bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ubiquitination of the transcription factor c‐maf is mediated by multiple lysine residues (LB188)
Authors: Zhang Zubin , Chen Guodong , Xu Xin , Tong Jiefei , Han Kunkun , Cao Biyin , Moran Michael , Mao Xinliang 
Published: June 2021

The transcription factor c‐MAF belongs to the family of basic leucine zipper transcription factors including c‐fos and c‐jun. Previous studies suggested that c‐Maf could be polyubiquitinated followed by degradation in the proteasomes, but the mechanism is not defined. Theoretically, any lysine residues in c‐MAF could be ubiquitinated. In the present study, we tried to find out the specific lysine residues mediating c‐MAF ubiquitination. Through a series of mutational screenings from lysine (K) to arginine (R), we found that any single lysine mutation (K to R) failed to prevent c‐MAF ubiquitination, and a single lysine residue failed to mediate c‐MAF ubiquitination, which indicated that multiple lysine residues were required for c‐MAF ubiquitination. Bioinformatics and computing analyses revealed that K85 and K350 could mediate c‐MAF ubiquitination, which was confirmed at the cell‐based assays. However, this duo was not the only pair because the K85R/K350R mutant could also be ubiquitinated. Functionally, both M12 (K85/K350) and W12 (K85R/K350R) mutants increased cyclin D2 promoter‐driven luciferase activity, but they were less potent than the lysine‐free counterpart (M14). Therefore, we demonstrated that c‐MAF ubiquitination is mediated by multiple lysine residues, of which K85 and K350 are sufficient but not the only duo in mediating the c‐MAF ubiquitination. Moreover, c‐MAF was found to be degraded by lysosomes. This study added a novel insight for c‐MAF functional modularity, suggesting that c‐MAF stability is strictly regulated.Grant Funding Source: National Natural Science Foundation of China
3D Culture of rat embryonic stem cells and rat induced pluripotent stem cells in a novel PGmatrix hydrogel (LB20)
Authors: Rajanahalli Pavan , Huang Hongzhou , Sun Xiuzhi , Weiss Mark 
Published: June 2021

Expanding embryonic stem cells (ESCs) in a synthetic 3D microenvironment in vitro while maintaining the undifferentiated state has been challenging. It would be advantageous to identify a substrate which could be modified such as by changing ligands that interact with cell surface receptors or by changing the substrate’s stiffness. We developed a synthetic PGmatrix hydrogel to include these design specifications and determined whether it could support the expansion of undifferentiated ESCs in a 3D microenvironment. Rat ESCs (rESCs) and rat induced pluripotent stem cells (riPSCs) were used for evaluation. The cells were grown both in 2D and 3D in PGmatrix hydrogel containing 2 inhibitors. Dynamic rheological tests of PGmatrix hydrogel in 2i medium showed a rapid increase of storage moduli (G’ = 600 Pa for 3mM in 1 hr) indicating gelation after mixing. The mechanical strength of hydrogel was full recovered after shear‐thinning into liquid phase.Also, the hydrogel reached a self‐supporting strength within 1000sec which is a reasonable rate to ensure cells were suspended in the hydrogel matrix before they reached the bottom of the plate. Both cell lines were cultured for more than 30 passages using regular trypsinization method to produce single cells at passage prior to plating. We harvested cells from the hydrogel for passaging by shearing the gel to liberate the cells. Phase contrast images of cells grown in 3D showed similar morphology which had rounded phase‐bright borders compared to cells grown on murine embryonic fibroblast (MEF) feeder layer. They retained pluripotent stem cell properties and showed positive staining for alkaline phosphatase, Oct‐4, Nanog, Sox‐2 and SSEA‐1. Karyotypes of pluripotent stem cells cultured in 3D were normal after 20 passages. Embryoid bodies (EBs) could be formed by hanging drop after the cells were trypsinized from 3D cultures. We plan to compare the gene expression profiles of 2D and 3D cells and determine if the 3D cultured cells can undergo differentiation into all the 3 germ layers and form teratomas. Growing ESCs in a fully synthetic 3D culture can significant advantages for directing differentiation and examining the interactions with surface receptors. Such knowledge will enable 3D culture for their use in drug development or for encapsulation of cells for clinical use.
Uncovering novel roles of tandem BRCA1 C‐terminal (BRCT) domains in DNA repair, genome stability and breast cancer (LB236)
Authors: Davis Jamaine 
Published: June 2021

Pax transactivation domain‐interacting protein (PTIP) has an important role in preserving genomic stability and is essential for cell survival following DNA damage, although the precise mechanism is undefined. PTIP consists of three pairs of tandem BRCA1 C‐terminal (tBRCT) domains required for assembly into multiple protein complexes. Mutations that disrupt BRCT domain binding correlate with increased cancer rates. To determine its precise role in DNA repair, we profiled the expression of PTIP in breast cancer cell lines. Surprisingly, we’ve discovered a novel truncated isoform of PTIP, containing the last four C‐terminal BRCT domains, (PTIP‐delta). RNAi gene depletion studies reveal a post‐transcriptional origin of PTIP‐delta. Kinetic profiling of PTIP synthesis indicates that a significant portion of PTIP is processed to PTIP‐delta within hours of translation. Recombinant forms of this PTIP‐delta retain the ability to form nuclear repair foci. Biophysical characterization uncovers new oligomerization interfaces of higher order structures composed exclusively of PTIP‐delta. These studies indicate that PTIP‐delta participates in the formation of multiple structurally and stoichiometrically diverse complexes during DNA repair. These studies implicate new and unusual functional roles of PTIP in DNA repair, genome stability and breast cancer.Grant Funding Source: NCI K 22
Significance of α‐subunit VISITDG sequence residues in the catalytic sites of Escherichia coli ATP synthase (LB247)
Authors: Ahmad Zulfiqar 
Published: June 2021

Background: F1Fo ATP synthase is the fundamental means of cellular energy production in animals, plants, and almost all microorganisms. ATP is generated by oxidative or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts. A typical 70 kg human with a relatively sedentary lifestyle will generate around 2.0 million kg of ATP from ADP and Pi (inorganic phosphate) in a 75‐year lifespan. ATP synthase is the smallest known biological nanomotor, found from bacteria to man. Being the smallest biological nanomotor, ATP synthase is in the forefront of nanomedicine. Nanomedicine results from nanotechnology where molecular scale nanomotors can be used to treat disease conditions. The question of paramount importance is how to build nanomachines that are compatible with living systems and can safely operate inside the body. Here we propose that ATP synthase is a workable base model for the development of nanomotors in nanomedicine usage. In order to use it as base model we must elucidate its catalytic properties. Phosphate (Pi) binding is a primary step in ATP synthesis, so understanding the molecular basis of Pi binding is an important goal. Methods: Mutagenic analysis of the catalytic site highly conserved α‐subunit VISIT‐DG sequence residues were performed to generates single and double mutants. Growth patterns were observed on limiting glucose and succinate plates. MgPi protection assay against NBD‐Cl induced inhibition was performed to reveal the direct or indirect role of residues in Pi binding. Results: Lately, we found that αSer‐347 of the VISIT‐DG sequence is the fifth important residue involved in phosphate binding as are the four catalytic site residues βLys‐155, βArg‐182, αArg‐376, and βArg‐246. Currently we are exploring the role of other VISIT‐DG sequence residues particularly αThr‐349 and αAsp‐350 in Pi binding.Grant Funding Source: supported by NIH/GM085771
Identification of Transcriptional Regulators for a Novel Glyoxylate Production Pathway in Methylobacterium extorquens AM1 (LB255)
Authors: Van Fosson Julie , Ngo Richard , Skovran Elizabeth 
Published: June 2021

Methylobacterium extorquens AM1 is of interest as a platform for production of biofuels and biodegradable plastics from methanol. Biofuels such as 1‐butanol can be derived from intermediates in the Ethylmalonyl‐CoA pathway; however, sequestration of these intermediates for biofuel production limits the cell’s ability to produce glyoxylate, a metabolite required to power carbon assimilation. A second glyoxylate production pathway, the Oxalyl‐CoA Reductase pathway, exists but is mainly operational during oxalate growth. Use of this pathway may compensate for loss of glyoxylate during 1‐butanol production but an understanding of pathway regulation is needed to enhance cell engineering efforts. Expression studies showed upregulation of this pathway by exogenous oxalate and glyoxylate and QscR was identified as a potential transcriptional activator using transposon mutagenesis. Subsequent studies showed direct binding to the Oxalyl‐CoA Reductase pathway promoter by QscR. Interestingly, growth with succinate prevented upregulation in vivo and neither oxalate nor glyoxylate affected binding in vitro, consistent with the presence of an additional regulator. Future studies will use transposon mutagenesis to identify mutations that cause an increase or decrease in fluorescence from an Oxalyl‐CoA Reductase pathway promoter fusion identifying any additional transcriptional regulators. These studies will aid our ability to engineer M. extorquens to use the alternative glyoxylate production pathway to supply cellular glyoxylate, freeing the Ethylmalonyl‐CoA pathway for biofuel and bioplastic production.Grant Funding Source: Supported by California State University Program for Education and Research in Biotechnology (CSUPERB) New Investigator Award and Louis Stokes Alliances for Minority Participation (LSAMP)
Salvage of Coenzyme A breakdown products by the STM4195 gene product in Salmonella enterica (LB276)
Authors: Ernst Dustin , Downs Diana 
Published: June 2021

Coenzyme A (CoA) is an essential cofactor required in all living organisms, where it functions primarily as an acyl‐group carrier and activator of carbonyl groups. The biosynthetic pathway generating CoA from pantothenic acid is universally conserved in prokaryotes and eukaryotes. Pantothenate is generated in a pathway that culminates in the condensation of pantoate and β‐alanine. Animals are unable to synthesize pantothenate and must salvage it, or other metabolic intermediates, to generate CoA. In Salmonella enterica, intermediates up to and including pantothenate can be fed exogenously to support CoA biosynthesis. Previous studies have shown that intermediates in CoA biosynthesis beyond pantothenate are not readily salvaged by S. enterica. The work here found that a degradation product of Coenzyme A, derived from boiling CoA, can bypass a genetic block in the final step of pantothenate biosynthesis. The data also showed that the STM4195 gene product is required to salvage this unknown metabolite, and also transports pantothenate precursors, ketopantoate and pantoate. STM4195 is a member of the sodium bile acid symporter family (SBF) of proteins. HPLC separation of a preparation of boiled CoA defined a peak with biological activity, and mass spectrometry and NMR approaches are being used to identify the compound. The results herein provide the first evidence for an active mechanism to transport ketopantoate and pantoate, and further demonstrate a role for STM4195 in the salvage of a unique CoA breakdown product.Grant Funding Source: Supported by NIH GM095837
Bro1 contributes to cargo selection during Multivesicular Body (MVB) sorting (LB283)
Authors: Dean Shirley , Davies Brian , Azmi Ishara , Payne Johanna , Katzmann David 
Published: June 2021

A key step in the lysosomal degradation of membrane proteins is sorting into the Multivesicular Body (MVB), a process wherein a region of the endosomal membrane buds into the lumen of the endosome to form an intraluminal vesicle (ILV). This sorting process is mediated by Endosomal Sorting Complexes Required for Transport (ESCRT‐0, I, II and III). Ubiquitination of MVB cargo and recognition of Ub‐cargo by ESCRT‐0, I and II permit entry into the MVB pathway; however, Ub is typically removed from the MVB cargo prior to cargo inclusion into the ILV. The coordination of deubiquination of MVB cargo with ESCRT‐III disassembly and ILV formation are not clear. Bro1 appears to play a critical role in coordinating cargo de‐ubiquitination and ILV formation through interactions with ESCRT machinery, the Ub peptidase, Doa4, ubiquitin‐modified cargo, and regulation of the ESCRT‐III‐associated AAA ATPase Vps4. These data suggest that members of the Bro1 family may be similarly impacting ESCRT‐dependent processes in a number of contexts.Grant Funding Source: NIH R01 GM07302
Does alternating dissection effect student performance on practical examinations (LB29)
Authors: Boehm Karl , Fraticelli Nicole , Jordan Rebecca , Riccardi Margaret , Houser Jeremy , Kondrashov Peter 
Published: June 2021

In the process of restructuring the Gross Anatomy course for first‐year osteopathic medical students a new lab model was introduced, with teams alternately performing the dissection of midline regions. To evaluate the effectiveness of the new model, we compared practical examination performance from 2 years based on whether or not they were in the dissecting or non‐dissecting lab group. Each practical question was assigned a body region and tissue type. A random intercept logistic regression model was fit to the data and the outcome variable was whether or not the student correctly identified the structure. The random effects were the students, each question asked during the practical exams and the assigned table number. The predictor variables included if the student had dissected the structure in an assigned lab, the region the anatomical structure is found and tissue type.Statistical analysis of the data indicates that there was no significant difference (p=.34) in student performance between the dissecting and non‐dissecting groups as a whole. Detailed comparisons indicated that in certain areas it might be more beneficial to have the whole class dissecting, as students who were not present for dissection of pelvic and abdominal neurovasculature did not perform as well on these questions. In conclusion students are capable of mastering the lab material in this curricular model.
Gestational weight gain in women receiving prenatal care in rural midwifery clinics (LB313)
Authors: Gonzales‐Pacheco Diana , Cox Kim , Murray‐Krezan Cristina 
Published: June 2021

Background: Gestational weight gain (GWG) impacts maternal and fetal outcomes and is the most significant predictor of postpartum weight retention. In 2009, the Institute of Medicine (IOM) released revised GWG recommendations on prepregnancy body mass index (BMI).Objective: To describe GWG in women receiving prenatal care provided by Certified Nurse Midwives (CNM) and compare their weight changes to the 2009 IOM GWG recommendations.Design: Secondary data analysis of GWG in pregnant women who participated in a pilot study comparing postpartum weight changes in women receiving group prenatal care vs. individual care provided by CNMs. Women were grouped by pre‐gravid BMI according to World Health Organization categories.Results: Data for 18 women were included in the analysis. Mean age was 25.17 + 4.97 years and mean gravidity was 1.94 + 1.2. For women in group care, median weight gain was 16.8 kg (Q1‐Q3: 13.6‐27.7 kg) vs. 12.3 kg (Q1‐Q3: 10.0‐22.6 Kg) for women in individual care (p= 0.56). By pregravid BMI, the group care cohort’s median weight gain was 16.4 kg (normal weight), 30.5 kg (overweight), and 15.1 kg (obese). For the individual care cohort, median weight gain by pregravid BMI was 18.1 kg (normal weight), 9.9 kg (overweight), and 11.4 kg (obese).Conclusions: Median weight gain between groups was not significantly different. Median weight gain by BMI category exceeded the 2009 IOM recommendations with the exception of the overweight individual care group.Grant Funding Source: Supported by the National Center for Research Resources and the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number 8Ul1TR000041
Lactation influences changes in volumetric bone mineral density and microstructure postpartum (LB315)
Authors: Brembeck Petra , Lorentzon Mattias , Ohlsson Claes , Winkvist Anna , Augustin Hanna 
Published: June 2021

Lactation decreases areal bone mineral density (BMD) during the first months postpartum and replenishment occurs especially after lactation has ceased. The aim was to study changes in volumetric BMD (vBMD) and microstructure of cortical and trabecular bone in tibia postpartum using high resolution peripheral quantitative computer tomography (HR‐pQCT). Seventy‐nine postpartum women and 19 controls were measured at baseline (0.5 mo after delivery), 4 and 12 mo thereafter. Lactation and body weight were registered. Women were categorized into 1=controls, and in relation to duration of lactation 2(0‐3.9 mo), 3(4‐8.9 mo), and 4(蠅9 mo). Significant changes were found only in group 3 and 4 in cortical vBMD, area and thickness, and trabecular area (P<0.05). At 4 months, cortical vBMD (3=‐0.3%, 4=‐0.5%), cortical area (3=‐0.7%, 4=‐1.5%) and cortical thickness (4=‐1.5%) decreased, and trabecular area increased (3=+0.1%, 4=+0.3%) compared to baseline. Still at 12 months, cortical vBMD (3=‐0.4%, 4=‐1.2%), cortical area (4=‐2.5%) and cortical thickness (4=‐2.5%) were lower than baseline, while trabecular area was higher than baseline in group 4 (+0.3%). In conclusion, longer duration of lactation is related to significant decreases in tibia cortical vBMD during the first year postpartum. There may be a cortical trabecularization, since cortical area decreases and trabecular area increases during lactation.Grant Funding Source: This study was supported by the Centre for Environment and Sustainability, Swedish Nutrition Foundation and the Swedish Research Council Formas.
Diet cost and risk of diabetes: does diet cost mediate the association between socioeconomic status and diabetes risk? (LB355)
Authors: Rehm Colin , Tinker Lesley , LaCroix Andrea , Wang Ching‐Yun , Drewnowski Adam 
Published: June 2021

Diet cost has been implicated as an important determinant of diet quality. No studies have evaluated whether diet cost is associated with diabetes risk. A prospective cohort study among 47,683 post‐menopausal women 49‐64 years using data from the Observational Study and the control arm of the Dietary Modification trial of the Women’s Health Initiative was conducted to evaluate the association between diet cost and diabetes risk. This study had two aims: 1) to examine the association between diet cost and diabetes and; 2) to determine the extent by which the socioeconomic gradient in diabetes was explained by diet cost. To estimate diet costs, a national food price database was linked to a food frequency questionnaire. Energy‐adjusted diet costs were log‐transformed to evaluate the impact of a relative increase in diet costs. The outcome was treated type‐2 diabetes. Cox proportional hazards models examined the association between diet cost and diabetes after adjusting for covariates associated with diet cost and/or diabetes (e.g., age, smoking, family history, physical activity, body mass index, and income among others). 2,174 new cases of diabetes were observed over 8 years of follow‐up. After adjusting for covariates, a 50% increase in energy‐adjusted diet costs (e.g., from $4/d‐$6/d) was associated with a 14% reduced risk of diabetes (hazard ratio 0.86; 95% CI 0.78, 0.94). A strong socioeconomic gradient in diabetes risk was also observed. Diet cost explained 15‐19% of the association between income/education and diabetes. As the first report to observe an association between diet cost and diabetes these results need to be compared to other studies. Examining upstream factors associated with risk of diabetes expands our understanding of the consequences of the contemporary food environment on health.Grant Funding Source: Supported by NIDDK R21‐DK‐085406; The WHI program is supported by contracts from the National Heart, Lung and Blood Institute, NIH.
Differential effects of whey protein concentrate and hydrolyzed whey/egg protein blends on post‐prandial markers of insulin signaling and skeletal muscle anabolism in rats (LB439)
Authors: Mobley C. Brooks , Fox Carlton , Pascoe Corrie , Ferguson Brian , Healy James , Lockwood Christopher , Roberts Michael 
Published: June 2021

We examined how gavage‐feeding the following dietary proteins (10 human eq. g dissolved in 1 ml of water) acutely affected skeletal muscle insulin signaling and anabolism markers 90‐ and 180‐min post‐feeding in male Wistar rats (~250 g) fasted overnight. Supplementation included: a) 80% whey protein concentrate (WPC, n = 15); b) 70% hydrolyzed whey + 30% hydrolyzed egg albumin (70W/30E, n = 15); c) 50W/50E (n = 15); d) 30W/70E (n = 15); and e) 1 ml of water with no protein (fasted, n = 14). Immunoblotting results from mixed gastrocnemius muscles revealed: 1) rats fed WPC, 70W/30E and 50W/50E typically presented an in phospho‐Akt (Thr308) compared to 30W/70E and fasting rats (p < 0.05); 2) markers of mTOR signaling (p‐mTOR Ser2481, p‐p70s6k Thr389, p‐rpS6 Ser235/236) were typically greater in rats fed 70W/30E and 50W/50E compared to WPC, 30W/70E and fasting rats (p < 0.05); and 3) 180‐min post‐feeding muscle protein synthesis (MPS) analysis demonstrated that, compared to fasted rats, WPC feeding increased MPS by 94% (p < 0.05), 70W/30E feeding increased MPS by 74% (p = 0.065) and 50W/50E and 30W/70E feedings non‐significantly increased MPS by 25% and 34%, respectively (p > 0.05). In summary, 70W/30E feeding elicits similar acute post‐feeding responses to that of WPC. However, replacing hydrolyzed whey with a higher percentage of hydrolyzed egg albumin attenuated these responses.Source of Research Support: 4LIFE Research, Inc.
A high‐throughput, fluorescence‐based cholesterol efflux assay (LB44)
Authors: Saddar Sonika , Kothambawala Tasnim , Chacos Nick , Saini Preeti , Tchaga Grigoriy , Yan Gordon 
Published: June 2021

Numerous studies have established a negative correlation between cellular cholesterol efflux and cardiovascular disease.Cholesterol efflux from peripheral tissues and cells by a process called Reverse Cholesterol Transport (RCT). RCT allows extrahepatic cells, including macrophage‐derived foam cells in arterial atherosclerotic plaque, to transport excessive cholesterol back to the liver for bile acid synthesis and excretion, thus lowering the peripheral lipid burden. Standard protocols for quantitation of cholesterol efflux involve labeling cells with tritiated cholesterol and measuring the release of labeled sterol in a 12 or 24 well plate. This protocol, however is not ideal for high‐throughput screening of large numbers of serum samples or screening compounds/small molecules for their effect on cholesterol efflux.We report here a simple, convenient and high‐throughput cell‐based assay using fluorescently‐labeled cholesterol in a 96‐well plate format. Our results demonstrate that the percentage cholesterol efflux elicited by 2% ApoB‐depleted normal human serum in J774 macrophage cells is comparable to those published in literature. Our assay method utilizes a positive control for this assay which induces efflux independent of any receptor, that is used as a measure of the reliability of the assay. This assay is highly reproducible with low inter‐assay variability (蠄 10%). We demonstrate that this is an efficient method of measuring cholesterol efflux in a cell‐based assay. The increased sensitivity of the assay coupled with its high‐throughput screening capability enables scientists to screen large libraries of reagents and serum samples and provides a valuable tool for drug discovery in the field of cardiovascular disease research.