bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 IRF4Transcriptional 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‐ &amp; 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 diseasesGrant 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
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 studiesGrant Funding Source: PI11/01885
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