bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determining Nitric Oxide‐induced Conformational Changes in Soluble Guanylate Cyclase using Lanthanide‐based Resonance Energy Transfer
Authors: Lopez Kyle Eric , Weichsel Andrzej , Montfort William R 
Published: November 2023

Nitric Oxide (NO) is a crucial signaling molecule involved in vasodilation and regulation of blood pressure. A key target for NO signaling is soluble guanylate cyclase (sGC), a heterodimeric protein that upon NO binding converts GTP to a secondary messenger, cGMP. NO binding at the ferrous heme of the beta subunit of the sGC dimer induces a conformational change in the protein resulting in increased catalysis and increased production of cGMP. Attempts to enhance the enzymatic activity of sGC by drugs tartegeting cardiovascular disease are hindered by a lack of information on the mechanism of this allosteric regulation and the structure of the protein. One method that can provide a better understanding of conformational changes in sGC is lanthanide‐based resonance energy transfer (LRET). This technique monitors luminescence of terbium ion (Tb3+) bound to a lanthanide‐binding tag (LBT) inserted into a protein sequence. We have designed several sGC constructs with an LBT tag at different locations in the protein in addition to 6‐His‐tags, which can bind transition metals and quench luminescence of the LBT‐bound Tb3+. Rates for Tb3+ luminescence decay upon quenching by Cu2+ bound to the 6‐His‐tag were used to calculate the distance between the metal ions using the Förster equation. Our preliminary conclusions from the LRET experiments are: (i) Tb luminescence decays with a bi‐exponential time course and a rate constant for the slow phase of ~2.3 ms; (ii) significant quenching of Tb luminescence is observed in the presence of millimolar concentrations of Cu2+; (iii) a calculated distance of ~23 Å between Tb and Cu ions in construct NT19_LBT2 suggests the C and N termini lie close to one another in the protein. Future experiments will probe how these distances change upon ligand binding and the distances between the LBT tags and a candidate drug with quenching fluorophore. These decay times and distances will lead to greater understanding of sGC structure and dynamics and will aid in the design of new drugs to treat cardiovascular disease through allosteric stimulation of sGC.Support or Funding InformationNIH MARC Training Grant T34 GM08718, AHA 14GRNT2008006, NIH R01 GM117357, Ironwood Pharmaceuticals
Detection of urinary short‐chain dicarboxylic acids by GC/MS as a measurement of oxidative stress and mitochondrial function in Alzheimer's Disease
Authors: Castor Katherine J , Shenoi Sonia , Rowshan Kiana , Applegate Sarah , Fonteh Alfred N , Harrington Michael G 
Published: November 2023

ObjectivesTo detect short‐chain dicarboxylic acids (DCAs) in urine from an aging cohort and determine whether they can be used as a measure of oxidative stress and mitochondrial function in Alzheimer's disease (AD).MethodsWe have developed a method to extract short‐chain DCAs from acidified urine using ethyl acetate, followed by separation, detection and quantification using gas chromatography coupled to isotope dilution negative ion chemical ionization mass spectrometry. Urine was obtained from volunteer study participants aged ≥ 75 years who have been classified in the following categories: cognitively normal (n = 59), mild cognitive impairment (n = 40), dementia of the AD type (n = 26), and other dementias (n = 10). Total DCA concentrations were normalized to creatinine and total protein levels in the urine and subjected to statistical analysis and correlations to established cerebrospinal fluid (CSF) biomarkers of AD (β‐amyloid and Tau protein levels).ResultsWe can detect C3‐C10 DCAs by our method with a linear range between ≤ 0.20 – 100 ng and have determined that even chain DCAs make up 65–75% of the total with the highest percentage coming from succinic acid (C4, 30–50%). A negative correlation was found between succinic acid and Tau protein levels (P < 0.001). Additionally, a negative correlation was found between total DCA concentration in cognitively normal volunteers and their ®‐amyloid CSF levels while total DCA levels did not differ between groups.ConclusionsMetabolic pathways such as oxidation and energy production that are associated with DCA metabolism may be involved in amyloid and Tau formation in normal aging but not in AD. This sensitive and specific method can be used to detect DCAs in biological fluids in disease biomarker studies and to determine pathological correlates.Support or Funding InformationResearch supported by the L.K. Whittier and Helen Posthuma Foundations
In Vivo Evaluation of Butyrate, a Histone Deacetylase Inhibitor Effect on Lipid Profile of Apo E−/− Mice Fed on Atherogenic Diet
Authors: Gray Marian L , Alatawi Abdullah , Alshaman Reem , Mathew Omana , Ranganna Kasturi , Yousefipour Zivar , Milton Shirlette , Chelliah Selvam 
Published: November 2023

A large body of evidence indicates diets high in fiber are correlated with lower incidences of adverse cardiovascular events including atherosclerotic vascular disease by cholesterol lowering effects. Although, butyrate, a natural product of dietary fiber fermentation produced in the colon appears to have a role in the cardio protective effects of fiber, its influence on hyperlipidemic conditions is not clear. In the present study, ApoE−/− knockout atherogenic mouse model fed on non‐atherogenic or atherogenic diets were treated with or without butyrate (50mg/kg, ip, six days/week; N=8 animals/group) for 18 weeks to determine the effect of butyrate on atherogenic diet related hyperlipidemic state. All animals were handled and treated by strictly following the Institutional guidelines approved by the Institutional Animal Care and Use Committee, there were no mortalities and animals were not under any stress. At the end of treatment, blood samples were collected for lipid analysis. The results indicate that chronic butyrate treatment causes no significant changes in overall lipid profiles both in groups fed on non‐atherogenic and atherogenic diet compared to their respective no butyrate treatment controls. However, butyrate treatment significantly reduced body weights of animals that were fed on atherogenic diet compared to the weights of animals fed on non‐atherogenic diet. The results appears to show that butyrate exerts a potential protective role in modulating vascular health by promoting lower body weight even with no change in atherogenic diet induced hyperlipidemic state. It is possible other mechanism(s) may be playing a role in butyrate‐induced lower body weights of animals fed with atherogenic diet.Support or Funding InformationThis study was made possible, in part, by research infrastructure support from grant number 2G12MD007605 from the NIMHD/NIH
Transcriptional Regulation of OLE1 expression in Saccharomyces cerevisiae is Dependent on Fatty Acid Species
Authors: Schriemer Clara , McDonough Virginia 
Published: November 2023

The OLE1 gene in Saccharomyces cerevisiae encodes the Δ9 desaturase, which inserts a double bond in saturated fatty acids to create unsaturated fatty acids (UFAs). OLE1 expression is controlled in part through the transcriptional regulators Mga2p and Spt23p in response to supply of UFAs. We investigated whether the regulation was consistent in response to varied UFA type and concentration. We found that in wild type cells, reporter gene assays show a stronger decrease in expression of OLE1 when fed 16:1Δ9 or 18:2Δ9, 12 as opposed to 18:1Δ9 or 17:1Δ10. Concentration of the fed fatty acid also impacted the regulation of OLE1 with higher levels of each UFA impacting expression to a greater degree. Fatty acid profiles of wild type cells show cells accumulate a higher concentration of 16:1Δ9 and 18:2Δ9, 12 than fed 18:1Δ9 or 17:1Δ10. This leads to the conclusion that the expression of OLE1 is dependent both on properties of fed fatty acids and the amount in the cell. While our initial hypothesis was that OLE1 is regulated in response to membrane fluidity, subsequent work does not support that idea. Conditions that would affect membrane fluidity (besides UFA species and amount), such as growth temperature and saturated or trans fatty acid supplementation do not regulate OLE1 in the direction predicted by fluidity changes. Recently our lab has isolated a mutant that is deficient in regulation of OLE1, called nro2 (no regulation of OLE1). The signaling mechanism for the NRO2 protein's action is unknown. Tests using the OLE1 promoter‐reporter gene constructs suggest that Nro2p responds more strongly to the fatty acids 16:1Δ9 and 18:2Δ9, 12, than 18:1Δ9 and 17:1Δ10. Efforts are currently underway to clone and identify NRO2.Support or Funding InformationNSF‐REU DBI‐0754293
Characterization of CoQ‐Synthome proteins and binding partners, their phosphorylation status, and regulation
Authors: Awad Agape M. , Whitelegge Julian P. , Srinivasan Chandra , Sondej Melissa A. , Clarke Catherine F. 
Published: November 2023

Coenzyme Q (also termed ubiquinone or Q) is an electron carrier in the mitochondrial respiratory chain that functions as an essential component in energy metabolism processes. Q transfers electrons from NADH and succinate to cytochrome c, a heme protein, and also serves as a vital lipid soluble antioxidant. Sufficient de novo Q biosynthesis is crucial for human health. Saccharomyces cerevisiae (baker's yeast) serves as an excellent model for studies on Q because of its powerful molecular genetics and its close homology to human Q biosynthesis and function. Coenzyme Q biosynthesis in S. cerevisiae (which makes Q6 with six isoprene units, versus Q10 in humans with ten isoprene units) takes place in the mitochondria and, currently, thirteen known mitochondrial proteins are responsible for facilitating this process—Coq1‐Coq11, Yah1 (ferredoxin), and Arh1 (ferredoxin reductase). Many of the proteins necessary for the biosynthesis of Q6 are associated together in a high molecular weight complex (termed the “CoQ‐Synthome”) that localizes in the inner mitochondrial membrane. Previous work indicated that the stability of the Q biosynthetic complex relied on the presence of Coq8, the putative kinase of the system. In addition, several Coq polypeptides are phosphorylated in a Coq8‐dependent manner, including Coq3, Coq5, and Coq7. Recent investigation has uncovered a physical association of Coq8 with Coq6, a flavin‐dependent monooxygenase and member of the CoQ‐synthome. Currently, we are investigating the role of phosphorylation, as it pertains to Q biosynthesis and regulation. Mainly, our research aims to: 1) investigate phosphorylation of Coq6 and associated Coq proteins, 2) conduct site‐directed mutagenesis of potential Coq6 phosphorylated residues and assess their effects on the content of Q/Q‐intermediates and yeast growth phenotypes, and 3) characterize the nature of the Coq6‐Coq8 specific interaction. Overall, Coq6 may be an ideal site for regulation since the monooxygenase activity of this protein occurs very early in the pathway, and generates potentially toxic catechol‐containing Q‐intermediates. Our objectives are to investigate this further and discover new information related to regulation by phosphorylation of the CoQ‐Synthome.Support or Funding InformationThis research was funded by NSF MCB 1330803.
Hypoxia suppresses fasting‐induced lipolysis in Caenorhabditis elegans
Authors: Han Ji Seul , Lee Jung Hyun , Kong Jinuk , Ji Yul , Kim Jae Bum 
Published: November 2023

Oxygen is an essential molecule in the regulation of energy homeostasis. Particularly, oxygen is required for beta‐oxidation and mitochondrial respiration to produce ATP. Thus, it appears that aerobic organisms may have adaptive mechanisms to modulate lipid metabolism in response to changes in oxygen availability. In this study, we have demonstrated that hypoxic condition suppresses fasting‐induced lipolysis in the intestinal cells of C. elegans. Similarly, hypoxia also suppressed PKA‐mediated lipolysis of mammalian adipocytes, indicating that there might be evolutionarily conserved regulatory pathways for metabolic regulation upon oxygen. In fasted worms, we have identified hypoxia inducible factor‐1(HIF‐1) as one of key factors in the suppression of PKA‐mediated lipolysis. HIF‐1 induction by inhibition of von Hippel‐Lindau tumor suppressor‐1(VHL‐1), and egg laying defective‐9(EGL‐9) prevented decreased neutral lipid contents upon fasting. Additionally, we observed that suppression of stimulated lipolysis is also mediated by HIF‐1 independent pathway including mitochondrial dysfunction. Together, our data have suggested that the decrease of lipolysis under hypoxic condition is an adaptive response to prevent accumulation of futile free fatty acids which may preserve stored lipid metabolites for energy homeostasis.Support or Funding InformationThis work was supported by National Creative Research Initiative Program (2011‐0018312) funded by the ministry of Education, Science, and Technology (MEST). Ji Seul Han, Jinuk Kong, and Yul Ji were supported by the BK21 program, Korea.
Structural Characterization of the Lipid Storage Droplet Protein Perilipin 5
Authors: Huggins Pearson N , Bailey Hannah M , Tansey John T 
Published: November 2023

The ability of eukaryotic cells to maintain a reservoir of neutral lipids is critical in order maintain the plasma membrane, assist in the production of hormones, and to act as a source of energy if needed. Neutral lipids are stored within a structure known as a lipid droplet coated by members of the PAT family of lipid droplet proteins embedded within its lipid monolayer. The PAT family is comprised of perilipins 1–5 which have been shown to play a roles in regulating lipid droplet metabolism and the droplet's interaction with lipases. This work seeks to analyze a member of the PAT family, Perilipin 5, using proteolytic mapping in order to determine its domain topology which may provide clues to its function. We hypothesized that delipidation of cellular extracts using either detergents or lipases would alter the conformation of perilipin 5. This would alter the proteolytic cleavage patterns, indicating independently interacting domains of the protein and cleavage sites that were protected from proteases by being buried in lipid. Cellular lysates of Chinese Hamster Ovary cell lines expressing perilipin 5 were used as a source of protein. Following proteolysis, samples were analyzed by western blotting. Triton‐X‐100 treated samples had more cleavage sites than controls or Tween 20 treated samples. Collectively, these data show at least two major and four minor cleavage products resulting from proteolysis and suggest that perilipin 5 has at least two distinct domains.Support or Funding InformationThe Bert and Jane Horn endowed fund of Otterbein University.
Real Time Four Dimensional Measurement of cAMP Gradients in Pulmonary Microvascular Endothelial Cells
Authors: Rich Thomas , Annamdevula Naga , Sweat Rachel , Mayes Samuel , Favreau Peter , Britain Andrea , Nakhmani Arie , Leavesley Silas 
Published: November 2023

In the last 30 years data from a variety of studies have suggested that localized cAMP signals underlie signal specificity in a variety of cells and tissues. For example, in pulmonary microvascular endothelial cells (PMVECs) cAMP produced by plasma membrane‐localized adenylyl cyclase is endothelial barrier protective, whereas cAMP produced by both endogenous and heterologously‐expressed AC is barrier disruptive. However, subcellular cAMP gradients have been difficult to measure in living cells. Most single cell approaches for measuring cAMP levels rely upon protein‐based probes. Current protein‐based probes for measuring cAMP in single cells are based upon Förster resonance energy transfer (FRET) between donor and acceptor fluorophores attached to a cAMP binding protein. Binding of cAMP triggers a conformational change in the protein that reduces FRET efficiency. Recently, FRET measurements using these probes have been used to assess cAMP gradients in 1D (x) preparations (e.g., neuronal processes). However, FRET probes have notoriously low signal‐to‐noise ratios, prohibiting accurate assessment of the distribution of cAMP (FRET efficiency) in 2D (x, y) or 3D (x, y, z). In fact, low signal‐to‐noise ratios have effectively prohibited 3D FRET assessment in living cells. Thus, the vast majority of FRET measurements are conducted in 2D. Here, we used a FRET probe utilizing Turquoise and Venus, donor and acceptor, transfected into rat PMVECs to determine whether state of the art hyperspectral imaging and automated image analysis approaches allow measurement of FRET in 3D. We demonstrate that cAMP gradients are readily observed in PMVECs. Interestingly, observed cAMP gradients occurred primarily along an apical to basolateral vector. Thus, cAMP gradients would not have been observable using standard 2D approaches for imaging FRET signals. Stimulation of cAMP production using either 0.1 or 1.0 μM isoproterenol or 50 μM forskolin triggered an increase in overall cAMP as well as increases in the apical to basolateral cAMP gradients. These data clearly demonstrate that standing spatial cAMP gradients occur within PMVECs, and that agonist treatment alters the spatial distribution of cAMP. These data also demonstrate that state‐of‐the‐art hyperspectral imaging approaches will allow investigators to better establish the distributions of intracellular signals within cells as a functions of time. The ability to simultaneously assess multiple signaling pathways at specified locations throughout living cells will allow us to disentangle complex interactions within spatially distributed feedback networks.Support or Funding InformationThis work is supported by P01HL066299, S10RR027535, S10OD020149, and the Abraham Mitchell Cancer Research Fund.
An Evaluation of Prenatal Prescribed (P) and Non‐Prescribed (N‐P) Supplement Labels
Authors: Saldanha Leila , Dwyer Johanna , Andrews Karen , Bailen Richard , Betz Joseph , Costello Rebecca , Ershaw Abby , Gahche Jaime , Gusev Pavel , Han Fei , Hardy Constance , Saravala Sushma , Pehrsson Pamela 
Published: November 2023

In the U.S. a prenatal supplement can be bought off store shelves as dietary supplements (DS), and some are sold behind the counter by prescription. Our objective was to evaluate P and N‐P product labels, and determine if there were differences in nutrient composition and amount for eight nutrients (vitamins A and D, folic acid, iodine, calcium, iron, zinc, and DHA) with data form two publicly‐available databases, Dietary Supplement Label Database (DSLD) and DailyMed. We identified 82 P and 132 N‐P supplements, complete with images of product labels. We used the questions and key label elements identified by the DHHS Office of Inspector General (OIG) in two 2003 reports Dietary Supplement Labels, and An Assessment and Key Elements to evaluate P and N‐P product labels. N‐P contained more vitamins and minerals than P products. The mean number ±SE of vitamins and minerals in the NP were 11±0.2 and 8±0.3 and in P was 9±0.9 and 4±0.1, respectively. Declared label amounts of vitamin A, vitamin D, iodine and calcium were higher in the N‐P products. Folic acid was higher in the P products. The amounts of iron, iodine, and DHA were similar in P and N‐P products. Since P products do not fall under any distinct FDA‐regulated category, there are no specific regulations that govern the labeling of these products. However, the labels did provide a list of ingredients and their amounts consistent with FDA's guiding principle that labels should be truthful and not misleading. 100% of the N‐P but only 43% of the P supplements used the DS labeling format to declare nutrients on the label. The remaining (57%) of P products used an alternative format. 100% of the N‐P labels listed the mandatory DS label information. While labels did provide the required information, such as nutrients, nutrient amounts and dosage, <10% provided the additional information listed in the 2003 OIG key elements for a DS label report. Optional label information would possibly be useful for women taking prenatal supplements, such as recommended daily dose, minimum (and maximum) duration of time for which a supplement should be taken, interactions and contraindications, and possible side effects. Since some supplements contain levels of nutrients that are higher than the recommended levels for pregnant and lactating women, women should be encouraged to discuss their choice and use of an N‐P prenatal supplement with their healthcare provider.Support or Funding InformationOffice of Dietary Supplements, NIH
Predictors of early feeding practices in the STRONG Kids 2 cohort
Authors: Donovan Sharon Marie , Musaad Salma M. 
Published: November 2023

Human milk is the ideal form of nutrition for the developing infant, however, current rates of breastfeeding in the U.S. do not meet public health recommendations. The decision to breastfeed (BF) is influenced by physiological and psychosocial factors. Herein, the factors that contribute to early infant feeding decisions were investigated in STRONG Kids 2 (SK2), a 3‐year longitudinal birth cohort in Champaign County, Illinois. Analyses were restricted to mothers who have data at 12 months (n=212), of which 83% are White, 4% are Hispanic or Latino and 5% are Black. Maternal prepregnancy weight, child birthweight and route of delivery are obtained by parental report. Mother and infant weight and length were measured at week 6 and months 3 and 12. Married women comprised 83% of mothers, 7% were cohabiting and 8% were single. Prior to pregnancy, 24% and 26% of women were overweight or obese, respectively. 29% of infants were delivered by C‐section (planned or unplanned). Vaginal delivery was highest among those who were under/normal weight (80%) vs. overweight (71%) or obese (56%) prepregnancy (P=0.006). At 6 weeks, 68% of infants were BF, 13% were formula‐fed (FF) and 18% were fed both human milk and infant formula (CF). BF was 67% at 3 mos and 41% at 12 mos. The % BF increased with maternal education from high school (43%) to some college (53%) to college graduate/post graduate work (77%) (P<0.001). The % BF was higher for under/normal weight (78%) and overweight (73%) mothers than for mothers who were obese at prepregnancy (56%) (P=0.0249). BF rate was also higher in mothers who delivered vaginally (75%) compared to planned (57%) or unplanned C section (50%) (P=0.0141). Single mothers were more likely to formula feed than breastfeed (50% vs. 39%), while the reverse was seen among married mothers (9% vs. 72%) (P<0.0001). Lastly, reasons for discontinuing BF or pumping milk by week 6 were investigated. The most common stated reasons were: “I didn't have enough milk” (81%); “Breast milk alone did not satisfy my baby” (67%); and “I had trouble getting the milk flow to start” (57%). In summary, in the SK2 cohort, prepregnancy obesity, C‐section delivery, lower levels of education and being a single mother were associated with lower BF rates. The most common reasons for stopping BF prior to 6 weeks postpartum centered on perception of insufficient milk production. These modifiable factors represent targets for future educational programs and intervention studies to improve BF initiation and duration.Support or Funding Information(Supported by the Dairy Research Institute and Gerber Foundation).