bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of differentially expressed proteins in Escherichia coli when exposed to L‐Telluromethionine
Authors: Broderick Kathleen Marie , Taylor Savannah , Boles Jeffrey O.
Published: June 2021

Analysis of the three‐dimensional structure of a protein using x‐ray crystallography can be improved by the incorporation of unnatural amino acids. Most naturally occurring atoms do not have sufficient mass to diffract electrons, thus, heavy atom derivatives of target proteins are required. An unnatural amino acid, L‐Telluromethionine (TeMet), which provides the necessary scattering power, has cytotoxic effects which reduce cell growth and uptake. A proteomic analysis of telluromethionyl Escherichia coli was carried out using the methionine auxotroph E. coli DL41(DE3) (pCock) expression system. Both telluromethionyl and methionyl exposed cultures were analyzed with the objective of identifying differentially expressed proteins. Analysis was carried out using 2D separation with cation/anion exchange chromatography serving in the first dimension and SDS polyacrylamide gel electrophoresis in the second. Tryptic digestion and peptide fragment analysis using nano‐ESI/qTOF/MS followed to allow peptide mass fingerprinting of differentially expressed proteins. Cell cultures were grown under typical conditions of recombinant protein expression with dihydrofolate reductase serving as the test vehicle. The biological roles of differentially expressed proteins is being used to improve methods of the TeMet bioincorporation.
Automated Generation of Motif Templates in Protein Function Prediction Software ProMOL
Authors: Osipovitch Mikhail , Bernstein Herbert J. , Craig Paul A.
Published: June 2021

ProMOL, an extension for molecular visualization system PyMOL, is structure‐based protein function prediction software. ProMOL implements routines for building motif templates based on active sites of proteins of known structure and function and screening unassigned structures for presence of the motif templates. Presently, motif generation in ProMOL requires user intervention in optimization of parameters for sensitivity and selectivity. An algorithm was developed to automate motif building and testing routines of ProMOL. The algorithm uses a set of empirically derived parameters for optimization, requires little user intervention, and provides a possibility for extending the ProMOL library of motif templates at a much higher rate. Presently, the library contains 181 motif templates. As a result of automating motif building and testing routines, 388 motif templates were generated and tested against their native, known homologous, and non‐homologous structures. The automatically generated motif templates exhibited comparable performance to the existing manually generated motif templates in terms of the rates of true positive and true negative results. The research is supported by 2R15GM078077–02.
Stearoyl CoA desaturase 1 (SCD1) and Seipin: Important players in hepatic steatosis?
Authors: Amine Lounis , Lalonde Simon , Rial Sabri , Prieur Xavier , Costet Phillipe , Magré Jocelyne , Mounier Catherine
Published: June 2021

Background

SCD1 is a lipogenic enzyme responsible for the formation of monounsaturated fatty acids, the main precursors of triglycerides. High SCD1 expression is associated with expansion of adipose tissue and ectopic fat accumulation (in various tissues). Seipin is the human Berardinelli‐Seip congenital lipodystrophy 2 gene product which regulates adipogenesis and lipid droplets (LD) formation but whose function remains to be elucidated.

Aim

In the present study we evaluated the role of SCD1 and seipin in LD formation in hepatic cells and the effect of SCD1 or Seipine deficiency in the lipogenesis and insulin pathways. The results were also validated in seipin‐deficient mice.

Methods

In human hepato‐carcinoma cell line (HepG2), the expression of the two proteins was either silenced. LD formation was monitored by confocal microscopy upon addition of oleate. Under these conditions was also evaluated the activation of insulin and lipogenesis signaling pathways.

Results

We observed that silencing SCD1 in HepG2 cells decreases the number and the size of LD, does not affect the lipogenic gene expression, increases insulin sensitivity (pAKT and pERK1). While silencing seipin has an opposite effect in the number of LDs. The mice develop a major hepatic steatosis. But the seipin deficiency does not affect lipogenesis but increases the expression of SCD1 and CD36 (in both HepG2 cells and liver of KO mice). Surprisingly, it increases significantly the insulin sensitivity in HepG2 cells

Conclusion

Our study demonstrated for the first time that low seipin expression in hepatic cells is directly associated with the development of hepatic steatosis. Seipin function appears to be inversely associated with SCD1 function. Finally, our work also suggests that variation of SCD1 and seipin expression influence insulin responses in hepatic cells.
Inflammatory Role of Cysteinyl Leukotrienes in Human Vascular Endothelial Cells
Authors: Duah Ernest , Kondeti Vinay , Adapala Ravi K , Sanford Joel , Thodeti Charles K , Paruchuri Sailaja
Published: June 2021

Cysteinyl leukotrienes (cys‐LTs) include leukotriene (LT) C4, LTD4, LTE4 are potent inflammatory lipid mediators that have been recently implicated in atherosclerosis. However, the molecular mechanisms by which cys‐LTs mediate atherosclerosis are not known. In the present study, we investigated the role of cys‐LTs in the regulation of endothelial functions and its contribution to the progression of atherosclerosis. We found that human endothelial cells (HUVECs) express cys‐LT receptors, CysLT1R and CysLT2R, with the expression of CysLT2R higher than CysLT1R. Interestingly, we found that activation of both CysLTRs induced calcium influx, ERK phosphorylation and cell proliferation in endothelial cells as measured by calcium imaging, western blotting and XTT assay, respectively. Importantly, LTD4 induced robust endothelial cell contraction and barrier dysfunction in endothelial monolayer. Finally, treatment of HUVECs with LTD4 induced the expression of inflammatory cell adhesion receptors such as ICAM1, VCAM and P‐selectin on endothelial monolayer with concomitant increase in HL‐60 cell attachment which is the hallmark of atherosclerosis. Taken together, our results suggest that cys‐LTs‐induce atherosclerosis by activating vascular endothelial cell inflammatory phenotype.
Myristate‐derived d16:0‐sphingolipids constitute an abundant cardiac sphingolipid pool with distinct synthetic routes and functional properties
Authors: Russo Sarah Brice , Tidhar Rotem , Futerman Anthony , Cowart L. Ashley
Published: June 2021

Previously, our laboratory demonstrated that sphingolipids (SL) mediated diet‐induced diabetic cardiomyopathy in mice (Russo et al. 2012. J Clin Invest). Intriguingly, affected hearts displayed accumulation of a novel pool of myristate‐derived d16:0 SL. However, the metabolism and biological functions of these lipids have yet to be defined. The present study sought to identify the metabolic routes through which these lipids were produced in the heart and to determine whether they were functionally distinct from canonical d18:0 SL. Data revealed that d16:0 SL were produced de novo within cardiomyocytes, comprised a major proportion of the cardiac simple SL pool, and increased in response to excess saturated fatty acids. Additionally, d16:0 and d18:0 SL were shown to be functionally distinct. In isolated adult cardiomyocytes, markers of apoptosis accumulated in response to increased d16:0 SL, but not d18:0 SL. In contrast, accumulation of d18:0 SL, but not d16:0 SL, promoted autophagy in these cells. Thus, these results reveal a previously unappreciated component of the cardiac sphingolipidome with functional differences from canonical sphingolipids.

This work was supported by NIDDK F30DK092125 (to S.B.R.), a Merit Award from the Department of Veterans Affairs and the NIH COBRE in Lipidomics and Pathobiology (both to L.A.C.).
Rescue of Sphingosine Kinase‐1‐knockout Phenotype by 2‐Acetyl‐5‐tetrahydroxybutyl Imidazole (THI) Following Murine Cardiac Arrest is Linked to Changes in Sphingosine‐1‐phosphate and Behenoylceramide Levels
Authors: Berdyshev Evgeny V. , Gorshkova Irina A. , Natarajan Viswanathan , Wang Huashan , Beiser David G. , Vanden Hoek Terry L.
Published: June 2021

Sphigosine‐1‐phosphate (S1P) signaling system is well documented to be critical for the resistance to different stress conditions. Both sphingosine kinase 1 (SphK1) and S1P lyase (S1PL) are crucial for maintaining S1P levels and regulating entire sphingolipid metabolism. Using our model of cardiac arrest and return of spontaneous circulation we tested the association of rescue of SphK1‐knockout poor survival after cardiac arrest by THI, an inhibitor of S1PL, with changes in S1P and ceramide levels. The LC/MS/MS analysis of circulating and cardiac S1P and ceramide content revealed a decrease in circulating and cardiac S1P levels in SphK1‐KO mice but also a unique association between the lack of SphK1 and changes in tissue and plasma N‐behenoylceramide (C22:0) content. The inhibition of S1PL in SphK1‐KO mice completely reversed poor animal survival after cardiac arrest, which was accompanied by a full reversal of dysregulated S1P and ceramide phenotype caused by the lack of SphK1. The SphK1‐KO phenotype and its reversal by S1PL inhibition were also characterized by dynamic changes in the expression of S1P receptors 1 and 2. Our data demonstrate the link between the lack of SphK1 and particular dysregulation in S1P and ceramide homeostasis which becomes critical for animal survival after cardiac arrest and can be reversed through S1PL inhibition.
The Dynamic Roles of Phospholipid and Triacylglycerol Metabolism in Regulation and Signaling in Yeast
Authors: Henry Susan Armstrong
Published: June 2021

For several decades, my laboratory has investigated the regulation of lipid metabolism, focusing on how cells sense metabolic activity in the pathways for the synthesis of phospholipids and triacylglycerol (TAG). We isolated mutants with defects in the expression of structural genes of phospholipid biosynthesis, including INO1, encoding inositol 3‐phosphate synthase, the ratelimiting enzyme in the synthesis of the phospholipid precursor, inositol. This led to the discovery of transcription factors and a promoter element responsible for transcriptional regulation in response to inositol. A major breakthrough in the field was the discovery that phosphatidic acid (PA), precursor to both phospholipids and TAG, serves as the signal for derepression of phospholipid biosynthetic genes in the absence of inositol. PA metabolism is influenced by the rates of synthesis and turnover of essentially all lipids that are derived directly or indirectly from it. Because of the interconnectivity of the pathways involving PA, removal or addition of inositol, triggers rapid changes in the levels of a wide spectrum of lipids, including many signaling lipids. We are currently exploiting these attributes of the cellular response to inositol to elucidate the roles of specific lipids, both in regulating lipid metabolism itself, and in activating stress response signaling across multiple membrane compartments.

Supported by NIH grant GM‐19629
Characterizing the stabilizing effect of Coq8p and the function of Coq9p in yeast Q biosynthesis
Authors: HE CUIWEN , Xie Letian , Tsui Hui Su , Allan Christopher M , Clarke Catherine F
Published: June 2021

Coenzyme Q is a mitochondrial lipid that is essential in cellular energy metabolism. Biosynthesis of Q in yeast requires nine proteins, Coq1p‐Coq9p, and deletion of any one of the yeast COQ genes leads to the decreased steady state of several other Coq polypeptides. However, when the Coq8 polypeptide is over‐expressed in some of the yeast coq null mutants, steady state levels of Coq polypeptides were restored to near wild‐type levels. We hypothesize that over‐expression of Coq8p may stabilize the Coq polypeptides by stabilizing the catalytic complex composed of Coq3p‐Coq7p and Coq9p. Using non‐denaturing 2‐dimensional blue native gels, we found that the Coq polypeptide complex dissociated upon the deletion of one of the COQ genes, but that over‐expression of COQ8 rescued the high molecular mass Coq polypeptide complex.

The function of Coq9p is still unclear. In the coq9 null yeast mutant overexpressing Coq8 (Coq8 OE), IDMQ6 accumulated when fed para‐aminobenzoic acid (pABA). We hypothesize that Coq9 is required for the deimination step from IDMQ6 to DMQ6. We generated a yeast strain with the COQ9 endogenous promoter replaced by a doxycycline‐ regulated promoter. We found that DMQ6 accumulated earlier and faster than IDMQ6 when there is a very small amount of Coq9p present. The results suggest that the deimination of IDMQ6 is faster than the turnover of DMQ6.
DHCR24: Unexpected new directions for a terminal step in cholesterol synthesis
Authors: Zerenturk Eser , Sharpe Laura J , Kristiana Ika , Brown Andrew J
Published: June 2021

Cholesterol is necessary for mammalian life, as an essential component in cell membranes, foetal development, and a precursor for steroid hormones. Hence, cholesterol levels must be tightly regulated. Previous research has focused on an early step in cholesterol synthesis: HMG‐CoA reductase, target of the cholesterol‐lowering statin drugs. However, less is known about other steps in the pathway. We investigated 3β‐hydroxysterol Δ24‐reductase (DHCR24), involved in the last step of cholesterol synthesis, and implicated in inflammation, oxidative stress and HCV infection. We show that DHCR24 is regulated at multiple levels, with potent effects on cholesterol synthesis. We found regulation through dual sterol regulatory elements (SREs) within the DHCR24 promoter, which work cooperatively to regulate expression, and show that endogenous side‐chain oxysterols, in particular 24(S),25‐epoxycholesterol (24,25EC), directly inhibit DHCR24 activity. These findings indicate DHCR24 may be an important regulatory step in cholesterol synthesis and maintaining cellular cholesterol homeostasis. This research is supported by the National Health and Medical Research Council and National Heart Foundation of Australia.
Conformational consequences of O‐GalNAc and O‐Man protein modifications: implications for the structural role of O‐GalNAc in the mucin‐like region of alpha‐dystroglycan
Authors: Live David , Foley Lachele , Borgert Andrew
Published: June 2021

The ramifications of protein glycosylation extend beyond the display of particular carbohydrate epitopes, to potentially inducing changes in the protein conformation. Both properties can impact physiological effects. In general only one form of glycosylation is found in a region of a glycoprotein, however a major exception is alpha dystroglycan (aDG). Its mucin‐like region carries glycans initiated by both O‐GalNAc and O‐Man. It is known that OGalNAc based modifications induce extended structures, which may be relevant to the shape of aDG and its role linking the cytoskeleton to the extracellular matrix. The conformational effects of O‐Man are not so clear, although these glycans are important for functional physiological properties, with aberrations in their structures related to forms of muscular dystrophy. To elucidate comparative conformational aspects of the two kinds of modifications, NMR studies have been carried out on a peptide segment from aDG, itself, and also modified with O‐Man and OGalNAc, prepared synthetically. These results are being complemented by extensive molecular dynamics simulations to further understand the consequences of the modifications on the protein flexibility. The O‐Man modification induces much less conformational restriction than that of O‐GalNAc. The results offer new insights into conformational effects of O‐glycosylation.
Study of Ras catalytic mechanism of intrinsic hydrolysis of GTP
Authors: Knihtila Ryan , Holzapfel Genevieve , Mattos Carla
Published: June 2021

Ras cycles between an active GTP bound form and an inactive GDP bound form. While Ras hydrolysis of GTP to GDP is intrinsically slow, we have discovered a GAP‐independent allosteric switch mechanism that results in ordering of switch II, placing the catalytic Q61 in the active site. This is expected to enhance the rate of hydrolysis through a mechanism where GTP acts as the general base, abstracting a proton from a nucleophilic water molecule, and shuttling it to a bridging water molecule that donates H‐bonds to Y32 in switch I and Q61 in switch II. This would promote a partial positive charge on the bridging water molecule, analogous to the arginine finger of GAP. In order to validate this mechanism we are solving the neutron crystal structure of H‐Ras to reveal the positions of hydrogen atoms in the active site. We have shown that perdeuteration of Ras results in an unchanged active site and have optimized crystal growth for neutron crystallography. We are also doing a series of hydrolysis experiments on wild type and mutant H‐Ras in the presence of NORE1A, an effector that stabilizes the active site in the same conformation seen with an activated allosteric switch in the presence of Raf‐RBD. A rate decrease in RasY32F will establish this residue as important in catalysis, while the RasQ61L mutant is expected to decrease the rate of reaction in the presence of NOR1EA as it does in the absence of effector proteins.
In vitro Characterization of the LytSR two component system in Staphylococcus aureus
Authors: Patel Kevin H , Golemi‐Kotra Dasantila
Published: June 2021

S. aureus, is a major gram‐positive pathogen, because of its remarkable capacity to develop resistance against different antimicrobial. Autolysins are known to regulate cell growth, cell division and biofilm formation. It has been shown that in Staphylococcus aureus, autolysis (PG hydrolases) and biofilm formation is regulated by the LytSR two component systems. In this study we investigated the LytSR signal transduction mechanism by performing kinetic characterization of the autophosphorylation of the histidine kinase LytS, and a subsequent phosphoryl transfer to its cognate response regulator LytR using radiolabel Adenosine‐5′‐triphosphate (ATP). For the purpose of the above in vitro experiments, we have cloned and purified LytS lacking its transmembrane domains and the full length LytR. We found that LytS has the ability to catalyze an ATP‐dependent autophosphorylation. Preliminary results of the pseudo first order kinetic reaction suggest that LytS is an active kinase. In addition, we show that activity of LytS depends on the concentration of Ca2+ions but not K+ ions. This research is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).
The VMP1‐Beclin 1 Interaction Regulates Autophagy Induction
Authors: Molejon Maria Ines , Ropolo Alejandro , Duette Gabriel , Santillan Maria , Boggio Veronica , Vaccaro Maria Ines
Published: June 2021

The vacuole membrane protein 1 ‐VMP1‐is an essential autophagy related transmembrane protein conserved from worms to mammals. VMP1 triggers autophagosome formation even under nutrient‐replete conditions. In the current study, we unveil the mechanism through which this protein induces autophagy in mammalian cells. We show that VMP1 autophagy‐related function requires its C‐terminus domain (Atg domain). This is achieved through its binding to the BH3 of Beclin 1 and this interaction concomitantly promotes the dissociation of Bcl‐2, an autophagy inhibitor, from Beclin 1. VMP1‐Beclin1 interaction leads to the formation of a complex with the Class III phosphatidyl‐3 kinase complex, a key positive regulator of autophagy. Our findings indicate that VMP1‐Beclin 1 interaction promotes the localization of other Atg proteins to the autophagosomal membrane. We show that the VMP1‐Beclin 1 complex promotes PI3P generation on autophagosomal membranes, acting upstream of the Atg16L1 complex during autophagy. Collectively, these findings reveal that VMP1, through its Atg domain, regulates autophagy induction in mammalian cells promoting the recruitment of Class III PI3K complex at the site of autophagosome formation.
Accelerated chronologic aging of S. cerevisiae induced by PUFA is associated to mitochondrial dysfunction, enhanced sensitivity to lipid peroxidation and ROS overproduction
Authors: Cortes‐Rojo Christian , Fernández‐Quintero Maricela , Aguilar‐Toral Roxana , Ballesteros‐Coria Kanek Alejandro , Montoya‐Perez Rocio , Mejia‐Zepeda Ricardo , Saavedra‐Molina Alfredo , de la Paz Jose Lucio Hernandez , Calderon Marissa , Madrigal‐Perez Luis Alberto
Published: June 2021

Correlative evidence from comparative studies has suggested that longevity in higher eucaryotes keeps an inverse correlation with the unsaturation degree of mitochondrial membranes. Since lipid peroxidation (LP) depends on the number of double bonds, it has been hypothesized that lower unsaturation degree of the membranes from longevous animals preserves mitochondrial function from the damage by ROS. To further test this hypothesis, we have studied if accelerated aging and impaired mitochondrial function by the incorporation of PUFA in the yeast is associated with increased sensitivity to LP and enhanced ROS production. When yeast incorporated C18:3 into mitochondrial membranes, it was observed accelerated aging along with increased ROS production, which was attributed to impairment in electron transfer between complex II and complex III. These effects were associated with a higher susceptibility to LP, although the cells become more resistant to this process at late stages of aging. These data confirm the observations made in higher eukaryotes about the primordial role of LP in the mitochondrial impairment associated to a lower longevity. This work was funded by a CONACYT (130638 to CCR) grant.
Genome Reduction In Yeast Involves Programmed Cell Death
Authors: Hurton Matthew David , Roblee Emily , Bennett Richard J , Austriaco Nicanor
Published: June 2021

Genetic reduction is of great significance in many biological pathways. In particular, aneuploid cancerous cells typically undergo genetic reduction to obtain a diploid state, a process accompanied by apoptotic programmed cell death. A tetraploid strain of Candida albicans, when grown on a diploid specific pre‐sporulation media, undergoes random chromosome loss, becoming diploid to near‐diploid in DNA content and undergoing significant cell death as part of the completion of a parasexual cycle. Cell death measurements were made for cells grown on pre‐sporulation media plates. Measurements of viability were made with a methylene blue stain, showing significantly lower tetraploid viability. Assays done to determine levels of reactive oxygen species present showed increased concentration in tetraploid cells undergoing genome reduction. Similar results were found in an assay for caspase These results indicate high ROS levels and high levels of caspase activity in the reducing tetraploid, suggestive of apoptosis‐like programmed cell death accompanying genome reduction. Similar results are seen in diploid Saccharomyces cerevisiae. Further research is currently being done with both tetraploid and diploid strands with apoptotic gene knockouts to further understand the mechanism of death, and understanding of these results could aid in understanding of cancerous and other biological mechanisms in humans.
Small intestine and colon crypts differ in impact of obesity, hyperinsulinemia and insulin‐like growth factor 1 receptor (IGF1R) loss on DNA damage‐induced apoptosis
Authors: Santoro Maria Agostina , Blue R Eric , Van Landeghem Laurianne , McNaughton Kirk K , Lund P Kay
Published: June 2021

Colorectal adenoma risk is linked to obesity, hyperinsulinemia and reduced apoptosis of normal colonocytes. We hypothesized that diet‐induced obesity (DIO)/hyperinsulinemia decreases apoptosis of genetically‐damaged intestinal stem cells (ISC) via IGF1R activation. Mice with villin‐cre mediated deletion of IGF1R in the intestinal epithelium and littermate controls with intact IGF1R were fed high fat diet (HFD) or low fat (LF) standard chow for 18 weeks, given 5 Gy abdominal radiation to induce DNA damage and euthanized 4 hours later, at the peak of apoptosis. HFD led to obesity, glucose intolerance and hyperinsulinemia, but no change in plasma IGF1. In small intestine of LF mice, IGF1R loss increased radiation‐induced apoptosis in the ISC zone. DIO also increased apoptosis in small intestine, but IGF1R loss had no additional effects. In colon, apoptosis was decreased in DIO group but unaffected by IGF1R loss. Plasma insulin levels significantly correlated with apoptosis in both small intestine and colon. Our studies provide novel evidence for region‐specific effects of DIO/hyperinsulinemia on apoptosis induced by DNA damage in small intestine vs. colon. DIO‐associated increases in apoptosis in small intestine are relevant to DIO‐associated intestinal dysfunction. DIO‐associated decreases in apoptosis of genetically damaged colonocytes may predispose to tumor initiation. Funding: NIH DK040247‐19
Assembly of apoptotic Bax oligomeric pore in mitochondrial outer membrane requires relocation of helices 5, 6 and 9 from the monomer core to the lipid bilayer and/or oligomer interfaces
Authors: He Feng , Zhang Zhi , Lin Jialing
Published: June 2021

Bax, a pro‐apoptotic protein of Bcl‐2 family, activated by BH3‐only proteins translocates from the cytosol to the mitochondria, forming oligomers to permeabilize the mitochondrial outer membrane (MOM) and initiate apoptosis. Recent studies reported that activated Bax protein inserts α helices 5, 6, and 9 into the MOM as a monomer, which then oligomerize to permeabilize the MOM involved in the formation of interfaces of helices 6 and 9. However, how helices 5, 6, and 9 control the membrane binding and assembly of interfaces toward large oligomers remains unclear. In this study we generated disulfide lockers in Bax monomer to eliminate the conformational changes that might be essential for Bax activation, and compared the conformation‐flexible with the conformation–restrained forms of Bax during the activation process in the MOM. The results show that a disulfide locker blocking α9 displacement from the monomer core prevents membrane binding. Disulfide locking α5 or α6 to the monomer core inhibits the formation of different interfaces that are required for assembly of a functional Bax oligomer in the MOM. The lockers also abolish insertion of the helices into the MOM and behave like a dominant negative Bax. Our data suggest that upon activation, activated Bax binds to membrane by the displacement of α9 as a monomer, through which, Bax forms dimers, small oligomers and large oligomers competent for cyt c release which are controlled by re‐arrangement of α5, α6, and α9, thereby providing the mechanistic insights of Bax‐governed cell death. (The work was supported by the NIH grant GM062964 to J.L.)
The anticancer effect of cytotoxin 1 from Naja atra Cantor venom is mediated by a lysosomal cell death pathway involving lysosomal membrane permeabilization and cathepsin B release
Authors: Kong Tianhan , Ming Wei , Qiu Yanfen , Liu Yamin , Dong Weihua
Published: June 2021

Cytotoxin family of cobra venom, also called cardiotoxins, can induce cancer cells necrotic and apoptotic cell death. Cytotoxin 1 (CTX1) from Naja atra Cantor venom is a 60 amino acid, 6698 Da protein with as yet untested anticancer efficacy. The toxicity of CTX1 on cancer cell lines (MCF‐7, P388, K562, and H22) and normal human cell line (16HBE) was tested. The rank order of CTX1 cytotoxicity was MCF‐7 >; P388 ¡Ö K562 >; H22 ¡Ö16HBE. Treatment with CTX1 greatly prolonged the survival of KM mice bearing P388 ascites tumors compared to cyclophosphamide treatment. Cell viability, apoptosis, and lysosomal permeability assays demonstrated that CTX1 induced dose‐and time‐dependent cell death, with most cells exhibiting the morphological and biochemical features of late apoptosis and necrosis. Mitochondrial membrane potential was lost in CTX1‐treated P388 cells. In addition, CTX1 induced an increase in both lysosomal membrane permeability and cathepsin B protease activity. These analyses reveal that CTX1 possesses significant and selective anticancer activity, likely by inducing programmed cell death through mitochondrial and/or lysosomal pathways.
Interleukin‐6 induces ErbBs receptor phosphorylation and utrophin expression in dystrophic myotubes.
Authors: Juretic Nevenka , Díaz Josefina , Jaimovich Enrique , Riveros Nora
Published: June 2021

Duchenne muscular dystrophy (DMD) is a neuromuscular disease originated by mutations in the dystrophin gene, leading to skeletal muscle fiber loss. A promising pharmacological treatment aims to increase the level of utrophin, a functional dystrophin homolog that could compensate dystrophin absence in DMD muscle fibers. It has been described that interleukin‐6 (IL‐6) and neuregulin‐1 (NRG‐1) induce utrophin expression in skeletal muscle.

We investigated a possible functional link among IL‐6, NRG‐1 and utrophin, in normal (C57) and dystrophic (mdx) skeletal muscle cells.

Using Western Blot and semiquantitative PCR, we demonstrated that IL‐6 (100 ng/ml) induces NRG‐1 receptor phosphorylation (ErbB2/ErbB3) in both cellular types, and a transient increase on utrophin mRNA levels in dystrophic myotubes. We observed that BAPTA‐AM (an intracellular Ca2+ chelator), GM6001 (a general metalloproteinase inhibitor), genistein (a general protein tyrosine kinase inhibitor) and PD‐158780 (an ErbB receptor tyrosine kinase inhibitor) treatment abolished utrophin mRNA induction.

Our results suggest that IL‐6 induces utrophin expression in dystrophic myotubes, through NRG‐1/ErbBs signaling activation, elicited by proteolytic processing of NRG‐1 and ErbBs phosphorylation. Understanding the mechanism involved on utrophin up regulation could help to the development of DMD new therapies.

FONDECYT 11100267
Core Concepts of Biochemistry and Molecular Biology
Authors: Tansey John T. , Baird Teaster , Cox Michael M. , Fox Kristin , Knight Jennifer , Sears Duane W. , Bell Ellis
Published: June 2021

Over the past two years, through a NSF RCN UBE grant, the ASBMB has held regional workshops for faculty members and science educators from around the country that focused on identifying: 1) Core Principles in Biochemistry and Molecular Biology, 2) Essential Concepts & Underlying Theories from Physics, Chemistry, and Mathematics, and 3) Foundational Skills that undergraduate majors in Biochemistry or Molecular Biology must understand to complete their major coursework. Using information gained from these workshops, as well as from the ASBMB accreditation working group and the NSF Vision and Change report, the Core Concepts working group is now developing a list of learning outcomes and objectives based on five core concepts (Evolution, Homeostasis, Matter and Energy Transformation, Information Flow, and Macromolecular Structure and Function) that will aid biochemistry and molecular biology educators in the development of assessment tools for the new ASBMB recommended curriculum. This work was supported by NSF‐0957205 RCN‐UBE to EB.
Incorporating Biotechnology in the High School Classroom: Professional Development for Biology Teachers
Authors: Bisogno Janet L , Koroly Mary Jo
Published: June 2021

Advancements in the biological sciences are increasingly dependent upon biotechnology. Although teachers and administrators support the inclusion of biotechnology in the classroom to prepare students for future careers, lack of school funding and minimal teacher exposure to current biotechnology skills have hindered incorporation. The purpose of this project was to develop professional development (PD) sessions for six life science teachers at one high school, utilizing biotechnology equipment and skills developed during programs offered by UF CPET.

During the 2011/12 school year, four PD sessions were delivered during regularly scheduled Professional Learning Community (PLC) time. The activities included pipetting, gel electrophoresis, blood typing, and ELISA. School‐based equipment lockers were developed and contained all required equipment and curriculum materials needed for each activity, modeled after the UF CPET locker program. Lockers were available for check out by the teachers, and classroom assistance was available as needed.

Evaluations from teachers indicated that they felt more comfortable incorporating biotechnology into their curriculum. Four teachers utilized the pipetting lab and gel electrophoresis lockers, three used the blood typing locker and one used the ELISA locker. For the current year, a fifth PD session and locker are planned to cover microarrays.

Sources of research support included ASBMB and UF CPET.
Using threshold concepts to improve learning and drive curriculum reform in biochemistry
Authors: Loertscher Jennifer , Minderhout Vicky , Green David , Lewis Jennifer
Published: June 2021

Threshold concepts are concepts that when mastered, represent a transformed understanding of a discipline, without which the learner cannot progress. They are therefore essential for learning. For example, equilibrium has been identified as a threshold concept in biology and is likely to be relevant for biochemistry. Preliminary data suggest that students bring incomplete knowledge of equilibrium into biochemistry courses and that biochemistry courses do not alter their understanding significantly. By focusing on threshold concepts, instructors can maximize the impact of instruction. We are beginning work with a community of biochemists, biologists and chemists, to 1) identify threshold concepts for biochemistry, 2) design classroom activities targeting these concepts, 3) disseminate classroom activities and assessment tools, 4) measure changes in student understanding. Our prior work linking assessment of student understanding of protein structure to curricular reform provides a model of a process that uses assessment and community input in a feedback loop to improve student learning.
Cutting genes with undergraduate scissors
Authors: Marintcheva Boriana , Young Kelly , Smith Crystal , MacDougall Lawrence , DaConceicao Silviane , Tandon Ankita
Published: June 2021

Bacteriophage T7 is a genetically and biochemically amenable model system offering great opportunities to train undergraduates in molecular biology and to empower them to pursue independent projects. Students enrolled in BIO497/Undergraduate Biological Research at Bridgewater State University were able to experience science as a process while engineering deletion mutants of T7 DNA metabolism genes.

PCR was used to amplify genes of interest from the T7 genome and to clone them using TOPO technology. The resulting plasmids were transformed in E. coli generating strains permissive for growth of phage deletion mutants. In parallel, multi‐step PCR was used to generate recombination cassettes carrying the sequence for cytidine monokinase (cmk), flanked by the natural neighboring sequences of each gene of interest. Cmk is essential for T7 growth, but dispensable for the host, therefore an ideal selection marker for recombinant viruses. In vivo recombination reaction was carried out resulting in the replacement of each individual gene of interest in the viral genome with the cmk sequence.

Throughout the experience students learnt multiple techniques, sharpened their problem solving and time management skills, wrote grant proposals, and presented their work locally and nationally. This project was generously supported by the Adrian Tinsley Undergraduate Research Program at Bridgewater State University.
Dietary intake associated with cognitive function of elderly Koreans
Authors: Chung Hae Kyung , Kim Eun‐Ji , Chung Ji‐Hye , Shin Ji‐Eun , Chang Moon‐Jeong , Kim Hye‐Young , Kim Mi‐Hye
Published: June 2021

Cognitive impairment of the elderly is modulated by nutrients (Vitamin B6, B12, folate and omega‐3), diet (Mediterranean) and nutritional supplement use. We aim to investigate the cognitive function of elderly Koreans in relation to their dietary practices. Elderly Koreans (n=316, 115 men and 201 women), aged ≥65y, were interviewed individually for sociodemographic characteristics, height, weight, FFQ, 24‐hour recalls, and a mini‐mental state examination evaluated by use of a 30‐point questionnaire in Korean (KMMSE). Frequency, Chi‐Square, and Anova tests were carried out using SPSS 12.0. Obesity was more prevalent in men (33%) and those living with spouse and children than women and those living alone. The mean KMMSE scores (± SD) were higher in men (27.55±3.37 vs. 25.53±4.88 in women), the educated, and the relatively younger group (27.77± 2.42 in <70 y vs. 22.67±5.24) in ≥90y) than in their counterparts. Although responses differ by gender, differences in KMMSE scores were associated with frequent consumption of bread, potato, fish, fish cake, pork, beef, radish, tomato, tangerine, apples, grapes, ice cream and rice wine. Intake of vegetables particularly hot peppers, cumbers, tomatoes and seaweed was closely associated with KMMSE scores in Korean elderly.

Grant Funding Source: Globalization of Korean Foods Project by Korea Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries (IPET)
Association between stressful live events and alcohol consumption among South Indians: findings of a populationbased cross‐sectional survey
Authors: Chui Kenneth , Thanikachalam Mohan , Mahalakshmi K , Vijayakumar Harivanza , Must Aviva , Bermudez Odilia , Chomitz Virginia , Thanikachalam Sadagopan
Published: June 2021

The types of stressor associated with alcohol consumption—a potential cause of malnutrition due energy displacement—have not been well described for the Indian population. We surveyed 8,080 South Indians (44.1% male; mean age 44.6±10.4 y) from 2008 to 2011 and collected information on demographics, current drinking status (current drinkers vs. non‐drinkers and ex‐drinkers), Hamilton Rating Scale for Depression, and Presumptive Stressful Life Events Scale (PSLES). Current drinkers comprise 10.9% of the participants and are mostly men (92.9% male). Sum of stressful life events in the past year predicts drinking status for men (OR=1.22, p<.001) but not women (OR=.97, p=.72), while the sum of lifetime stressful events is non‐predictive. However, both sums predict having moderate or more severe depression (ORs for yesteryear's event: men 1.56; women 1.42. ORs for life‐long event: men 1.44; women 1.46, all p<.001). Factor analysis of the 51‐item PSLES identified 22 factors, with the top 5 being negative changes in social interactions; retirement/lack of offspring; marriage of a daughter; marital/sexual problems; and schooling/examinations. Of the 5 factors, only negative social changes (ORs: men 1.20, p<.001; women .89, p=.43) and marital/sexual problems (ORs: men 1.71, p<.001; women 1.02, p=.89) predict current drinking among males. Future interventions should be targeted by sex and stressor type.
Blood lead, iron deficiency and IQ in Uruguayan first‐graders
Authors: Kordas Katarzyna , Barg Gabriel , Roy Aditi , Manay Nelly , Queirolo Elena I
Published: June 2021

Cognition in children with both iron deficiency (ID) and elevated blood lead levels (BLL) is not well studied. In 1st‐grade children (n=150, age 6.4±0.6 y, 56.7% boys) from Montevideo, Uruguay we investigated associations among BLL ≥5μg/dL, serum ferritin (SF) <12μg/L), and IQ. Mean IQ, SF and BLL were 92.9±17.0 points, 15.0±14.1 μg/L, 4.7±2.2 μg/dL, respectively. Of the study children, 48.8 and 30.2% had BLL ≥5μg/dL and ID, respectively, 13.4% had both. BLL and SF were not correlated. In unadjusted linear regressions, BLL and SF were not independently associated with IQ; neither were BLL ≥5μg/dL nor ID. However, children with both elevated BLL and ID had lower IQ 11.7 points lower than their ID‐only peers (p=0.07). Preliminary analyses suggest that elevated BLL and ID may result in particularly poor cognitive function in school children.

Sponsor: NIEHS 1R21 ES16523–01
The relationship between whole grain consumption and fiber intake in Americans: Results from NHANES 2009–10
Authors: Albertson Ann , Joshi Nandan , Bapna Arohi , Fernandes Sneha
Published: June 2021

Evidence indicating the role of whole grain (WG) foods in the reduction of risk for various cancers, coronary heart disease and overweight continues to grow. Total dietary fiber (TDF) is one nutrient often associated with whole grain. One‐day food intake from the NHANES 2009–10 (n=9042) is used to examine this relationship for Americans 2 yrs. and older. Results indicate that adults ≥19 yrs. in the highest tertile of TDF consumption had significantly greater WG intake than adults in the lower 2 tertiles. Americans 2 yrs. and older meeting WG recommendations (>;3oz. eq.) also had significantly higher TDF intakes (27.7g versus 13.6g for no WG group). Among those who met WG recommendations, highest contributors of percent daily fiber intake were Fruits (15.1%), Vegetables (14.0%), Yeast bread rolls (11.6%), Pastas, cooked cereals, rice (11%) and RTE cereals (10.7%). Among individuals with zero whole grain intake, RTE cereal contributed little TDF (2%). Among RTE cereals, WG and No Bran cereals was the highest contributor to dietary fiber (6.22%) as compared to No WG and with Bran (2.85%), WG and Bran (0.92%), No WG and No Bran (0.71%), among those who met WG recommendations. Whole grain consumption should continue to be encouraged for all Americans.
Dietary Patterns and prostate cancer risk: a population based cohort study in elderly Swedish men
Authors: Ax Erika Helena , Cederholm Tommy , Grundmark Birgitta , Bill‐Axelson Anna , Becker Wulf , Holmberg Lars , Zethelius Björn , Garmo Hans , Sjögren Per
Published: June 2021

The aim of this study was to assess the relationship between adherence to a modified Mediterranean Diet Score (mMDS) and a relatively low carbohydrate‐high protein score (LCHP‐score) and prostate cancer (PCa) incidence, in elderly Swedish men. In Uppsala Longitudinal Study of Adult Men, a Swedish population‐based cohort (n=1138, mean age 71±1), diet was determined by 7‐ d food records, and non‐adequate energy reporters were excluded. PCa cases were identified in the Swedish Cancer Register. Risk of PCa was analyzed with Cox proportional hazard regression. Exploratory analyses of nutrients identified by the World Cancer Research Fund as probable modifiers of PCa risk were conducted. During follow‐up (median 13.3y) 72 cases of PCa were identified. The mMDS was not associated with PCa. Adherence to the LCHP‐score was inversely related to PCa, with adjusted hazard ratios (HR) of 0.55 (0.32–0.96) for medium‐, and 0.47 (0.21–1.04) for high‐, compared with low adherence. Inclusion of selenium intake in risk models attenuated the association between the LCHP‐score and PCa, high vs. low adherence HR 0.70 (0.28–1.73).

In this study, a moderately low carbohydrate‐high protein dietary pattern was associated with lower PCa incidence. A higher intake of selenium associated with the LCHP‐score was indicated to have contributed to observed relations.
Modeling an MMA‐derived inflection point for serum vitamin B12 (SB12) in the US
Authors: Bailey Regan L , Durazo‐Arvizu Ramon , Carmel Ralph , Green Ralph , Pfeiffer Christine M , Sempos Christopher T , Yetley Elizabeth A
Published: June 2021

Uncertainties exist about cut‐points to define suboptimal B‐12 status. We sought to identify an inflection point at which the SB12 relationship to MMA changes by applying various statistical to describe the prevalence of suboptimal B‐12 status. SB12 and MMA were available for adults (≥ 19 y; n=12,683) from NHANES 1999–2004, a nationally‐representative, cross‐sectional survey. After controlling for kidney function, age, and fasting, six models were evaluated. The SB12 inflection points determined by each model varied considerably (range 124–311 pmol/L). A linear‐splines model was most robust statistically and indicated two inflection points (124 and 286 pmol/L), generating 3 groups. Group 1 was small (1%) but notably deficient (SB12 < 124 pmol/L; n=131) with the highest MMA (mean=726; median=287 nmol/L) and homocysteine (mean=17; median=12 μmol/L). The dominant (66%) group 3 (SB12 ≥ 286 pmol/L; n=8,569) had apparently adequate B12 status. An intermediate but sizeable group 2 (n=3,983; 33%) emerged with a B12 to MMA relationship distinct from the others. The derivation of MMA‐defined inflection points for SB12 is highly dependent on the model that is chosen. Epidemiologic studies will benefit from recognizing the variability among statistical models, considering these new subsets, and evaluating the model that best integrates statistical, biological, and practical considerations.
Plain water intake of Korean adults: Korean National Health and Nutrition Examination Surveys 2008–2010
Authors: Kim Jihye , Yang Yoon Jung
Published: June 2021

Objective

The objective of this study was to examine plain water intake of Korean adults according to anthropometric, dietary, and food consumption characteristics.

Methods

This study was conducted using data from the 2008– 2010 Korean National Health and Nutrition Examination Survey. The subjects were 5,917 men and 8,511 women aged 20–64 years. Water intake was estimated by asking the question “How much water do you usually consume per day?” in the questionnaire. Dietary intake was estimated by 24‐hour dietary recall. A food frequency questionnaire including 63 food items was also administered. Lean body mass and percentage of body fat were measured by dual‐energy X‐ray absorptiometry.

Results

The mean plain water intake for men and women were 6.3 cup/day and 4.6 cup/day, respectively (1 cup = 200ml). Water intake increased as lean body mass, waist circumference, and body mass index levels increased, except for percentage of body fat. As energy and alcohol intakes increased, water intake increased. However, as energy‐adjusted fat and carbohydrate consumption increased, water intake decreased in men. Water intake increased as energy‐adjusted protein and energy‐adjusted fiber consumption increased in women. As total weight of food intake and total volume of food intake increased, water intake increased. Water intake according to consumption of vegetables was increased in men and women. In the male subjects, water intake increased as frequencies of green tea, alcoholic drink, and all beverages were increased. In the female subject, water intake increased with increased frequencies of green tea, milk, soy milk, and alcoholic drink and decreased frequencies of coffee and soda.

Conclusions

Our results suggest that people who had a higher BMI and ingested more food consumed more water. Plain water intake was influenced by the particular types of food ingested such as grains, meat, vegetables, fruits, and beverages.
Molecular Basis for Dystroglycan Binding to Laminin‐G Domain‐Containing Ligands
Authors: Campbell Kevin P. , Yoshida‐Moriguchi Takako , Hara Yuji , Inamori Keiichiro
Published: June 2021

Dystroglycan (DG) is a highly glycosylated extracellular matrix (ECM) receptor. The like‐acetylglucosaminyltransferase (LARGE) is responsible for posttranslational modification of alpha‐dystroglycan (α‐DG) which is required for its function. Reduced ligand binding activity of α‐DG due to perturbed glycosylation is a common pathologic feature among several different forms of muscular dystrophy (called dystroglycanopathies). We now report that LARGE is a bifunctional glycosyltransferase, with both xylosyltransferase (Xyl‐T) and glucuronyltransferase (GlcA‐T) activities. We examined the enzymatic activity of LARGE with respect to Xyl‐α‐pNP as well as GlcA‐β‐MU as the acceptors and UDP‐Xyl and UDP‐GlcA as donors, using a secreted form of LARGE that lacks its transmembrane domain (LARGEdTM). We have identified the enzymatic function of LARGE, which produces a polysaccharide with repeating units of [‐3Xyl‐α1,3GlcA‐β1‐] onto these acceptors. We further show that this modification confers binding ability on α‐DG to laminin‐G domain‐containing ECM ligands: laminin, agrin, and neurexin. α‐DG binds the LG4 and 5 domains of laminin alpha chains, and it is suggested that three basic patches contribute to these interactions. The LARGE‐synthesized, negatively charged glycan on α‐DG likely binds to laminin through electrostatic associations with these basic patches.