bluuebunny/crossref_metadata_2025_split huggingface dataset:
Saccharomyces cerevisiae G2 checkpoint and double‐strand break (DSB) repair genes synergistically suppress the formation of homology‐directed translocations
Authors: Fasullo Michael Thomas 
Published: June 2021

Checkpoint and DNA repair mutants exhibit more genetic instability compared to wild‐type yeast. We previously observed that G2 checkpoint and rad51 mutants exhibit elevated rates of spontaneous translocations. We measured rates of spontaneous translocations in diploid mutants defective in checkpoint and/or DSB repair genes. Directed translocation events were measured by selecting for His+ recombinants that result from mitotic recombination between two truncated his3 genes. The rate of spontaneous translocations in wild type is 4.4 x 10−8. Whereas rad51 and rad9 mutants exhibited fourfold and sevenfold higher rates of spontaneous translocations, rad51 rad9 double mutants exhibited 39‐fold higher rate. Compared to rad51 rad9, we observed similar rates in rad55 rad9 and rad57 rad9 mutants. While xrs2, rad50, and mre11 mutants exhibited 10‐20 fold higher rates, compared to wild type, xrs2 rad9, rad50 rad9, and mre11 rad9 exhibited similar rates compared to rad51 rad9. Interestingly, rate of spontaneous translocations in mec1‐21, a S phase checkpoint mutant that retains some G2 checkpoint function, was 23‐fold higher than wild type, while rates in mec1‐21 rad51 double mutants were 100‐fold higher. Pulse field gel electrophoresis confirmed that recombinants contain translocations. The results illustrate that both DSB repair pathways and G2 checkpoint pathways suppress translocation frequencies.Grant support: CA70105 from the National Cancer Institute
Expression and characterization of Francisella tularensis fatty acid biosynthetic genes FabD and FabH in E. coli
Authors: Chmielowski Jennifer N , Bohn Kelsey C , Wen Lisa , Huang Jenq‐Kuen , Timsina Yam N , Chand Phani C , Pathak Ashish K 
Published: June 2021

Francisella tularensis is an extremely infectious airborne pathogen, and has long been considered as a potential biological weapon. The intent of this research is to study a key regulatory enzyme of type II FAS of Francisella tularensis, namely FabH, as a novel drug target against tularemia. FabH, also known as 3‐oxoacyl‐acyl carrier protein synthase III, catalyzes the initial condensation and elongation reactions in fatty acid synthesis. Genes encoding the Francisella tularensis of FabD and FabH were custom synthesized and inserted in pET15b expression vector by Celtek Genes. Each recombinant protein was overexpressed by IPTG induction. Both recombinant FabD and FabH proteins were purified by affinity chromatography on a Ni‐NTA column. FabH activity was measured by monitoring the conversion of [14C]‐acetyl‐CoA and malonyl‐ACP substrates (produced by FabD in the coupled assay) to a radiolabeled acetoacetyl‐ACP. The active enzyme has been successfully used in screening a class of 46 newly synthesized indole‐2‐carboxylic acid derivatives for its inhibition. Three compounds have shown IC50 values in the range of 2‐6 µM against Francisella tularensis FabH.Supported in part by the University Research Council, Western Illinois University.
A Pyruvic Acid Analog Primarily Targets GAPDH To Promote Cancer Cell Death
Authors: Ganapathy‐Kanniappan Shanmugasundaram , Geschwind Jean‐Francois , Kunjithapatham Rani , Buijs Manon , Vossen Josephina Anna , Tchernyshyov Irina , Torbenson Michael S , Cole Robert N , Syed Labiq H , Vali Mustafa 
Published: June 2021

The objective of the current study is to elucidate the principal mechanism underlying the anti‐glycolytic effect of the pyruvic acid analog, 3‐bromopyruvic acid (3‐BrPA). Up regulation of glycolysis is a common event witnessed in majority of tumors. 3‐BrPA induces tumor cell death by depleting intracellular energy (ATP) through inhibition of glycolysis. Previous reports claimed 3‐BrPA as an energy blocker targeting the enzyme, type II hexokinase. However, substantial experimental evidences on the primary targets, essentially involved in the 3BrPA cytotoxicity, are lacking. Here, we used a combination of dose‐response studies coupled with proteomics in multiple cancer cell lines to identify the molecular targets of 3‐BrPA in an unbiased fashion. Radiolabeled 3BrPA studies showed glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) as the primary chemical and biological target at a dose that induced cell death. Loss of function studies through RNA interference (RNAi) confirmed the necessity of GAPDH for cell survival. Hence, we provide evidence for the first time that GAPDH is the primary molecular target of 3‐BrPA.This study was supported by NIH/NCI grant RO1 CA 100883‐01 (J.F.G), Charles Wallace Pratt Research Fund and the Abdulrahman Abdulmalik Liver Cancer Research Fund.
Identification of a novel PPARα‐interacting protein
Authors: Bagattin Alessia , Hugendubler Lynne , Mueller Elisabetta 
Published: June 2021

PPARα (peroxisome proliferator‐activated receptor α) is a ligand‐activated transcription factor involved in the regulation of fatty acid β‐oxidation. It is mainly expressed in tissues with elevated energy metabolism like liver, heart, kidney, skeletal muscle and brown fat. Upon ligand binding, PPARα heterodimerizes with the retinoid X receptor α (RXRα). The PPARα‐RXRα complex binds to specific PPAR response elements (PPRE) in the promoter regions of target genes enhancing their transcription. PPARα is activated by fibrates, synthetic compounds that induce peroxisome proliferation in rodents and are used as hypolipidemic drugs in humans. Many studies aim at the identification of novel agonists and cofactors that can modulate PPARα activity. We identified ACOX1 (Acyl Coenzyme A Oxidase) as a PPARα‐interacting protein by pull‐down using PPARα‐GST fusion proteins and 35S‐labeled‐in vitro translated ACOX1. ACOX1 is able to increase the transcriptional activity of PPARα in transient transfection experiments in U2OS (human osteosarcoma) cells. ACOX1 is the first and rate‐limiting enzyme of the peroxisomal fatty acid β‐oxidation and its expression is induced by PPARα. These results suggest the possibility of a novel regulatory loop between PPARα and a metabolic enzyme.Supported by the Intramural Research Program of NIDDK, NIH.
A role for MGA2, but not SPT23, in activation of transcription of ERG1 in Saccharomyces cerevisiae
Authors: Rice Caitlin , Stukey Joseph , McDonough Virginia 
Published: June 2021

A major role of the Saccharomyces cerevisiae MGA2 gene product is regulation of unsaturated fatty acid production, by controlling transcription and mRNA stability of OLE1, the gene encoding the ?‐9 fatty acid desaturase. Lipid composition studies indicated that the mga2? strain contains elevated relative amounts of squalene when compared to wild‐type cells. The deletion of the MGA2 homologue, SPT23, does not similarly impact squalene amounts. To explore the role of MGA2 in the regulation of sterol synthesis, ERG1 transcription was studied using an ERG1 promoter‐lacZ reporter gene construct. We report here that in addition to Mga2p's role in regulation of unsaturated fatty acids, MGA2 is required for full basal expression of ERG1. Mga2p was found to differentially regulate activation of ERG1 and OLE1, as unsaturated fatty acids did not affect ERG1 expression levels as has previously been shown to control Mga2p's activation of OLE1. The spt23? strain shows similar ERG1 expression to wild‐type cells, while the mga2?/spt23? strain shows reduced ERG1 expression, comparable to the mga2? mutant alone, suggesting that the role of regulation of ERG1 transcription is unique to Mga2p. Finally, particular exogenous unsaturated fatty acid supplementation was shown to influence cellular relative squalene amounts, which was less dependent on genotype.CR was supported by a Deans Research Award
A novel approach for investigating glycan involvement in embryonic cellular interactions. II. lectin studies
Authors: Naminik Arjang , Oppenheimer Steven B. 
Published: June 2021

Here we investigate the effects of 5 lectins at concentrations ranging from 0.0005 mg lectin ‐ 0.5 mg lectin per ml low calcium sea water on specific cellular interactions in the NIH model sea urchin embryo Lytechinus pictus using a microplate assay. 3 separate experiments were conducted with 12 microwells in each experiment at each lectin concentration, yielding a total of 36 wells with hundreds of embryos assessed for each lectin concentration. Lectins(or no lectin controls) were added at 24 hrs of development and the effects on archenteron/elongation/binding to the blastocoel roof were recorded after an additional 24 hrs. Phytolacca americana (specific for N‐acetyl‐beta‐D‐glucosamine residues) inhibited archenteron elongation/attachment to the blastocoel roof in a concentration dependent manner when controls and treated embryos were compared (p less than 0.0001 comparing complete archenterons in controls versus experimentals). Pisum sativum (specific for alpha‐D‐glucose and alpha‐D‐mannose) and Ulex europaeus UEA 1 (specific for L‐fucose) lectins both caused exogastrulation, where not only does the archenteron not attach but it everts from the embryo proper (p value of less than 0.0001 comparing exogastrulation in controls versus experimentals). Helix pomata (specific for N‐acetyl‐alpha‐D‐galactosamine) and Glycine max (specific for N‐acetyl‐alpha‐D‐galactosamine and D‐galactose) had no effects on the embryos, like the controls.The results indicate that specific lectins that bind to specific saccharides influence a model cellular interaction. Most important, however, is that the microplate approach used here is able to quantitatively assess specific probed events in living embryos (Supported by NIH NIGMS SCORE (S0648680), RISE, MARC , the Joseph Drown Foundation and the Sidney Stern Memorial Trust).
Phenylalanine 508 forms an intra‐domain contact crucial to CFTR folding and dynamics
Authors: Serohijos Adrian Wendil Ruba , Hegedus Tamas , Aleksandrov Andrei , He Lihua , Cui Liying , Riordan John R , Dokholyan Nikolay V 
Published: June 2021

Cystic fibrosis is the most prevalent genetically inherited disease in the US. In 90% of CF cases, the disease is caused by the deletion of phenylalanine‐508 (Phe508) from the first nucleotide‐binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP‐binding cassette (ABC) transporter. Prior experimental studies suggest that Phe508 deletion cause the aberrant folding of NBD1 and the misassembly of various CFTR domains. To understand the role of Phe508 in channel function and of the impact of its deletion, we constructed a 3D structural model of CFTR using the structure of another ABC transporter, Sav1866. The CFTR model predicts, and we experimentally validated, that Phe508 mediates an interaction between NBD1 and the fourth cytoplasmic loop in the second membrane‐spanning domain. This explains the observed sensitivity of CFTR assembly to many disease‐associated mutations in CL4 and NBD1. Other cytoplasmic‐membrane domain contacts were also experimentally confirmed. Electrophysiological measurements and molecular dynamics simulations also show that the CFTR membrane‐cytoplasmic interfaces regulate channel gating and are highly dynamic. Aside from advancing our understanding of CFTR structure and function, this study identifies a focused region in CFTR that can be targeted in drug development.Funding is provided by NIH (JRR), CFF (NVD), and AHA (AWRS).
Characterization of EGFR pathway using small molecule inhibitors and In‐Cell ELISA technology
Authors: Narahari Janaki , Hughes Douglas E , Webb Brian 
Published: June 2021

In‐Cell ELISA technology represents an emerging new technique for studying intracellular signaling proteins in context of the whole cell using post‐translational modification (PTM)‐specific antibodies. It offers a convenient and fast cell‐based assay methodology for analyzing signal transduction pathways. Typically, Western Blots are used to monitor post‐translational protein modifications (PTMs), using specific antibodies. However, western blotting involves time‐consuming steps such as lysate preparation, gel loading and electrophoresis, membrane transfer and probing of the membrane with the appropriate antibody. Western blots are also only semi‐quantitative and have very low throughput. PTMs associated with protein activation can be easily monitored by In‐Cell Elisa method in a 96‐ or 384‐well microplate format, which allows for assay scalability and automation while conserving valuable reagents like cell culture regents and stimulators/inhibitors.Development of reagents for In‐Cell Elisa, compatible with either Colorimetric or Fluorescent detection of proteins, are described here. We demonstrate the utility of In‐Cell Elisa technology for simultaneous analysis of different proteins involved in Epidermal Growth Factor signaling using various small molecule activators and inhibitors. Results comparing our In‐Cell Elisa technology to more commonly used western blots technology will be demonstrated.
Flavan‐3‐ols Transport Across Blood‐Brain Barrier
Authors: Faria Ana , Pestana Diogo , Cruz Luís , De Freitas Victor , Azevedo Isabel , Mateus Nuno , Calhau Conceição 
Published: June 2021

This work aimed to evaluate the transmembranar transport of flavonoids across RBE‐4 cells (rat capillary cerebral endothelial cells). HPLC‐DAD/MS was used to detect the various phenolic compounds in studied samples (apical and basolateral media), and their metabolites. The metabolites of the tested polyphenols were synthesized to be used as patterns.Flavan‐3‐ols passed across RBE‐4 cells (epicatechin>B4 procyanidin>catechin) in a time dependent manner. This transport was stereoselective, involving one or more membrane transporters. Additionally, it was found that the cells were capable to metabolize these compounds, particularly by conjugation with glucuronic acid, since this metabolite was detected. An UDP‐glucuronosyltransferase inhibitor was used (β‐estradiol) and found to facilitate the transport through the BBB.In conclusion: (i) the synthesis of flavonoid metabolites allowed the detection of these compounds; (ii) the studied flavan‐3‐ols were capable of crossing the BBB, possibly reaching the central nervous system, which correlates with some neuroprotective effects described in literature for these class of flavonoids; (iii) these cells have enzymes capable of metabolizing these compounds and to conjugate them with glucuronic acid.Financial support from iBeSa and FCT (PTDC/QUI/65501/2006, SFRH/BD/28160/2006 and SFRH/BD/30915/2006) is gratefully acknowledged.
Compensation of calories from one cup or six cups of 94% fat free popcorn, but not potato chips, as a before meal snack
Authors: Nguyen Von , Summers Amber , Brosnahan Jen , Zukley Linda , Lowndes Joshua , Reimers Kristin , Angelopoulos Theodore J , Rippe James M 
Published: June 2021

Strategies to enhance compliance to reduced calorie diets are needed to reduce the health burden of obesity. Contrasting evidence exists over the ability of pre meal snacking to control satiety and control ad libitum energy intake. This study measured caloric compensation when popcorn or potato chips are consumed as a before meal snack.Subjects (n=35) consumed a standardized breakfast and a standardized pre‐load 30 minutes prior to an ad libitum. Conditions were delivered in a randomized order and consisted of 200 mL of water, one cup of 94% fat free popcorn (15 kcal), 6 cups of 94% fat free popcorn (100 kcal), and one cup of potato chips (150 kcal). Plate weights were obtained before and after lunch to measure ad libitum consumption. Caloric intake from lunch and the preloads were combined and compared to the non‐caloric water preload condition to assess compensation. Palatability of food served for lunch was also rated.Caloric intake in the water control, one cup popcorn, and six cups popcorn conditions were not significantly different (716 ± 279, 698 ± 286, 739 ± 294 kcal), demonstrating calorie compensation for up to 100 kcal of low fat popcorn. In contrast, combined caloric intake the potato chip condition (803 ± 278 kcal) was significantly greater compared to the water control and both popcorn conditions (p<0.001). No difference in lunch palatability was observed across the preload conditions.The results of this study suggest that consuming lower energy density snacks such as 94% fat free popcorn prior to a meal may be an effective strategy to satisfy hunger without increasing total calorie intake when a snack is consumed before a meal.
Intestine specific expression of DGAT1 reverses the resistance to diet‐induced obesity phenotype of DGAT1‐deficient female mice
Authors: Lee Bonggi , Zhu Jiabin , Cheng Ji‐Xin , Buhman Kimberly K. 
Published: June 2021

Mice deficient in DGAT1 (DGAT1−/−), a key enzyme in triacylglycerol (TG) biosynthesis, are resistant to high fat (HF) diet‐induced obesity (DIO). DGAT1−/− mice have no defect in quantitative absorption of dietary fat; however, their enterocytes store abnormal amounts of TG in cytoplasmic lipid droplets (CLDs) and TG appears in blood at a slower rate. Therefore, we examined whether this intestine phenotype contributes to resistance to DIO in DGAT1−/− mice. We generated mice with intestine specific DGAT1 overexpression using the villin promoter and then crossed these mice with DGAT1−/− mice to generate mice expressing DGAT1 only in intestine (DGAT1 IntONLY). When fed a HF diet, DGAT1 gene expression is higher in DGAT1 IntONLY mice compared with wild‐type mice and expression is absent in DGAT1−/− mice. In contrast, DGAT2 gene expression is similar in all mouse lines. Enterocytes of HF fed DGAT1 IntONLY mice have very little to no TG storage in CLDs and TG appears in blood at a faster rate than DGAT1−/− mice. In contrast to DGAT1−/− mice, DGAT1 IntONLY mice are not resistant to HF DIO. Our findings demonstrate that intestine specific DGAT1 expression complements both the intestine and body weight phenotype of female DGAT1−/− mice. It suggests that DGAT1 stimulates dietary TG secretion out of enterocytes and that altering this cellular function affects whole animal energy balance.Grant Funding SourceAmerican Heart Association, Purdue Research Foundation
The effect of phosphorus manipulation on in vivo glycogenesis and lipogenesis of rats fed a high fructose diet.
Authors: Obeid Omar A , Mattar Lama , Hwalla Nahla 
Published: June 2021

ObjectiveHigh phosphorus intake was reported to be associated with the development of metabolic syndrome. This effect is though to be related to its capacity to induce lipogenesis and uric acid production. Fructose is also known to have phosphorus sequestering capacity, but it is not clear whether phosphorus manipulation of the diet would affect fructose metabolism and thus its capacity to induce metabolic syndrome.MethodsAdult male Sprague‐Dawley rats were maintained for four days on high fructose diet with different added quantities of phosphorus (0%, 0.015%, 0.15% and 1.5%). Food intake and body weight were monitored throughout the experiment. At the end of the feeding period, overnight fasted rats were fed a test meal and injected with 3H2O to measure in vivo rates of glycogen and lipid synthesis.ResultsPostprandial lipogenesis of both epididymal fat pad and liver was found to increase with increased phosphorus content of the diet. At the same time, postprandial hepatic glycogenesis increased with increased phosphorus content of the diet. But, plasma uric acid concentration was similar among the groups.ConclusionIncreased phosphorus content of high fructose diet seems to stimulate the lipogenic capacity of both liver and adipose tissue. This in turn may promote the development of hypertriglyceridemia and thus metabolic syndrome.
Intensive nutritional support in genetically identical triplets of nine months old with primary and severe malnutrition
Authors: Vasquez‐Garibay Edgar Manuel , Padilla‐Marquez Gabriela , Ibarra‐Gutierrez Maria Isabel , Ramirez‐Magaña Olga Yolanda , Romero‐Velarde Enrique 
Published: June 2021

ObjectiveTo characterize the gain in velocity of growth of nine months old, genetically identical triplet infants with severe and primary protein energy malnutrition after a six weeks‐periods of intensive nutritional supportSubjectsMother: 18 years old, 28 weeks of gestational age and father of 20 years old. First Triplet: Apgar 7‐9, birth weight 890g with respiratory distress. Second triplet: Apgar 8‐9, multiple congenital abnormalities in thoracic members that were diagnosed as arthrogryposis, birth weight 1100g. Third triplet: Apgar 3‐8 with respiratory distress. They were genetically identical. First and second triplets received milk infant formula lactose free, and the third triplet soy formula. At two, four and six weeks they received liquids (mL/kg/d) (189, 236 and 256), energy kcal/kg/d (141, 189, and 205), and proteins g/kg/d (3.2, 4.1 and 4.4) respectively.ResultsInitial weight at admission: first triplet 3540g, second 3320g, third 3935g. The cost of weight gain (g) was 4.3, 3.7 and 4.3 kcal/kg/d and 0.10, 0.08 and 0.10 g/kg/d of proteins respectively.ConclusionThe weight increase among triplets was identical in the six weeks‐periods. The velocity of increase of arm circumference, arm muscular area and arm fat area was lower in the second triplet with arthrogryposis.
Improved Type 2 Diabetes Control Outcomes as a Result of a Culturally‐appropriate Diabetes Education Program
Authors: Salto Lorena , Beeson Lawrence , DeLeon Marino , Cordero‐MacIntyre Zaida 
Published: June 2021

O: Assess the impact of a culturally‐appropriate diabetes education program in a Hispanic group.M: 39 subjects (16 M & 23 F), 29‐75 y, participated in a 3‐month study. Blood glucose, chol levels, anthropometric and DXA values were obtained at baseline and 3 m. Paired t‐tests were used to compare baseline and 3‐m means. Spearman's rho was used to determine the linear relationship between laboratory results and dietary changes (Southwestern FFQ).R: Baseline FBG mean was 167.9 mg/dL, baseline HbA1c mean was 8.5%, insulin mean was 13.7 uU/mL. HbA1c % decreased (8.5% ±2.5 vs. 7.6%±1.7 p=.003) and insulin increased (13.7 uU/mL± 11.3 vs 16.7 uU/mL± 13.3 p=.05). HDL (49.74 mg/dL±10.4 vs. 43.9±9.9 p=.012), LDL (120.6 mg/dL±32.2 vs. 110.3±34.8 p=.013) and total chol (191.3 mg/dL±34.3 vs. 177.9±40.9 p=.005) decreased. Waist circumference (100.1 cm±11.9 vs. 98.6±12.6 p=.044) and DXA total fat (16790.8g ±8.332.1 vs. 16454.6g ±8119.0 p=.033) decreased. At 3 m, there were significant correlations between a decrease in HDL and % cal from total fat (r=372, p=.02), monounsaturated fat (r=.404, p=.011) and carbohydrates (r=.339, p=.035).C: The program had a beneficial effect on dietary changes and blood sugar control.