bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformational Stability of SIKE and a Mimic of a Maximally Phosphorylated Form
Authors: Graham Sasha , Kim Seung‐Hwan , Bell Ellis , Bell Jessica
Published: November 2023

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure or stability of SIKE or the effects of phosphorylation. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. To determine the effects of phosphorylation on the conformational stability of the protein, SIKE and SIKE‐S6E (where the 6 phosphorylatable serines have been mutated to the phosphomimic, glutamate) have been used in Fluorescence based Thermal Shift assays and chemical denaturation studies using a comparison of Urea and Guanidine Hydrochloride denaturation to examine the relative contributions of charge interactions in the overall stability of the two molecules. Results of direct experiment are being compared with Molecular Dynamics simulations using homology models created using Phyre2 for SIKE, SIKE S6E and a form with six phosphoserines built into the model.

Support or Funding Information

This work was supported by NIH grant R21A1 107447 to JKB
Defining carbohydrate binding of glucan phosphatases via Affinity gel electrophoresis
Authors: Auger Kyle , Raththagala Madushi , Wilkens Casper , Svensson Birte , Gentry Matthew
Published: November 2023

In plants, starch is the energy storage molecule that is readily broken down when needed. In animals, glycogen is the molecule that is used for energy storage. Both molecules are comprised of α‐1, 4 linked glucose polymer chains and α‐1,6 glucose branches that are tightly compacted. Our lab has determined the x‐ray crystal structures of both plant and human glucan phosphatases and their enzymatic mechanisms. Despite this progress, we lacked the techniques to quickly and efficiently quantify their glucan phosphatase affinities for different substrates. The main objective of this study was to determine a technique to measure carbohydrate binding quickly and efficiently. We established a protocol to reproducibly and quantitatively measure the binding of the enzymes to glucans utilizing Affinity Gel Electrophoresis (AGE). The results show that the various glucan phosphatases possess differing abilities to bind to different glucan substrates. The plant glucan phosphatase SEX4 possesses a 50 fold higher affinity for the glucan amylopectin than LSF2, while SEX4 only possessed a 3 fold higher affinity for the glucan amylose than LSF2. Mutations were made to the various domains of the plant and animal glucan phosphatases to determine which regions of the enzyme are most necessary for binding.
UDCA Prevents 17α‐Ethinylestradiol‐induced Cholestatic Injury via Inhibiting AMP‐activated Protein Kinase Activation
Authors: Li Xiaojiaoyang , Liu Runping , Yuan Zihang , Jiang Zhenzhou , Zhang Luyong , Zhou Huiping
Published: November 2023

Introduction

Cholestasis, as a common liver injury in clinic, increases the risk of other malignant hepatobiliary diseases such as hepatic carcinoma and cholangiocarcinoma. Estrogen‐induced cholestasis occurred in many susceptible women due to pregnancy or hormone therapy. Previous studies from our group and others suggest that AMP‐activated protein kinase (AMPK) induced phosphorylation of farnesoid X receptor (FXR), a key nuclear receptor involved in regulating bile acid homeostasis represents an important underlying mechanism of estrogen‐induced cholestasis. Most recent clinical study indicated that ursodeoxycholic acid (UDCA) monotherapy is effective in the treatment of intrahepatic cholestasis of pregnancy. However, the functional molecular/cellular mechanisms of UDCA‐mediated protective effect against estrogen‐induced cholestasis remains unclear and is the focus of this study.

Methods

The sandwich‐cultured rat primary hepatocytes and 17α‐ethinylestradiol (EE)‐induced rat cholestasis models were used. The protein levels were determined by Western blot analysis. The mRNA levels of targeted genes were determined by real‐time RT‐PCR. Immunofluorescent staining was used to detect canalicular structure of liver.

Results

EE significantly suppressed the expression of FXR and bile acid transporters via activation of AMPK signaling pathways. In rat primary hepatocytes, UDCA significantly inhibited EE‐induced AMPK activation and down‐regulation of FXR and bile acid transporters. UDCA rapidly and dose‐dependently induced phosphorylation of protein kinase A (PKA), which was correlated to its effect on inhibition of EE‐induced AMPK activation and down‐regulation of FXR expression. H89, a PKA inhibitor, blocked UDCA‐induced inhibitory effect on EE‐induced AMPK activation and downregulation of FXR and bile acid transporters. Furthermore, UDCA inhibited EE‐induced AMPK activation and downregulation of FXR and bile acids transporters in vivo rat cholestasis models.

Conclusion

UDCA protected hepatocytes from EE‐induced cholestatic injury via inhibiting AMPK activation. Our data also suggested that activation of PKA may also contribute to UDCA‐mediated protective effects.

Support or Funding Information

This poster presentation was supported by the Fundamental Research Funds for the Central Universities. And this study was supported by National Natural Science Foundation of China Grants (81320108029).
Quantification by ELISA of the Levels of DNA Topoisomerases I, IIα, and IIβ in the Nuclei of HuT 78 Cells Treated with Interleukin‐2
Authors: Foglesong Paul David , Nair R. Sajana , Gupta Nikita K.
Published: November 2023

The activities of DNA topoisomerase I, DNA topoisomerase IIα, and DNA topoisomerase IIβ are stimulated in three transient peaks in nuclear extracts of HuT 78 cells, a human T cell lymphoma cell line, within 12 hrs after treatment of the cells with 1,000 units of interleukin‐2 (IL‐2)/ml. This study examined if the changes in the specific activities of these three enzymes was due to variations in the amounts of the proteins. The amounts of each of these enzymes in the nuclear extracts were determined by quantitative ELISAs using specific monoclonal antibodies, and the results were compared to the specific activities of the enzymes. There is no direct correspondence in the amounts of DNA topoisomerase I or DNA topoisomerase IIβ with their specific activities in the 12 hours following IL‐2 treatment of the cells. Therefore, increased synthesis/degradation of these proteins cannot explain the observed variations in their specific activities in the nuclear extracts of HuT 78 cells treated with IL‐2. However, the amount of DNA topoisomerase IIα increased after treatment with IL‐2 with a peak observed at 6 hours after IL‐2, but the amounts of the enzyme did not correlate with its specific activities. These results indicate that the mechanism for the observed activation of DNA topoisomerase I and DNA topoisomerase IIβ is not synthesis/degradation of the proteins, but rather post‐translational modification. However, the changes in the activities of DNA topoisomerase IIα in the extracts appear to be due to both increased synthesis/degradation of that enzyme and also post‐translational modification. It is hypothesized that phosphorylation of these proteins by protein kinases activated by IL‐2 is the primary mechanism for the observed variations in their specific activities.

Support or Funding Information

This work was supported by grants from the William & Ella Owens Medical Research Foundation and University of the Incarnate Word.
Glutathione S‐Transferase Expression in Mice (Mus musculus) Exposed in utero to Atrazine
Authors: Hafner Megan , Martin Debra
Published: November 2023

Atrazine is a widely used herbicide on crops in the USA and has been found in ground water and streams where it does not easily breakdown. Although the EPA has set the annual safe concentration of atrazine in water systems to be 3 ppb, levels above this have been measured leading to concerns about environmental affects on health. In mammals, the liver is the organ that helps to breakdown environmental toxins, such as atrazine. One of the proteins of interest in the liver is glutathione S‐transferase (GST) because of its ability to target xenobiotics. In this study, GST levels were determined by Immunoblots for liver and serum samples of new born mice (Mus musculus) pups who were exposed to 0, 3, and 30 ppb atrazine in utero (n= >20 per group). Results demonstrated that there was a decreasing trend in liver GST levels as exposure to Atrazine in utero increased suggesting potential liver damage. This was not supported by the serum samples since they also showed a significant decrease in GST level as exposure to Atrazine in utero increased.

Support or Funding Information

R&D Systems

Saint Mary's University of Minnesota
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 Information

NIH 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

Objectives

To 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).

Methods

We 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).

Results

We 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.

Conclusions

Metabolic 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 Information

Research 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 Information

This study was made possible, in part, by research infrastructure support from grant number 2G12MD007605 from the NIMHD/NIH
A novel LysoPhosphatidylcholine Acyl Transferase Activity is Expressed by Peroxiredoxin 6
Authors: Fisher Aron B , Dodia Chandra , Sorokina Elena , Li Haito , Zhou Suiping , Raabe Tobias , Feinstein Sheldon
Published: November 2023

The phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6) has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysopholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for choline as the lysophospholipid head group and for palmitoyl CoA as the fatty acyl substrate. The calculated kinetic constants for acylation were Km 18 mM and Vmax 30 nmol/min/mg protein; the Vmax was increased 25 fold by phosphorylation of the protein while Km was unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl PC indicated that lysoPC generated by Prdx6 PLA2 activity remains bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA2 and LPCAT activities for the remodeling pathway of phosphatidylcholine synthesis or for repair of membrane lipid peroxidation.
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 Information

NSF‐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 Information

This 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 Information

This 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.
Evidence of a Perilipin‐5 Splice Variant
Authors: Ranzau Brodie L. , DuBreuil Daniel M , Hubbell Theresa , Tansey John T
Published: November 2023

The large diversity of proteins found within animals are made possible through alternative splicing of mRNA. This has been shown to occur within the perilipin family of lipid storage droplet proteins in both perilipins 1 and 3. In the course of our studies on perilipin 5, we observed a signal on an immunoblot corresponding to an approximately 35 kDa protein. We have hypothesized that this is a splice variant of perilipin 5 that we have termed perilipin 5b. The objective of the current study is to confirm the existence of this splice variant and characterize the protein it encodes. Western blots indicate that perilipin 5b is present in C2C12 cultured myoblasts and in heart and liver tissue from fed and fasted mice. Highest levels of the protein were present in heart tissue from fed mice. Analysis of murine genomic DNA sequences reveals stop codons found in the introns between exons 7 and 8 and 8 and 9. Failure to splice out either of these introns could give rise to a protein of approximately 35 kDa. Reverse trancriptase PCR yields products that are consistent with a splice variant. Finally, analysis of the murine genbank EST library yields several transcripts that indicate a read through event into an intron has occurred. Collectively these data indicate that, like other family members, perilipin 5 undergoes differential splicing to generate multiple protein products.
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 Information

The 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 Information

This work is supported by P01HL066299, S10RR027535, S10OD020149, and the Abraham Mitchell Cancer Research Fund.
Ellagic acid mediated attenuation of store operated calcium entry alters cytokine expression in Jurkat T cells
Authors: Kaul Shashank , Murphy Matthew , Morrill Jacob , Mathias Clinton , Thomas David , Bose Diptiman D
Published: November 2023

Calcium is a ubiquitous second messenger regulating a myriad of cellular processes including proliferation, gene expression, and apoptosis. Agents that can modulate Ca2+ homeostasis are being extensively explored as potential chemotherapeutic agents. Ellagic acid (EA), a polyphenolic compound found in many fruits and plant extracts, has been known to possess anticarcinogenic and anti‐inflammatory properties. EA is thought to modulate Ca2+ homeostasis by enhancing sarcoplasmic endoplasmic reticulum ATPase pump (SERCA) activity, however effects of EA on Ca2+ signaling is not clearly understood. In order to study this relationship, we measured changes in Ca2+ transients in Jurkat T‐cells pretreated with EA (30 μM, 12 h). EA‐treated Jurkat T cells were challenged with SERCA inhibitor, thasipargin (TG) (2 μM). The amplitude of TG‐induced Ca2+ transient in the EA‐treated cells was 30% lower than vehicle control cells. The depletion of ER‐stores signals activation of store‐operated Ca2+ entry (SOCE) channels, resulting in Ca2+ influx. EA‐pretreatment induced a concentration‐dependent decrease in store‐operated Ca2+ entry in Jurkat T‐cells. Since expression of certain cytokines is directly dependent on Ca2+ influx, we measured the levels of IL‐2 and INFγ in EA‐treated Jurkat T cells. Indeed, EA‐treated resulted in a significant decrease in IL‐2 and INFγ expression. These results link EA‐mediated attenuation of Ca2+ influx to altered cytokine expression. These results indicate the anti‐inflammatory effects of EA could be attributed to its ability to attenuate store operated Ca2+ influx.
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 Information

Office 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).
Applying the HEI to Multiple Levels of the Food Stream
Authors: Krebs‐Smith Susan M. , Reedy Jill , Schap TusaRebecca E. , Subar Amy F. , Wilson Magdalena M.
Published: November 2023

An individual's food choices are influenced by a myriad of contextual factors, including what types of food are available to them where they live, work, and shop. Food availability, in turn, is influenced by many social, economic and other forces acting at multiple levels. The food stream refers to the flow of foods from agricultural production, through processing and distribution channels, to the food that ends up on our plates. Characterizing the quality of foods available at all “levels” along the food stream allows researchers to develop a better understanding of influences on consumer behavior.

The Healthy Eating Index (HEI) is a measure of diet quality, independent of quantity, which can be used to assess compliance with the Dietary Guidelines for Americans. The HEI is a valuable tool for characterizing quality of foods across levels of the food stream because it employs a universal set of standards which are density‐based. This presentation will demonstrate the application of the HEI to foods available, offered, sold, or consumed from various levels of the food stream. Levels vary widely in the overall diet quality and in the patterns of dietary quality, as shown by radar graphs. For example, typical grocery basket purchases generally receive higher scores on overall diet quality than the set of foods marketed through grocery store circulars, yet the pattern of HEI component scores for grocery baskets revealed lower quality scores for total protein foods and refined grains than the grocery store circulars.

The benefits of using the same index across multiple levels of the food stream can be realized with the HEI, especially if the requisite supporting databases are made available. Collectively, such studies could add to our understanding of the food stream, the influence of different levels on food available to consumers, and the potential impact of environmental and policy changes at each level.
Tailored nutrition intervention based on the transtheoretical model using technology convergence :application in child obesity prevention and management program
Authors: Hwang Ji‐Yun , Lee Ji Eun
Published: November 2023

The available technology such as smartphone applications (apps) and websites can be used to improve and intervene eating behaviors and food choices for obesity prevention and management in children although it is also thought to be a contributor to the obesity epidemic. Easy access to intervention and support with self‐monitoring of behaviors is important for obesity management. This study is to develop nutrition contents of apps aimed at modifying key nutrition and eating behaviors related to weight gain in young children including consumption of fruit and vegetables, sugar‐sweetened drinks, fast food, and snacks. The developmental process involved selecting and deciding on key theories of behavioral changes, collecting relevant guidelines, textbook contents, and educational materials, and developing nutrition messages based on transtheoretical models and educational contents for caregivers and teachers. Based on each stage of transtheoretical model and current eating pattern, 3,261 feedback massages for apps and 60 nutrition contents for website were developed. Qualitative feedback about feedback messages for apps and contents for websites was also obtained by teachers and professionals and quality control and regular updates were found to be main concerns for long‐term and effective use. Smartphone apps may be an innovative medium to deliver brief eating behavior changes directly to individuals, with potentially favorable cost‐effectiveness.

Support or Funding Information

This research was supported by the Civil research projects for solving social problems through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning (NRF‐2014M3C8A2A01070970).
Using university students for collecting population‐based diet records in urban and rural Mongolia
Authors: Choi Jeannie , Bromage Sabri , Ganmaa Davaasambuu
Published: November 2023

Study objective

The objective of this study was to assess food intake and dietary quality of healthy adults across the geographic and seasonal expanse of Mongolia using 3‐day weighed diet records collected by trained local medical and public health students.

Background

Implementation of conventional dietary assessment tools, often designed for highly literate and technologically advanced societies, can pose challenges for developing nations. Despite these challenges, nationally‐representative dietary assessment in developing nations is of utmost importance in evaluating food security, informing nutrition policy, and improving disparities. Improving nutrition in Mongolia, which faces the highest rates of age‐standardized mortality attributable to dietary risks, will require accurate measurements of national dietary intake data which have until recently been unavailable.

Methods

We trained over 200 local medical and public health students in Mongolia to collect dietary data using a locally‐developed 3‐day weighed diet record protocol. Students were trained in a classroom setting which included a didactic and hands‐on component, and were supervised in the field. After data collection, records were electronically‐transcribed, and foods condensed to form a smaller set of food groups, mean consumption of which was calculated for different seasonal, regional, and gender strata of the study population. Metrics of dietary diversity were calculated and compared statistically across strata.

Results

We find evidence of significant geographic, seasonal, and gender‐based trends in food group consumption and dietary diversity that warrant serious reconsideration of the country's national nutrition strategy.

Conclusions

Use of trained medical and public health students provided an extremely cost‐effective approach for collecting prospective diet records at epidemiological scale in a limited‐resource setting where they would otherwise be infeasible. The results gleaned from our study have important implications in the larger context of Mongolia's recent experience of economic recession, urbanization, and climate change, and will suggest future directions for compilation of local food composition data, nutritional analysis of diet, and development of a multi‐nutrient food fortification strategy for ensuring adequate micronutrition across the country.

Support or Funding Information

Current operating budget includes funds from: World Health Organization Sight and Life; Harvard School of Public Health Nutrition Round Table; SB was supported in part by NIH grant 5T32ES007069; Past funders: Millennium Challenge Corporation; Vitamin D Society
Dietary compensation in randomized nutrition trials: Can we identify and adjust?
Authors: Carpenter Catherine L , Robbins Wendie , Henning Susanne M
Published: November 2023

Background

Nutrition, an essential lifestyle factor that influences health and development of disease, exhibits several methodologic shortcomings in analyses of health‐related parameters. Human nutrition studies range from ecologic assessments to large‐scale multi‐center randomized trials, with randomized trials considered the most informative. Dietary compensation, a common methodologic factor that influences all nutrition trials, can be described as underlying changes in dietary consumption patterns resulting from administration of nutritional interventions. Randomized trials that compare nutritional supplementation in one group to usual diet in another are fairly common. Unless the study is conducted in a controlled in‐patient hospital setting, the addition of nutrients to an underlying diet may result in changes to the underlying diet. Effects resulting from nutritional supplementation may therefore be confounded by underlying dietary changes from consumption of other foods that occur in addition to changes from the nutritional intervention.

Aims and Methods

To assess dietary compensation, we utilized data from a previous three‐month phase II randomized controlled trial that was conducted on 117 young adult men attending a major university. The intervention group received 75 grams of whole‐shelled walnuts per day compared to controls who avoided tree nuts. Both groups consumed an ad libitum diet. We primarily determined whether walnut supplementation would affect male fertility parameters. To assess whether compensation was occurring, we conducted 24‐hour recalls every 2 weeks using the National Cancer Institute (NCI) ASA24 (Automated‐Self‐Administered 24‐hour Recall) assessment.

Results

Weight gain is one potential type of compensation that occurs in many trials with supplementation. Results indicate that men in the walnut group did not gain weight compared to the control group (p=0.90). The walnut supplementation group, by consuming 75 grams of walnuts, received almost 500 additional calories per day, suggesting that if weight remained constant, some compensation occurred in the walnut group. However when we assessed compensation patterns utilizing a modified repeated measures analysis of variance model, almost all nutrient patterns did not significantly differ between the intervention compared to the control group. Total fat compensation, marginally significantly different between intervention and control, was added as a covariate to the main effect model.

Conclusion

The method we developed for dietary compensation in the walnut supplementation and male fertility trial enabled us to identify dietary compensation and adjust for potential influences from dietary compensation.

Support or Funding Information

California Walnut Commission