bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the novel H82R mutation in CGI‐58 that causes Neutral Lipid Storage Disorder in humans (LB153)
Authors: Hassanien Sarah , McMahon Derek , Kurz Daniel , Shah Dharika , Dinh Anna , Brasaemle Dawn
Published: June 2021

Comparative gene identification‐58 (CGI‐58) interacts with and co‐activates adipose triglyceride lipase (ATGL). The H82R mutation in human CGI‐58 causes a neutral lipid storage disorder (NLSD) characterized by ichthyosis and excessive triglyceride storage in many types of cells. We studied the comparable H84R mutation in mouse CGI‐58, and H84A mutated CGI‐58, to ask how CGI‐58 function is impaired. The ectopic expression of wild‐type (WT) CGI‐58 in human NLSD fibroblasts reduced excessive triglyceride storage to normal levels, whereas H84R CGI‐58 was significantly less effective. Additionally, neither H84R nor H84A CGI‐58 co‐activated ATGL in an in vitro triglyceride hydrolase assay. A co‐immunoprecipitation assay demonstrated that H84R and H84A CGI‐58 bound ATGL as well as WT CGI‐58. Immunofluorescence microscopy revealed that H84R and H84A CGI‐58 localized to ectopic perilipin 1A on lipid droplets of cultured NIH3T3CARΔ fibroblasts as well as WT CGI‐58. Moreover, the addition of forskolin and isobutylmethylxanthine to the cells triggered the dispersion of all three variants of CGI‐58 from the perilipin scaffold into the cytoplasm. Thus, H84R CGI‐58 shows appropriate subcellular localization in cells expressing perilipin 1A and binds to ATGL, yet does not co‐activate hydrolase activity. Future experiments will explore additional mechanisms for deficient CGI‐58 function.

Grant Funding Source: Supported by NIH R01 DK054797.
Lipid raft abnormalities and subsequent protein trafficking effects in Niemann‐Pick type C1 (LB158)
Authors: Kuech Eva‐Maria , Shammas Hadeel , Maalouf Katia E. , von Koeckritz‐Blickwede Maren , Das Anibh M. , Naim Hassan Y.
Published: June 2021

Many proteins and lipid components are organized within membrane microdomains, known as lipid rafts (LR). LR are cholesterol‐ and sphingolipid‐rich membranes resistant to non‐ionic detergents. In Niemann‐Pick type C1 (NPC1), a neurovisceral lysosomal storage disease, entrapment of cholesterol and sphingolipids occurs and may lead to alterations in LR composition. We investigate the LR status in fibroblasts derived from a patient with NPC1 and analyze the trafficking profiles of a typical LR‐associated membrane protein, dipeptidylpeptidase IV (DPPIV).

Sucrose density gradients of Triton X‐100 cellular lysates revealed a substantial shift of the LR‐marker flotillin2 in NPC fibroblasts from the floating LR fractions to the detergent‐soluble membranes. Notably, the cell surface expression of DPPIV increased significantly by 2‐fold in NPC1 cells, while the overall protein level of DPPIV remained constant. FACS analysis corroborated the increase of DPPIV at the cell surface. This accumulation is the result of delayed lipid rafts‐dependent endocytosis in NPC1 cells as assessed by (i) a significant reduction in the level of internalized biotin‐labeled DPPIV and (ii) imaging studies revealing a reduced localization of DPPIV with EEA‐1 in NPC1 cells. Strikingly, N‐butyl‐deoxynojirimycin (NB‐DNJ), an inhibitor of glucosylceramide synthase, restored significantly LR levels and endocytosis of DPPIV. We conclude that altered composition or reduced levels of LR constitute a potential mechanism in the pathogenesis of NPC1 and NB‐DNJ treatment restores a balanced membrane lipid composition and the associated biochemical anomalies.
Involvement of a type 2 ribonuclease H in kinetoplast maxicircle DNA replication in Trypanosoma brucei (LB165)
Authors: Misra Smita , Mittal Mukul , Singha Ujjal , Chaudhuri Minu , Chaudhuri Gautam
Published: June 2021

Ribonuclease H (RNase H, EC # 3.1.26.4) catalyzes the hydrolysis of RNA from a DNA/RNA hybrid. This ubiquitous enzyme interacts with several different DNA‐modifying enzymes in the cell and participates in the replication, recombination and repair of nuclear and mitochondrial DNA molecules. We report here that the type 2 RNase H of the parasitic protozoan Trypanosoma brucei is a predominantly mitochondrial enzyme and is essential for the survival of both the procyclic and the blood stream forms. Yeast 2‐hybrid and co‐IP analyses for the mitochondrial DNA replication‐associated proteins interacting directly with RNase HIIC revealed binding of RNase HIIC with the mitochondrial DNA ligase Kα which is involved in kinetoplast DNA replication. Interestingly, we found that RNase HIIC also binds with the DNA recombinase DMC1 which is also located in the mitochondrion of the parasite. Chromatin immunoprecipitation data indicated binding of both RNase HIIC and DMC1 to the maxicircle DNA. We found that the maxicircle DNA and transcripts derived from it are decreased significantly in the cells with decreased levels of RNase HIIC. This enzyme is required for the recovery from DNA damage caused by caffeine, hydroxyurea and methylmethane sulfonate, as the RNase HIIC knocked down cells are more sensitive to the lethality of these DNA damaging agents. We conclude that RNase HIIC collaborates with DNA ligase Kα and DMC1 to participate in the replication and repair of mitochondrial maxicircle DNA in these parasite cells. This research was supported in parts by NIAID grants 5R01AI042327, 1R21AI076757‐01A1 and NIGMS grant #3 S06 GM008037‐34‐S2 to GC.
Functional Studies of Orphan Enzymes by LC‐MS Based Metabolomics: Oxidation of Endogenous N‐Arachidonoylserotonin by Human Cytochrome P450 2U1 (LB174)
Authors: Xiao Yi , Siller Michal , Goyal Sandeep , Yoshimoto Francis , Wei Shouzou , Guengerich Frederick
Published: June 2021

Cytochrome P450 (P450) 2U1 has been shown to be expressed (at the mRNA level) in human brain and thymus. In order to elucidate the physiological function of this enzyme, an LC‐MS‐based metabolomics and isotopic labeling approach was used to identify endogenous substrate of human P450 2U1. In addition to fatty acid oxidation reactions, oxidation of endogenous N‐arachidonoylserotonin was characterized. Subsequent NMR, mass spectrometry, and comparison with a synthesized chemical standard showed that the main product was the result of C2 oxidation of the indole ring, in contrast to other P450s that generated different products. The product (2‐oxo) was 4‐fold less active than N‐arachidonoylserotonin in inhibiting fatty acid amide hydrolase. The oxidation rate of N‐arachidonoylserotonin was similar to that of arachidonic acid, one of the previously identified fatty acid substrates of P450 2U1. The presence of N‐arachidonoylserotonin in human brain and the oxidation of N‐arachidonoylserotonin by P450 2U1 suggest a possible role in human brain and possibly other sites. (Supported in part by USPHS R37 CA090426)
Quantitative Phosphoproteomic Analysis of RIP3‐dependent Protein Phosphorylation in the Course of TNF‐induced Necroptosis (LB175)
Authors: Zhong Chuan‐Qi , He Wan‐ting , Xu Xiaozheng
Published: June 2021

Tumor necrosis factor (TNF)‐induced cell death in murine fibrosarcoma L929 cells is a model system in studying programmed necrosis (also known as necroptosis). Receptor interacting protein 3 (RIP3), a serine‐threonine kinase, is known to play an essential role in TNF‐induced necroptosis ; however, the phosphorylation events initiated by RIP3 activation in the necroptotic process is still largely unknown. Here, we performed a quantitative mass spectrometry‐based analysis to compare TNF‐induced changes in the global phosphoproteome of wildtype (RIP3+/+) and RIP3‐knockdown L929 cells at different time points after TNF treatment. A total of 8,058 phosphopeptides spanning 6,892 phosphorylation sites in 2,762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 hours, respectively. By comparing the phosphorylation sites in wildtype and RIP3‐knockdown L929 cells, 174, 167 and 177 distinct phosphorylation sites were revealed to be dependent on RIP3 at the 0.5, 2 and 4 hour time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3 dependent phosphorylation in LPS‐stimulated peritoneal macrophages and TNF‐treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report is highly relevant to the study of TNF‐induced necroptosis of L929 cells.
Structural Characterization of a Phospho‐Switch in the Metastasis Suppressor RKIP (LB189)
Authors: Skinner John , Ong Heng , Rosner Marsha , Sosnick Tobin
Published: June 2021

RKIP is a member of the phosphatidylethanolamine binding protein (PEBP) family with over 400 members. Previous work showed that phosphorylation of S153 was sufficient to alter RKIP binding partners, effectively upregulating the MAP kinase and β‐adrenergic receptor signaling pathways (Granovsky and Rosner, Cell Res., 18:452, 2008). Yet little is known regarding the mechanism by which RKIP phosphorylation regulates these key signaling pathways. Here we propose a three state allosteric model to explain RKIP function. Using NMR, we demonstrate that the RKIP phospho‐switch is associated with a major structural change that can be instigated by breaking a single salt bridge. We also demonstrate that RKIP can dimerize as a result of the phospho‐switch due to formation of disulfide linkages and the functional significance of this oligomerization will be presented. Together these results reveal a novel phospho‐switch that regulates RKIP function and has broad applicability to other PEBP family members throughout evolution.
The Functional Significance of Phospho‐Rab GTPases (LB194)
Authors: Lepore Dante , Collins Ruth
Published: June 2021

Intracellular membrane trafficking is an essential and conserved process throughout eukaryotes. To preserve organelle identity in a complex cellular system, control mechanisms must exist to oversee the production and targeting of intracellular vesicles. Key control elements are the Rab GTPases, which utilize a nucleotide‐bound cycle to act as master regulators for each discrete trafficking step. Several studies, including global proteomic screens, have identified multiple Rabs as phosphoproteins, but the functional consequence(s) of this modification are unknown. The Saccharomyces cerevisiae Rab GTPase Sec4p is negatively regulated by phosphorylation, but the molecular mechanisms and proteins responsible for this modification are unknown. Using Sec4p as a model for Rab phosphorylation and unique antibodies able to specifically detect modified Sec4p, we have shown that phosphorylation is tightly regulated in conjunction with cell cycle progression and spatial location within the cell. Currently, we are performing an overexpression screen to elucidate the kinase that modifies Sec4p as well as the biochemical pathway responsible for activating Sec4p phosphorylation. We hypothesize that Rab phosphorylation may be a novel regulatory mechanism through which additional cellular processes can impinge upon membrane trafficking.

Grant Funding Source: National Institute of Health
Sec62 as a novel gating modulator for protein translocation and ion permeability of the mammalian Sec61 complex (LB196)
Authors: Schorr Stefan , Linxweiler Maximilian , Greiner Markus , Jung Martin , Cavalie Adolfo , Zimmermann Richard
Published: June 2021

In mammalian cells the endoplasmic reticulum (ER) plays a key role during the biogenesis of secretory and membrane proteins and acts as a major regulator for intracellular Ca2+ signaling. Sec62, a component of the ER‐protein translocase, acts during post‐translational translocation of small precursor proteins and in ER‐Ca2+ leakage‐control. Our project aims to elucidate the molecular mechanisms how it regulates the translocation machinery concerning these two fundamental and seemingly opposing functions. We used siRNA mediated depletion of target proteins from human cells followed by functional assays for protein transport and live cell calcium imaging.

Depletion of Sec62 in combination with proteasomal inhibition led to accumulation of the precursor of the ER resident protein ERj3 at the cytosolic face of the ER membrane. Control experiments using different siRNA as well as a plasmid rescue approach confirmed the specificity for Sec62. Follow‐up experiments revealed a pattern of dependencies upon translocon components which is characteristic for a post‐translational translocation pathway.

Decrease of Sec62 level led to an increased ER calcium leakage that could be attributed to Sec61α. We observed that Sec62 binds the N‐terminus of Sec61α in a Ca2+ sensitive manner and identified a crucial putative EF‐hand in the C‐terminal part of Sec62.

These experiments demonstrated for the first time that human Sec62 is important for the early steps of translocation of a large precursor protein indicating that Sec62 participation is not restricted to small proteins as suggested in previous studies.

Additionally, Sec62 acts as a modulator, shifting the translocon from the open to the closed state after translocation is completed in order to maintain the ion impermeability of the ER membrane.Further experiments will allow us to determine the recently proposed role of Sec62 for various cancer entities.
The Effect of Maté on Enzymes Involved in the Recovery from Oxidative Stress (LB199)
Authors: Sadik Amina , Chang Theodore
Published: June 2021

In comparing the effect of Maté to that of green tea, it has been shown that Maté has higher anti‐oxidative properties. We have shown that the extract of Yerba Maté, paraguariensis, leaves does indeed have strong anti‐oxidative properties through the evaluation of its scavenging activity and its ability of assist cardiomyocytes in their recovery from induced oxidative stress using both spectroscopy, a colorimetric assay and flow cytometry. In order to elucidate the mechanism behind this action, we quantified the enzymes known to be expressed naturally by cells under oxidative stress such as glutathione peroxidase, superoxide dismutase, and catalase. These enzymes were conjugated to antibodies and stains detectable via flow cytometry. Their expression was evaluated in presence and absence of oxidative stress. The obtained results indicated that Maté does not increase the expression of the genes coding for these enzyme but instead acts as a free radical scavenger, decreasing the amount of reactive oxidative species in the cellular environment, thereby decreasing the need for enzymes usually involved in the recovery from antioxidative stress. All the enzymes quantified showed a decrease in their expression in a dose‐dependent manner.
Upon discovering the inverse relationship of the activity of these enzymes to the concentration of Maté used to treat the cells, we concluded that Maté exerts it antioxidative capacity and therefore assists cardiomyocytes in their recovery from induced oxidative stress without or with very little involvement of the enzymes known to remove oxidative species from the cellular environment. These findings show that Maté is an effective antioxidant that prevents damage to the cardiovascular system.

Grant Funding Source: Touro University Nevada
Identification of the functional binding domains of eukaryotic initiation factor eIF4G with 3’ Cap‐independent translation element of Barley Yellow Dwarf Virus (LB203)
Authors: Zhao Pei , Goss Dixie
Published: June 2021

Different from canonical translation initiation, some plant viral RNAs lack a 5’cap (an unusual 7‐methyl guanosine linked by a 5’to 5’ triphosphate). They utilize a cap independent translation element (CITE) to efficiently start translation. Barley yellow dwarf virus (BYDV) has a translation element (BTE) located in its 3’UTR mRNA. BTE binds with eukaryotic initiation factor eIF4G (one subunit of heterodimer of eIF4F) and recruits other initiation factors to assemble the ribosome preinitiation complex for synthesis of viral proteins. BYDV is one of the world‐wide economically most important viruses causing severe crop loss. How BTE mediate BYDV translation is still unclear. By measuring fluorescence anisotropy of labeled RNA, here we report 3’BTE binds with a truncated fragment of eIF4G, eIF4G601‐1196 (contained amino acid residue from 601 to 1196). The equilibrium dissociation constants (Kd) of this binding is 39.5±3.5 nM. Adding eIF4E, the cap binding protein, can increase the binding ability of eIF4G601‐1196 with 3’BTE. eIF4G601‐1196•4E complex binds with 3’BTE 5.8 folds tighter than eIF4G601‐1196 alone, with the equilibrium dissociation constant 6.78±1.49nM. Further circular dichroism spectra (CD) and thermodynamic studies will characterize the binding of the eIF4G with 3’ BTE. These results suggest eIF4E plays an indirect role in translation initiation, even for an uncapped virus.

Grant Funding Source: NSF MCB 1157632
Regulation of long noncoding RNA by RCK/p54 (LB205)
Authors: Huang Jo‐Hsi , Chen Yen‐Chun , Chu Chia‐Ying
Published: June 2021

RCK/p54, or DDX6, is a member of the conserved DEAD‐box RNA helicase family, and is encoded by the DDX6 gene in human. Early studies of Dhh1, the yeast homolog of RCK/p54, have shown that that Dhh1 is a core component of cellular decapping machinery and a general translational repressor. In human cells, it has been shown that that RCK/p54 is essential for both miRNA‐mediated and AU‐Rich‐Element‐dependent translational repression. In addition, depletion of RCK/p54 disrupts the formation of P‐bodies, the putative foci of mRNA decay and storage. These results suggest that RCK/p54 is required for translational repression and mRNA turnover. Studies in C.elegans, Drosophila, and Xenopus have also shown that DDX6 homologs function in storage of maternal messages during early development. These findings implicate that RCK/p54 participates in multiple post‐transcriptional gene modulation mechanisms.

Long noncoding RNAs (lncRNAs) has recently emerged as essential players in the control of cell fate and the regulation cellular physiology. Extensive studies have been launched to decode the functions of lncRNAs, but fewer studies described the regulation of lncRNAs to date. In this study, we tried to investigate whether lncRNAs, like mRNAs, are subjects to regulation by RCK/p54. Here, we combine RCK/p54 RIP‐seq and RCK/p54‐depletion RNA‐seq for an integrative analysis. Our result provides evidences that RCK/p54 directly interacts with lncRNAs and regulates their function.
Role of protein phosphatase 2A in modulating autophagy and mitophagy (LB220)
Authors: Rice Monica , Dagda Ruben , Strack Stefan
Published: June 2021

Protein phosphatase 2A (PP2A) is a holoenzyme composed of a scaffolding/catalytic dimer that binds to a regulatory (B) subunit which dictates subcellular targeting and substrate specificity of PP2A. The PP2A holoenzymes that modulate autophagy and mitophagy in neurons are not known to date. Bβ2 is a regulatory subunit that redirects PP2A from the cytosol to the mitochondrion to induce mitochondrial fission and apoptosis of neurons. Given that mitochondrial fission precedes mitophagy, we surmised that Bβ2 regulates mitophagy in neurons. To address this hypothesis, we used a combination of cell imaging and biochemical approaches to quantify autophagy and mitophagy in SH‐SY5Y cells transiently expressing the cytosolic regulatory subunit Bβ1 and the mitochondrial regulatory subunits Bβ2 and B56α. Western blot analyses showed that all three PP2A holoenzymes decrease the levels of the autophagosome marker microtubule‐associated protein 1 light chain 3 (LC3‐II) and prevent further increases in LC3‐II as induced by bafilomycin, a drug used to measure autophagic flux. Although all PP2A holoenzymes decrease the number of GFP tagged LC3 (GFP‐LC3) puncta per cell, confocal analyses show that Bβ2 increases the colocalization GFP‐LC3 puncta with mitochondria. Similar results were observed in COS‐7 cells. Our results suggest that PP2A decreases autophagic flux while PP2A/ Bβ2 promotes mitophagy.

Grant Funding Source: P20 GM103554
Structural basis for the replication of genomic RNA of porcine enteric sapovirus by VPg (LB243)
Authors: Min Hye Jung , Hwang Hyo‐Jeong , Kim Jeong‐Sun , Lee Chul Won
Published: June 2021

Structural basis for the replication of genomic RNA of porcine enteric sapovirus by VPg Hye Jung Min, Hyo‐Jeong Hwang, Jeong‐Sun Kim, and Chul Won Lee Department of Chemistry, Chonnam National University, Gwangju 500‐757, Korea. Viral protein genome‐linked (VPg) plays a critical role in the life cycle of vertebrate and plant positive‐sense (+) RNA viruses by acting as a protein primer for RNA synthesis. The replication of the viruses is primed by the VPg protein by attaching to the 5’‐end of the viral RNA genome. Here we show the solution structure of VPg of porcine enteric sapovirus (PSV) determined by multi‐dimensional NMR spectroscopy. The structure of VPg comprises three helices (denoted α1, α2, and α3) which were tightly packed together through hydrophobic interactions formed by several hydrophobic residues to form a three‐helix bundle structure. The orientation and length of three helices of PSV VPg are similar to those of feline calicivirus (FCV) VPg with similar core structure formed by conserved hydrophobic residues. The tyrosine residue (Tyr956) within the conserved motif for nucleotide acceptor is exposed to the solvent surface to facilitate nucleotidylation by viral RNA polymerases. These results may provide the molecular basis for the replication of positive‐RNA viruses.
Sarcolipin interaction with SERCA is distinct from Phospholamban; only Sarcolipin can promote uncoupling of the SERCA pump (LB244)
Authors: Shaikh Sana , Sahoo Sanjaya , Sopariwala Danesh , Bal Naresh , Periasamy Muthu
Published: June 2021

Sarcolipin (SLN) and Phospholamban (PLB) modulate the activity of the Sarco‐Endoplasmic Reticulum Ca2+ ATPase (SERCA) pump, in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting the uncoupling of SERCA ATP hydrolysis activity from its Ca2+ transport function. However, the mechanistic details of this uncoupling are unknown. In order to define how SLN promotes uncoupling of SERCA, we compared the interaction of SLN and SERCA1, with that of PLB. We used the cross‐linker (1, 6‐bismaleimidohexane (BMH)) to detect dynamic protein interaction during the SERCA cycle. We also performed Ca2+ uptake and ATP hydrolysis assays to study the effect of SLN on SERCA. Our studies reveal that 1) SLN affects the Vmax of SERCA Ca2+ uptake but not the pump affinity for Ca2+ 2) SLN can bind to SERCA in the presence of high [Ca2+] whereas PLB can only interact with the ATP bound, Ca2+ free E2 state and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants we additionally show that PLB and SLN can bind to the same groove but interact with different residues on SERCA. These data suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca2+ causes uncoupling of the SERCA pump and increased heat production.

Grant Funding Source: Supported by NIH and AHA
Residues near the active site of UCH enzymes that may contribute to isopeptide binding in ubiquitin‐linked substrates (LB257)
Authors: Chaney Joseph , Ronau Judith , Paul Lake , Das Chitta
Published: June 2021

Ubiquitin carboxyl terminal hydrolase (UCH) enzymes release small peptide and protein leaving groups from the C‐terminus of ubiquitin. Although the interaction with ubiquitin is fairly well understood in these enzymes, recognition of the isopeptide moiety of the substrate at the active site has not been well characterized. The crystal structure of TsUCH37 (T. spiralis) bound to suicide substrate, UbVMe, reveals a conserved tryptophan residue that may play a role in stabilizing the isopeptide linkage in the active site. To study the contribution of this tryptophan residue, the equivalent residue in human UCH37, W55, was mutated to alanine and phenylananine. The phenylalanine mutant retained most of its ubiquitin‐AMC (Ub‐AMC) hydrolysis activity as compared to the wild type enzyme, but the alanine mutant was substantially impaired. The loss of activity with the alanine mutant is not due to any alteration in the three‐dimensional fold of the enzyme. Considering the distance of this tryptophan residue from G76 of ubiquitin (~5 Å), this observation suggests that W55 may be involved in binding to the AMC portion of the Ub‐AMC substrate, which may indicate that it makes contact with the isopeptide moiety in ubiquitin‐linked substrates. This is being further investigated in other members of the UCH family, such as BAP‐1 and UCHL3.

Grant Funding Source: Supported by National Institute of Health
Evaluation of PCR‐ELISA and Kato‐Katz techniques for schistosomiasis mansoni cure assessment (LB279)
Authors: Siqueira Liliane , Gomes Luciana , Oliveira Eduardo , Enk Martin , Rabello Ana , Coelho Paulo Marcos
Published: June 2021

Introduction: The importance of following up therapeutic intervention among individuals diagnosed with schistosomiasis is based on the fact that the expected cure rate for Praziquantel varies around 90% and that non‐cured patients continue to maintain transmission in endemic settings. Methodology: The aim of the study was to evaluate parasitological and molecular biological techniques for the assessment of cure after chemotherapy. The study was carried out in a low transmission area, Pedra Preta, Montes Claros, Minas Gerais, Brazil. To establish the infection rate, a combination of parasitological exams was performed. Four stool samples from 201 inhabitants were analyzed according to the Kato‐Katz method (18 slides) and a commercial test, the TF‐Test®. All patients with positive parasitological exams were treated with a single dose of Praziquantel, 50 mg/kg for adults and 60 mg/kg for children. To establish the cure rate out of the 72 positive patients, 69 were followed up after 30 days, 67 after 90 days and 61 after 180 days of treatment. For the direct comparison between the Kato‐Katz method and the PCR‐ELISA technique, 500mg of the same stool sample of each participant was analyzed, resulting in 12 Kato‐Katz slides per sample. The data analysis involved the determination of the cure rate defined by both diagnostic methods. Results: The cure rate 30 days after treatment by the Kato‐Katz technique was 100% and one positive individual was detected by the PCR‐ELISA (cure rate of 98.5%). Three positives individuals were detected by the PCR‐ELISA 90 days after treatment (cure rate of 95.5%), not identified by analyzing 12 Kato‐Katz slides. The cure rate after 180 days after treatment was 98.4% by Kato‐Katz (1 out of 61) and 96.5% by PCR‐ELISA (2 out of 61), with the final cure rate of 95.1% (3 out of 61). It is worth to note here that 30, 90 and 180 days after treatment the cure rate determined by 2 Kato‐Katz slides was 100%. Conclusions: In situations of very low parasite load, such as after therapeutic interventions; the assessment of cure may require increased number of Kato‐Katz slides or a test with higher sensitivity as PCR‐ELISA.
Identification of sharpin as a molecule regulating leukocyte transmigration from siRNA screens (LB281)
Authors: Auvinen Kaisa , Pouwels Jeroen , Jalkanen Sirpa , Ivaska Johanna , Salmi Marko
Published: June 2021

Migration of leukocytes into tissues is a hallmark of an acute inflammation. The migration is regulated by molecular interactions between adhesion molecules on endothelium and their counterparts on leukocytes. Here we have used siRNA‐based screens to search for new molecules regulating leukocyte transmigration through endothelium. The functional significance of the hits was confirmed using time‐lapse microscopy in in vitro flow chamber assays, in which human leukocytes were perfused with a defined shear stress over a confluent monolayer of cultured endothelial cells. In peripheral blood mononuclear cells a cytosolic protein, SHARPIN, was localized to uropods of migrating leukocytes. Moreover, using siRNA silencing we found that after the transmigration, the leukocytes without SHARPIN trapped close to transmigration site while control leukocytes moved onwards. In endothelial cells the use of siRNA to silence one ephrin type‐B receptor, reduced granulocyte transmigration through endothelium while overexpressing of it increased transmigration. As a conclusion we have found new molecules in leukocytes and in endothelium that regulate leukocyte transmigration.
Medical Illustration Students in the Gross Anatomy Lab (LB30)
Authors: Young J'Nelle , Tobolic Tess , Ward Rachel , Althaver Nathan , Tubbs Ryan
Published: June 2021

Michigan State University’s College of Human Medicine utilizes a prosection‐based approach to preclinical anatomy curriculum. Carefully prosected cadavers have proven to be excellent learning tools in the gross anatomy laboratory but an immense amount of work goes into producing the specimens. To aid in maintaining a robust specimen inventory, MSU offers an undergraduate prosection course. This self‐directed course includes descriptive protocols designed to guide students through challenging prosection projects. To supplement the protocols, students use conventional dissector texts and atlases to find suitable images and directions for the various steps of each project. In an effort to streamline the protocols and offer prosection students one high yield resource for their prosections, medical illustration students from nearby Kendall School of Art and Design are creating customized anatomical images to compliment current protocols. Recently, CHM has partnered with Kendall School of Art and Design to create and pioneer a medical illustration program. With the proper prerequisites, Kendall students are eligible to take MSU’s undergraduate prosection course. Kendall students who have completed the prosection course serve as laboratory assistants (LAs). These students are ideal for this project as they had previously completed the course and experienced the challenges associated with the written protocols. The LAs attempt to identify the most challenging steps in a protocol and enrich prosection protocols with step‐by‐step illustrations. This project introduces a novel approach to dissection instruction and explores the role of medical illustration students in the anatomy lab.
Effects of two popular weight loss programs on changes in android and gynoid body composition in women (LB302)
Authors: Simbo S , Coletta A , Mardock M , Lockard B , Bryd B , Jagim A , Kresta JY , Baetge C , Jung YP , Koozehchian M , Khanna D , Kyul H , Oliver JM , Greenwood M , Rasmussen C , Kreider R
Published: June 2021

The purpose of this study was to determine if different diet and exercise programs differentially affect android and/or gynoid body composition changes in sedentary women. 54 sedentary women (34±8 yrs, 35±6 kg/m2) were randomized to participate in the Curves (C) or Weight Watchers (W) weight loss programs for 16‐wks. Participants in the C program followed a 1,200 kcal/d diet for 1‐wk and 1,500 kcal/d diet for 3 wks (30:45 CHO:PRO). Subjects then ingested 2,000 kcals/d for 2‐wks (45:30 CHO:PRO) and repeated this diet while participating in the Curves with Zumba program 3‐d‐wk. Remaining subjects followed the W point‐based diet program, received weekly counseling, and were encouraged to exercise. DEXA body composition with android (A) and gynoid (G) measurements were analyzed at 0 and 16 wks. MANOVA revealed a significant time (p<0.001) and group x time (p=0.004) effect. Both groups had similar changes in A‐FM (W ‐419±760, C ‐624±700 g, p=0.31), A‐BF (W ‐2.1±10, C ‐4.9±6 %, p=0.24), and G‐FFM (W ‐639±1,853, C ‐9±1,247 g p=0.16). Differences were seen in A‐FFM (W ‐313±614, C 49±403 g, p=0.02), G‐FM (W ‐395±1,657, C ‐1,398±1,728 g, p=0.03), G‐BF (W ‐0.6±8, C ‐4.2±6 %, p=0.06), and the lean index to height (W ‐2.4±4.6, C 0.5±1.5 kg/m2, p=0.005). Results indicate that different types of diets can differentially affect changes in A and G body composition

Grant Funding Source: Supported by Curves International (Waco, TX)
Dietary changes during a successful weight loss trial in postpartum women (LB314)
Authors: Huseinovic Ena , Winkvist Anna , Bertz Fredrik , Brekke Hilde
Published: June 2021

Dietary strategies that facilitate weight loss following pregnancy are poorly examined. Our aim was to examine dietary changes during a weight loss intervention in 61 overweight and obese postpartum Swedish women randomized to a 12‐wk dietary (D), exercise (E), combined (DE) or control (C) treatment at 10‐14 wk postpartum. A 4‐d weighed diet record was conducted at baseline, post intervention (12‐wk) and 9 mo later (1‐y). Differences in dietary intake between women receiving Dietary treatment (D+DE, D‐groups) and No Dietary treatment (E+C, ND‐groups) were examined using linear regression. The D‐groups achieved a 1‐y weight loss of 10%. At baseline, 21% had an intake of 500g fruit and vegetables/d while sweets and salty snacks contributed to 20% of total energy intake (E%). At 12‐wk, D‐groups had reduced their intake of sweets and salty snacks, caloric drinks and increased their intake of vegetables more than did ND‐groups (all p<0.01). Further, 45% of the D‐groups and 17% of the ND‐groups demonstrated an intake of 500g fruit and vegetables/d (p<0.05) while sweets and salty snacks contributed to 8 vs 21 E% (p<0.01). At 1‐y, D‐groups had maintained an increased intake of vegetables compared to ND‐groups (p<0.05). Reduced intake of sweets and salty snacks and increased intake of vegetables were distinct dietary changes among women receiving dietary treatment and achieving sustainable weight loss following pregnancy.

Grant Funding Source: Supported by the Swedish Research Council and the Swedish Research Council for Health, Working Life and Welfare.
Serum albumin varies by tumor stage in cutaneous malignant melanoma (LB347)
Authors: Datta Mridul , Schwartz Gary
Published: June 2021

High albumin adjusted serum calcium has been reported in several cancers but has not been studied in malignant melanoma. We examined the relationship between serum albumin, total and corrected calcium in malignant melanoma in a cross‐sectional study. Two thousand three hundred six patients with cutaneous melanoma were identified between Jan. 2000 and Dec. 2010 and 644 met our inclusion criteria. At the time of diagnosis, total serum calcium was non‐significantly lower in stage 4 (9.34 mg/dL) vs. stages 1‐3 (9.48‐9.51 mg/dL). Serum albumin was significantly lower in stage 4 (3.92 g/dL; p蠄0.05) than stages 0‐3 (4.09‐4.15 g/dL). Higher albumin corrected serum calcium was observed with increasing melanoma stage (9.45‐9.61 mg/dL). Men but not women showed a significant decrease in serum albumin in stage 4 when compared with stages 1‐3 (p = 0.02). Patients >75 years had significantly lower serum albumin (3.98±0.33 vs 4.16±0.35 g/dL; p<0.001), but higher adjusted calcium (9.64±0.49 vs 9.53±0.43 mg/dL; p=0.03) compared to patients 蠄 75 years. Serum albumin may serve as a biomarker to evaluate disease progression in cutaneous malignant melanoma patients.

Grant Funding Source: Dr. Datta is supported by the Comprehensive Cancer Center of Wake Forest University Cancer Control Traineeship ‐ NCI/NIH Grant# #R25CA122061.
Inhibitory effects in 3T3‐L1 differentiation of anticancer kimchi (LB351)
Authors: Kang Soon Ah , Kim Mi‐Ja , Kim Tae‐Soon , Jang Ki‐Hyo , Cho Eun Ju , Park Kun‐Young
Published: June 2021

Kimchi is famous health food in the world. It has a high concentration and a wider range of functional and bioactive substances.

Many researchers reported that Kimchi has many health benefits such as antioxidant, anti‐cancer, antimicrobial, and anti‐diabetic effects. This study was evaluated using in vitro system anti‐obesity effects of anticancer Kimchi extract were tested in vitro using 3T3‐L1 cells. Oil red o staining of 3T3‐L1 preadipocyte cells treated with 25, 50, and 100 μg/mL anticancer Kimchi extract decreased significantly by 41.4, 61.4, and 71.7%, respectively (p<0.05) compared to those in standard Kimchi treated cells. Furthermore, anticancer Kimchi extract affected the down‐regulation expression patterns of genes such as peroxisome proliferator‐activated receptor γ(PPARγ), adipose‐specific fatty acid binding protein(aP2), glycerol‐3‐phosphate dehydrogenase 1(GPD1), and fatty acid synthase(FAS), significantly (p<0.05). Therefore, anticancer Kimchi may be considered for use in preventing fat accumulation.

Grant Funding Source: Ministry of Food, Agriculture Forestry
Decrement of triglyceride accumulation of Coriander (LB352)
Authors: Kang Soon Ah , Kim Mi‐Ja , Kim Jong‐Ae , Kim Hyun‐Sook , Jung Kwang_Gyun , Kim Seong‐Hee , Kim Jin‐kyung
Published: June 2021

Cells were fixed and stained with Oil Red O to assess lipid accumulation. Total lipid and triglyceride contents increased during the cell differentiation. Coriander (Coriandrum sativum L.) is widely cultivated in Russia, Central Europe, North Africa and Asia. It is valued for its medicinal uses. In this study, the antioxidant activities of Coriander were evaluated using in vitro system including DPPH, ABTS, and ORAC methods. Total phenolic contents of Coriander extract were 3.6 μmole quercetin equivalent/g raw sample and EC50 of Coriander extract for scavenging DPPH and ABTS radicals were 5.7 and 8.0 μmole ascorbic acid equivalent/g raw sample, respectively. Triacylglycerol content in 3T3‐L1 preadipocyte cells treated with 25, 50, and 100 μg/mL Coriander extract decreased significantly by 37.0, 55.7, and 69.8%, respectively (p<0.05) compared to those in untreated cells. Also, Coriander extract induced the down‐regulation of adipogenesis‐related genes such as PPARγ, GDP1 and FAS. The Coriander extract showed phenolic compounds with antioxidant ability and had in vitro decrement of triglyceride accumulation on 3T3‐L1 adipocyte cells.

Grant Funding Source: Korean government (MEST)
Effects of lycopene supplementation on oxidative stress markers in adipose tissue of obese rats (LB389)
Authors: Luvizotto Renata , Nascimento André , Wang Xiang‐Dong , Yeum Kyung‐Jin , Ferreira Ana Lúcia
Published: June 2021

Background: Obesity has been associated with oxidative stress, and NADPH oxidase system is known as main source of reactive species. Lycopene is a potent antioxidant. However, it is not known the influence of lycopene on NADPH‐oxidase activity in adipose tissue of obese rats. Objective: To analyze the effects of lycopene supplementation on oxidative stress markers, including NADPH‐oxidase activity, in adipose tissue of diet‐induced obese rats. Methods: Male Wistar rats were first fed with hypercaloric diet (HD, n=14) for 6 weeks and afterwards, these rats were randomly assigned to receive HD (n=7) or HD with lycopene (10 mg/Kg BW/d; HD+L, n=7) by gavage for additional 6 weeks. Adipose tissue was collected for evaluation of NADPH‐oxidase activity and other oxidative stress markers, by HPLC system and immunoassay, respectively. Results: In obesity, lycopene treatment for six weeks decreased protein carbonylation (HD: 5.24 ± 0.77 vs HD+L: 3.78 ± 0.50) in visceral adipose tissue, and 4‐HNE in visceral (HD: 0.43 ± 0.04 vs HD+L: 0.36 ± 0.02) and retroperitoneal (HD: 0.40 ± 0.07 vs HD+L: 0,31 ± 0,02) adipose tissue. Absence of difference in NADPH oxidase activity was found between HD and HD+L groups. Conclusion: Lycopene supplementation decreased lipid peroxidation and protein carbonylation and did not interfere in NADPD‐oxidase activity in adipose tissue. These data suggest that lycopene may be an effective strategy in reducing some oxidative stress markers in obesity.

Grant Funding Source: Supported by FAPESP #10/06100‐9 and #10/19746‐4
Research on Preparation Process of Collagen Polypeptide from Pig Skin by Two‐Step‐Hydrolysis (LB397)
Authors: Zhang Zhisheng , Li Guijie , Shu Ying , Ren Hui , Liang Chunhui , Jiao Xueqin , Zhao Renbang , Tang Xia , Zhao Congzhi , Chen Feng
Published: June 2021

In this study, neutral proteinase, alkaline proteinase, and papain were used to hydrolyze fresh pig skin respectively. The effects of pH value ,temperature, enzyme content and substrate content on hydrolysis were studied by the single factor test, and orthogonal design, by assessing the parameter ‐ degree of hydrolysis (DH), was adopted to determine the optimum condition of hydrolysis. The optimal hydrolysis condition of neutral proteinase was: pH 6.5, 45°C, enzymatic content 0.5%, and substrate content 15%; while The optimal hydrolysis condition of alkaline proteinase was: pH 11, 55°C, enzymatic content 0.5%, and substrate content 35%; and the most suitable hydrolysis parameters of papain were: pH 5, temperature of 60°C, 0.9% of enzyme content and 30% of substrate content. The ability of hydrolysis was sequenced as: alkaline proteinase > papain > neutral proteinase. On the basis of the optimum condition of each single proteinase, any two of the proteinases were chosen to hydrolyze fresh pig skin by two‐step‐hydrolysis. The results showed that any combination of the enzymes has a higher DH value than its single component and the adding sequence inflects DH value. Overall, the two‐step‐hydrolysis of papain plus alkaline shows the highest DH value (22.31%).

Grant Funding Source: Supported by Science and Technology Department of Hebei Province, project no. 14227114D
Development of a prognostic breast cancer diagnostic: Quantitative analysis of HER2 status using a multiplex bead assay on homogenized formalin fixed paraffin embedded breast tumor tissue (LB484)
Authors: Lerch Melissa , Strathmann Frederick , Baird Geoffrey
Published: June 2021

Immunohistochemistry is typically used to classify breast cancer into molecular subtypes based on the presence or absence of three protein biomarkers: estrogen receptor (ER), progesterone receptor (PR), and receptor tyrosine‐protein kinase erbB‐2 (HER2). Tumor cells that overexpress HER2 specifically respond to the drug trastuzumab (Herceptin). HER2 can be measured semi‐quantitatively by IHC, but in up to 20% of cases, IHC results are equivocal, meaning that additional testing (fluorescence in situ hybridization [FISH]) is required to distinguish clinically meaningful HER2 overexpression from normal HER2 levels. Aiming to ameliorate this situation, we developed a diagnostic assay that allows direct quantitative determination of HER2 protein concentration from formalin fixed paraffin embedded (FFPE) tumor tissue with a multiplex bead assay. We hypothesized that substantial preanalytical and analytical factors inherent in IHC methodology were contributing to the high rate of equivocal results, and that more accurate and precise quantitative analysis of HER2 protein concentration would serve as a gold standard method for HER2 assessment, obviating the need for reflexive FISH. In this method, tumor tissue from three 10‐micron histologic sections is first subjected to lysis and protein extraction using a buffer similar to that used in IHC for epitope retrieval. Numerous HER2 antibody clones were evaluated to identify a matched pair capable of detecting HER2 in these FFPE tissue lysates, using a sandwich immunoassay geometry. The final multiplex bead‐based assay was validated on tissue culture controls and clinical samples.
Exosomes Pinched from Mesenchymal Stem Cells Overexpressing GATA‐4 Serve as Reservoir for Anti‐apoptotic miRs for Cardioprotection (LB524)
Authors: Yu Bin , Kim Ha Won , Wang Yi‐Gang , Ashraf Muhammad , Xu Meifeng
Published: June 2021

There is an abundant evidence that exosomes play an important role in intracellular signaling and exert regulatory function through carrying bioactive molecules. This study is aimed to determine whether cardioprotection by mesenchymal stem cells (MSCs) overexpressing GATA‐4 (MSCGATA‐4) is initiated by exosomes which deliver anti‐apoptotic miRs to regulate the target proteins in recipient cells. Exosomes were isolated and purified from MSCGATA‐4 and the control MSCs (MSCNull). The cell injury was investigated in primary cultured rat neonatal cardiomyocytes (CM) and in rat heart. CM were incubated with exosomes from MSCGATA‐4 (ExoGATA‐4) and MSCNull (ExoNull) under hypoxic environment for 48 hrs. CM survival was significantly increased; the cell apoptosis and LDH release were reduced in CM treated with ExoGATA‐4 compared to ExoNull. ExoGATA‐4 maintained the mitochondrial membrane potential (ΔΨm) CM. Real‐time PCR indicated that the anti‐apoptotic miRs (e.g. miR‐19a and miR‐451) were highly expressed in MSCGATA‐4 and in ExoGATA‐4. Si‐miR‐19a transfection significantly abolished ExoGATA‐4 mediated cardioprotection. Furthermore, miR‐19 was upregulated and BIM and PTEN were decreased in ExoGATA‐4 treated CM. A rapid internalization of ExoGATA‐4 by CM was observed via time‐lapse images. Direct transplantation of ExoGATA‐4 into the boarder of ischemic myocardium following ligation of left anterior descending coronary artery (LAD) significantly improved cardiac contractile function and reduced infarct size. ExoGATA‐4 were detected inside damaged cells where the expression of miR‐19a was the highest. It is concluded that ExoGATA‐4 mediated cardioprotection through anti‐apoptotic miRs which reduced apoptosis in the ischemic cells.

Grant Funding Source: Supported by National Institutes of Health grants HL105176 and HL114654 (M. Xu).
In Vitro knock out of Inducible Cyclicamp Early Repressor(ICER) in zebrafish cell lines through the use of the CrispR/Cas9 system (LB530)
Authors: Hasani Aliaskar , Molina Carlos
Published: June 2021

ICER is a dominant negative transcriptional regulator induced by cAMP and it binds to CREB or CREM in order to repress cAMP‐mediated gene expression. Inhibin alpha, Inha, has been shown to regulate gonadal stromal cell proliferation and suppress tumor activity. The multi‐functions of ICER: as a neuronal plasticity, tumor suppressor, regulator of apoptosis, and etc., have led to the characterization of the relevant gene in various organisms like Homo‐sapiens (humans), Mus, musculus (mouse) and Gallus gallus (chicken) (NCBI). Recently the ICER sequence had been continuously expressed in Danio rerio (zebra fish) through use of the vectors pFlag and TOPO. ICER, which is regulated by the signal path of cAMP, represses the expression of CREM gene is its isoforms. This fact proves to be a favorable influence in the regulation of cancer. Using zebrafish as models, we utilized the Clustered Regular Interspersed Short Palindromic Repeats (CRISPR) system with the Cas9 in order to knock out the ICER promoter gene. These were done in two cell lines, embryo fibroblast and tailfin. Our assay results showed the disappearance of the ICER gene, and we will do further experimentation on our cell lines.