have C3~C8 straight chain fatty acid residues. They contribute to fruity and floral sensory properties to the jujube wine. Trans‐linalool oxide is the only terpene while 9‐Tetradecenoic acid is the only acid compound found in the jujube wine. The alcohols compounds contribute to sweety and creamy flavor while n‐hexanol is responsible for resin flavor and green grass odor.

Grant Funding Source: Surpported by Research Center of Primary Product Processing and Engineering of Hebei Province, Baoding
Regulation of branched‐chain amino acid (BCAA) catabolism: factors inactivating branched‐chain alpha‐ketoacid dehydrogenase (BCKDH) kinase (LB428)
Authors: Kondo Yusuke , Itami Yuya , Zhen Hongmin , Kitaura Yasuyuki , Shimomura Yoshiharu
Published: June 2021

BCAAs (leucine, isoleucine, and valine) are essential amino acids. The BCAA catabolism is regulated by BCKDH complex, which is located at the second step of the catabolic pathway. The BCKDH complex is controlled by a covalent modification. BCKDH kinase (BDK) is responsible for inactivation of the complex by phosphorylation, and BCKDH phosphatase (BDP) is for activation of the complex by dephosphorylation. It has been demonstrated that two forms of BDK exists in rat liver mitochondria: a form bound to BCKDH complex and a free form. Only the bound form is suggested to be in active. The regulation of BCKDH complex has been examined under many physiological conditions and the results suggest that BDK is the primary regulator of BCKDH complex by the mechanism in which the amount of the bound form is inversely correlated with the activity state of the complex. In the present study, we addressed to identify factor(s) inactivating BDK and show here the results suggesting that there are new factors that inactivate BDK in rat liver mitochondria.

Grant Funding Source: Sponsored by Ajinomoto Amino Acid Research Program 2012
UK National Diet and Nutrition (NDNS) Survey: ad‐hoc cross‐sectional survey to sustainable Rolling Programme (RP) for surveillance (Y6‐9 2013‐17) (LB467)
Authors: Ray Sumantra , Nicholson Sonja , Roberts Caireen , Page Polly
Published: June 2021

NDNS provides the only authoritative source of representative data on dietary habits and nutritional status (plus routine metabolic and CVD risk data) for the UK population. Following the first dietary and nutritional survey of British adults (1986/87), NDNS operated as a series of stand‐alone cross‐sectional surveys by age group (pre‐school, young people, adults, older adults). After government review, it moved to a cross‐sectional RP model in 2008 for more rapid data generation, greater ability to track temporal changes and to respond to policy needs. Now funded to 2017, 500 adults and 500 children are sampled each year. Along with boosted samples in Scotland, Wales and Northern Ireland, this enables in‐depth analysis using combined data years and trend data every 2‐3 years. An estimated 4 day food diary was determined to be low burden and cost‐effective compared to the 24H Recall and replaced the previous 7 day weighed diary as the measure of dietary intake for the RP. As in past NDNS, blood and urine samples are analysed and stored for future research. NDNS remains unique in the inclusion of a doubly‐labelled water sub‐study as an objective biomarker to validate energy intake estimated from reported food consumption. Combined results for NDNS RP 2008‐12 will be published in 2014/15. NDNS data provide robust assessment of nutritional deficiency or excess at different stages of life in the UK population, progress against health recommendations/targets and exposure monitoring. Methods and headline results are published via web‐based reports; data are available via UK Data Archive. NDNS is carried out by NatCen Social Research, Medical Research Council Human Nutrition Research and University college London. UK health departments fund NDNS and the complementary UK sodium (salt) surveys and Dietary Survey of Infants and Young Children.

Grant Funding Source: “Supported by” the UK Department of Health and the UK Food Standards Agency
Control of Cardiac Fibroblast Phenotype by the Meox2/Zeb2 Signalling Switch (LB47)
Authors: Jahan Fahmida , Northcott Josette , Cunnington Ryan , Cheung David , Rattan Sunil , Filomeno Krista , Dixon Ian , Wigle Jeffrey
Published: June 2021

The fibroblast to myofibroblast phenoconversion, which is induced by TGF‐β, is a crucial step during cardiac fibrosis. Activated myofibroblasts increase extracellular matrix (ECM) synthesis, which impairs contraction that results in myocardial stiffening, and eventually heart failure. Meox2, a regulator of cell proliferation and senescence, can block TGF‐β mediated epithelial to mesenchymal transition (EMT). In contrast, Zeb2, a repressor of Meox2, enhances EMT in concert with TGF‐β. However, their roles in the fibroblast to myofibroblast phenoconversion remain elusive. We have shown that Meox2 is more highly expressed in fibroblasts than in myofibroblasts. Conversely, Zeb2 expression is lower in fibroblasts but higher in myofibroblasts. This suggests that Meox2/Zeb2 may regulate the fibroblast phenotype during cardiac fibrosis. We have shown that ectopic expression of Meox2 represses a set of myofibroblast markers. We have successfully knocked down Meox2 protein in primary cells. By confocal microscopy, Meox2 protein was found to be confined to the nuclei of fibroblasts whereas a cytoplasmic distribution was seen in myofibroblasts. By immunoblotting, Zeb2 expression was found to be higher in the nuclei of myofibroblasts. To test if ectopic Zeb2 expression enhances this phenoconversion, we have recently generated an adenoviral Zeb2 expression construct. We anticipate that the Meox2/Zeb2 signalling switch plays a critical role in cardiac fibrosis and findings from this study may provide a basis for developing Meox2 or Zeb2 based novel anti‐fibrotic drugs in the future.

Grant Funding Source: Supported by Canadian Institutes of Health Research & Heart and Stroke Foundation of Canada