bluuebunny/crossref_metadata_2025 huggingface dataset:
Fis1 and Drp1 cooperate in mitochondrial homeostasis (756.8)
Authors: Hill Blake , Manlandro Cara Marie , Cleland Megan , Kennedy Nolan , Bakkum Amber
Published: June 2021

Defects in mitochondrial fission and fusion cause numerous human disorders yet how these defects affect mitochondrial function and contribute to disease is unknown. Mitochondrial fission requires the dynamin‐related GTPase Drp1 and a lethal mutation (A395D) was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham et al., 2007, N. Engl. J. Med. 356, 1736‐1741). Another Drp1 mutation (C446F) was reported to cause severe cardiomyopathy in mice (Ashrafian et al., 2010, PLoS Genet 6, e1001000). We are determining how these mutants affect mechanoenzyme activity and function. For A395D, we found impaired localization, recruitment, assembly, and GTP hydrolysis. For C446F, we found impaired GTP hydrolysis and assembly. Thus, these mutants impair higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria. Curiously, these mutants show enhanced in vitro interactions with the mitochondrial protein Fis1, a possible recruiter of Drp1. To determine the consequences of disrupting these interactions, we devised an unbiased and general method to rapidly identify residues critical to protein interfaces and applied this technology to yeast Fis1 interactions. Of the >3000 Fis1 alleles screened, ~9% selectively disrupted interactions with one of the three protein partners including yeast Drp1. To test the functional consequences, each allele was parsed into its corresponding point mutation and tested for mitochondrial fission. Of 211 yeast Fis1 mutants tested to date, 97 resulted in nonfunctional fission indicating that our method identifies residues essential for mitochondrial fission. Orthologous mutations were introduced into human Fis1 and also found to impair interactions with Drp1 and mitochondrial morphology consistent with a role for Fis1 in mitochondrial homeostasis.

Grant Funding Source: RO1‐GM067180
Activation of ceramide‐metabolizing pathway in mitochondria from diabetic heart (758.3)
Authors: Novgorodov Sergei , Riley Christopher , Gudz Tatyana
Published: June 2021

Mitochondrial dysfunction is an important contributor to the development of cardiac dysfunction in diabetic cardiomyopathy. Sphingolipids are key mediators of cell‐stress responses and effectors of mitochondrial function. The objective of the study was to investigate mechanisms underlying sphingolipid‐mediated mitochondrial dysfunction. The alterations of mitochondrial sphingolipid metabolism in cardiac mitochondria from mice with streptozotocin‐ induced type‐1 diabetes were examined. Sphingolipid analysis revealed profound increases in dihydroceramides in both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. However, ceramide levels have not changed. Analysis of sphingolipid‐metabolizing enzyme gene expression indicated that, of the 42 genes investigated, only four genes exhibited an up‐regulation of > 2 fold: acid ceramidase, dihydroceramide synthase 2, desaturase 1 and galactosidase. Increased dihydroceramide levels along with activation of desaturase1 and dihydroceramide synthase 2 are indicative of an activation of de novo ceramide biosynthesis in diabetic heart. However, the lack of an increase in ceramide suggests concomitant up‐regulation of ceramide‐metabolizing pathways. Indeed, knocking down mitochondria‐resident neutral ceramidase (NCDase) drastically increased ceramide level in both SSM and IFM mitochondria from diabetic hearts. Ceramide increase in mitochondria from diabetic NCDase KO mouse hearts correlated with concomitant decrease in mitochondrial calcium retention capacity and suppression of mitochondrial respiration. These data attest that diabetes increases ceramide flux in cardiac mitochondria, which is controlled at least in part by mitochondrial NCDase.

Grant Funding Source: These studies were supported by grant from The American Diabetes Association (#7‐12‐IN‐28) to SN.
The regulation of apoptosis by RNA processing factors in Saccharomyces cerevisiae (762.4)
Authors: Chirigos Allison , Schwotzer Erica , Luo Yi , Whitworth Gregg
Published: June 2021

We have identified a novel link between mRNA processing and the regulation of apoptosis in the yeast Saccharomyces cerevisiae: the mRNA export factor Npl3, and its cytoplasmic kinase Sky1, are both necessary for the induction of apoptosis in response to environmental stress conditions. In the normal nuclear export cycle, Sky1 phoshorylation of serine 411 in the RS domain of Npl3 is associated with cytoplasmic release of the mRNA cargo. Provocatively, deletion of either factor (sky1Δ, npl3Δ), or mutation of the phosphorylation site (npl3‐S411A), abrogates induction of apoptosis in response to cation toxicity (CuSO4, LiCl). We are currently using HPLC‐coupled mass spectroscopy to quantify Npl3 phosphorylation levels at both the canonical site and adjacent residues which have been suggested as possible phosphorylation targets by high‐throughput mass spectrometry sequencing. Additionally, we are using high‐throughput epistatic‐analysis of double mutants to construct a genetic interaction network which probes the role of NPL3 and SKY1 in apoptotic stress conditions in the context of well described regulators of both the metacaspase‐mediated (YCA1) and mitochondrial (AIF1) pathways. In total, our data suggest a role for the Npl3/Sky1 export machinery in the mitochondrial pathway.

Grant Funding Source: EJS, AJC, YL & GW supported by the HHMI Fellows Program & Grant Initiative; GW by Lenfest Grants.
Structural mechanisms of starch dephosphorylation (769.2)
Authors: Gentry Matthew , Meekins David , Guo Hou‐Fu , Husodo Satrio , Vander Kooi Craig
Published: June 2021

Starch is comprised of glucose chains long enough to interact and self‐assemble into tightly packed helices that make starch water‐insoluble. Plants utilize reversible phosphorylation of glucose residues within starch to disrupt the helices and render the granule surface accessible to hydrolyzing enzymes.

Glucan dikinases solubilize starch outer glucan chains by phosphorylating different positions on glucose units and allow starch degradation to initiate. However, β‐amylase activity is inhibited when a phosphate group is reached and glucan phosphatases must then act. We initially discovered glucan phosphatase activity and now define the structural basis of dephosphorylation by glucan phosphatases.

We determined the structure of two Arabidopsis starch glucan phosphatases Starch Excess 4 (SEX4) and Like Sex Four2 (LSF2) both with phospho‐glucan bound and without glucan. SEX4 utilizes a carbohydrate binding module to position the oxygen of the C6 carbon into its catalytic cleft. LSF2 binds maltohexaose‐phosphate using an aromatic channel within an extended phosphatase active site and positions the C3 carbon into its active site.

These structures are the first of glucan‐bound glucan phosphatases, and they provide insights into the molecular basis of this agriculturally‐ and industry‐relevant enzyme family, and their unique mechanisms of catalysis, substrate specificity, and interaction with starch.

Grant Funding Source: NSF CAREER MCB1252345, NIH R01NS070899, KSEF 2268‐RDE‐014
Communication between the zinc and tetrahydrobiopterin binding sites in nitric oxide synthase (774.1)
Authors: Chreifi Georges , Li Huiying , McInnes Craig , Suckling Colin , Poulos Thomas
Published: June 2021

The nitric oxide synthase (NOS) dimer is stabilized by a Zn2+ ion coordinated to symmetry related Cys residues exactly on the dimer axis. Each of the two essential tetrahydrobiopterin (H4B) molecules in the dimer interacts directly with the heme, and each H4B molecule is about 12 Å from the Zn2+. We have solved the crystal structures of the bovine endothelial NOS (eNOS) dimer oxygenase domain bound to three different pterin compounds and they reveal an intimate structural communication between the H4B and Zn2+ sites. The structure of one of these compounds, WSG1002, bound to eNOS with varying levels of Zn2+, showed that WSG1002 and Zn2+ cannot bind to eNOS simultaneously. WSG1002 both directly and indirectly disrupts hydrogen bonding between key residues in the zinc binding motif, resulting in destabilization of the dimer. Addition of excess Zn2+ stabilizes the Zn2+ site which decreases binding of WSG1002. These results have directed our attention to differences between eNOS and the other two mammalian NOS isoforms, neuronal NOS (nNOS) and inducible NOS (iNOS), that provide a structural basis for why the order of dimer stability is eNOS>nNOS>iNOS.

Grant Funding Source: Supported by NIH
Identification of missing genetic variants in primary congenital glaucoma (776.8)
Authors: Ganguly Sriparna , Mukherjee Ankur , Chakrabarti Subhabrata , Kaur Inderjeet , Mandal Anil , Senthil Sirisha , Kabra Meha , Majumder Partha
Published: June 2021

Background and Objective: Variations in the cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) gene and mutations in the latent transforming growth factor beta binding protein 2 (LTBP2) gene cause autosomal recessive Primary Congenital Glaucoma (PCG). However, in India, only 45% of PCG patients harbor alterations in the CYP1B1 gene. The present study aimed to identify novel genetic variations contributing to unexplained PCG cases in India.

Methods: 112 unrelated Indian PCG patients in whom neither chromosome carried a CYP1B1 or an LTBP2 mutation, and 106 matched controls, were recruited for this study. DNA isolated from each case and control was screened for a total of 906,600 single nucleotide polymorphism (SNP) loci using an Affymetrix 6.0 whole genome genotyping array. The marker data were first used for an association analysis followed by multiple test corrections, after an initial adjustment for population stratification. A second approach undertaken in this study was detection of regions of homozygosity (ROH) across all autosomes that are significantly found more often in cases than in normal controls.

Results: Eleven SNPs in 5 genes and intergenic regions showed statistically significant association with PCG. These genes are neuronal growth regulator 1 (NEGR1) [1p31.1], glutamate receptor, ionotropic, kainate 4 (GRIK4) [11q22.3], plexin domain containing 2 (PLXDC2) [10p12.31], YY1 associated factor 2 (YAF2) [12q12] and copine IV (CPNE4) [3q22.1]. The results of the ROH analysis have indicated the presence of two regions of homozygosity that cover two important genes, namely kelch‐like family member 26 (KLHL26) [19p13.11] and teashirt zinc finger homeobox 2 (TSHZ2) [20q13.2].

Conclusions: The present study has identified likely locations of new putative genes underlying PCG. These genes have previously not been associated with any type of glaucoma, but the products of these genes appear to be involved in pathways related to eye development and physiology.

Grant Funding Source: Department of Biotechnology
Metallomic study of mercury in fish muscle (Semaprochilodus sp.) from Amazon ‐ Brazil (778.3)
Authors: Moraes Paula , Queiroz João , Braga Camila , Leite Aline , Buzalaf Marília , Zara Luiz , Okoshi Marina , Padilha Pedro
Published: June 2021

The aim of this study is to present the metallomics results obtained for the protein fractionation in muscle samples of “jaraqui” (Semaprochilodus sp) from JIRAU Hydroelectric Power Plant ‐ Madeira River/Amazon, Brazil. The protein spots obtained after the fractionation (2D‐PAGE) were cut from the gel and analyzed by graphite furnace atomic absorption spectrometry (GFAAS) and the protein spots in which were detected mercury were characterized by mass spectrometry (ESI‐MS/MS). The GFAAS determinations indicated that 65% of the mercury linked to protein fraction with molar mass (Mm) lower than 50 kDa. The mercury concentration present in five selected spots was between 12 and 40 mg kg‐1. Based on these concentrations was possible to estimate approximately one to three mercury atoms per molecule of protein spots. The ESI‐MS/MS analysis allowed to characterize the follow proteins: Hemoglobin subunit beta (Mm= 16.60, pI= 6.00); Hemoglobin subunit beta‐A/B (Mm= 16.95, pI= 8.90); Parvalbumin alpha (Mm= 12.40, pI= 3.20); Parvalbumin beta (Mm= 13.10, pI=3.60) and Parvalbumin 2 (Mm= 11.82, pI= 3.40).

Grant Funding Source: Financial Support: Fapesp 2009/54856‐8 and ESBR PD‐6631‐0001/2012
Expression, purification and functional characterization of influenza hemagglutinin II (HA2) protein containing transmembrane (TM) domain and ectodomain (781.1)
Authors: Ratnayake Punsisi Upeka , Weliky David
Published: June 2021

Developing better vaccines against influenza requires investigating the viral infection process. Influenza hemagglutinin protein consists of two polypeptide chains HA1 and HA2. Conformational changes occurring in HA2 subunit initiate the fusion of viral membrane to endosomal membrane. The objective of this project was to assess the structure and function of the HA2 transmembrane domain while it is embedded in the lipid bilayer, during viral fusion process. Hydrophobicity and limited membrane space are two factors that limit membrane protein expression in cells. Therefore, expression of nonnative proteins in E‐coli causes formation of inclusion bodies. HA2 consisting of ectodomain and different lengths of TM without fusion peptide (FP) were expressed. Next, the proteins were purified from inclusion bodies followed by refolding. Also, HA2 ectodomain with and without FP were expressed and purified. Folding of all proteins was verified using circular dichroism spectroscopy. The activities of various proteins were analyzed using a lipid mixing assays at pH 5.0 and pH 7.0. No significance difference in the fusion activity was observed at pH 5.0 between the constructs with different lengths of TM domain. However, in the presence of FP, a significant increase of fusion activity was observed leading to conclude that fusion peptide is the main contributor in the fusion process.

Grant Funding Source: Supported by National Institute Health (NIH) research grant 5R01AI047153‐13
The aging muscle stem cell niche as a therapeutic target (79.2)
Authors: Gilbert Penney , Gupta Neeraj , Cosgrove Benjamin , Blau Helen
Published: June 2021

Impaired muscle strength and regeneration are major problems of aging. We found that skeletal muscle stem cells (MuSCs) from aged mice are two‐thirds less effective in regenerating muscle than young MuSCs, even in a young microenvironment. Culture of aged MuSCs in hydrogel niches in conjunction with a pharmacological inhibitor of p38 mitogen‐activated protein kinase, enhanced MuSC proliferation and decreased the proportion expressing p16Ink4a. The capacity of this population to regenerate damaged muscles was similar to young MuSCs following transplantation into mice, evaluated by non‐invasive imaging, stem‐cell repopulation, and serial transplantation assays. The rejuvenation of the aged muscle stem cell population was attributable to expansion of a functional subpopulation, as revealed by time‐lapse microscopy. Molecular analyses identified a mechanism whereby substrate mechanics contributes to the modulation of MuSC stemness. Notably, strength was restored when the rejuvenated population was delivered to damaged muscles of aged mice. These findings suggest that a localized autologous muscle stem cell therapy post‐injury could benefit aged individuals.
Determining the function of the Synechococcus elongatus PCC 7942 O‐GlcNAc transferase (791.1)
Authors: Olszewski Neil , Sokol Kerry
Published: June 2021

O‐GlcNAc modification, the posttranslational addition of a single O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) to a serine and/or threonine, regulates many processes in plants and animals. Although O‐GlcNAc transferases (OGTs) are predicted to exist in many bacteria species, the function of these OGTs is largely unknown. When the OGT of the photosynthetic cyanobacterium Synechococcus elongatus PCC 7942 was deleted the resulting mutant was viable and had no growth rate defects. The mutant however did have several defects, including cell aggregation that led to the cell settling out of culture, higher free cellular phosphate levels and altered organization of both thylakoids and polyphosphate bodies. These phenotypes were rescued by re‐introduction of the wild‐type OGT, but were not fully rescued by OGTs with mutations affecting the predicted catalytic domain indicating the phenotypes are due to deletion of the OGT. During the course of this research a mutant that was suppressed for the settling phenotype arose spontaneously. Whole genome sequencing has identified a single new mutation in this strain and experiments are underway to determine if this mutation is responsible for suppression of the settling phenotype and to determine if it affects the other phenotypes associated with deletion of the OGT.
A chemical genetic approach to understand inositol polyphosphate signaling (794.6)
Authors: Dul Barbara , Bratz Jeffrey , Fiedler Dorothea
Published: June 2021

Cells regulate their growth in response to nutritional and mitogenic signals through signaling pathways that are well defined and tightly controlled by second messengers. It is critical that these messengers respond quickly and balance cellular responses. Inositol polyphosphates (IPs) have been found to play an important role in signaling and highly conserved from yeast to higher organisms. Genetic studies in yeast ascribe important functions for IPs in many cellular processes, including telomere maintenance and nutrient sensing. IP deficient mice exhibit several defects in insulin secretion and are resistant to age and diet induced obesity. We are interested in understanding the mechanisms by which the various IPs function within cells and have begun to study this pathway with a chemical genetic approach. This approach relies on designing orthogonal protein/ligand pairs ‐ by introducing a mutation in the kinase active site and using a size complementary ATP analog ‐ and has successfully been applied to numerous protein kinases. We found that this approach works with yeast inositol kinases Arg82, Ipk1, and Kcs1. Because yeast screens remain the most versatile tool for assessing functional genomic interactions at an organismal level, we exploit the yeast system to investigate IP signaling in transcriptional regulation. We anticipate that a number of these fundamental signaling roles will be maintained in higher organisms.

Grant Funding Source: NIH
Role of G‐protein coupled estrogen receptor in estrogen‐mediated protection from cardiomyocyte hypertrophy (795.6)
Authors: Burton Tyler , Waikel Rebekah , Malone Payton
Published: June 2021

Independent, preceding studies have reported decreased risk and progression of cardiac disease in pre‐menopausal women as compared to men. 17‐β estradiol has been implicated as the molecular variable underlying this sex‐specific observation and is primarily thought to signal through nuclear receptor ER‐β. Our study seeks to understand the regulatory role of the extracellular G‐Protein Coupled Estrogen Receptor (GPER) in estrogen‐mediated protection from cardiac hypertrophy. Primary, neonatal cardiomyocytes were cultured in a sex‐dependent manner. Cellular response to the hypertrophic agent, phenylephrine, in the presence and absence of GPER activity, was assessed. Cell surface area reduction was observed in response to GPER activation, independent of nuclear estrogen receptor activity. Related studies have predominantly made use mixed cultures. Interestingly, sex‐specific cardiomyocyte cultures differ in cycling and ligand responses. The significance of sex‐specific cellular characteristics is currently being assessed. Differential expression levels identified specific miRNAs as downstream mediators of GPER, which are consistent with our previous sex‐specific Next Generation Sequencing data from hypertrophic and wild‐type hearts.

NIH 5P20RR016481‐12, 8P20GM103436‐12, R15HL108192‐02

Grant Funding Source: NIH 5P20RR016481‐12, 8P20GM103436‐12, R15HL108192‐02
Phosphatidylinositol (4,5)‐bisphosphate regulates psychostimulant behaviors through its interaction with the dopamine transporter (803.2)
Authors: Hamilton Peter , Belovich Andrea , Khelashvili George , Saunders Christine , Erreger Kevin , Javitch Jonathan , Sitte Harald , Weinstein Harel , Matthies Heinrich , Galli Aurelio
Published: June 2021

Phosphatidylinositol (4,5)‐bisphosphate (PIP2) is known to regulate the function of ion channels and transporters. The human dopamine (DA) transporter (hDAT) is a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). AMPH’s addictive properties are mediated, at least in part, through elevation of extracellular DA by inducing DA efflux through the DAT. Thus, the objective of our study is to understand how to precisely manipulate the DAT to prevent DA efflux without altering its physiological function of DA uptake. This understanding is paramount to the development of pharmacological therapies for AMPH abuse. Here, we demonstrate that PIP2 directly binds to the hDAT. This binding occurs through electrostatic interactions with positively charged hDAT N‐terminal residues and is shown to facilitate AMPH‐induced, DAT‐mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT‐PIP2 interactions and AMPH‐induced DA efflux, without altering the hDAT physiological function of DA uptake. We evaluated, for the first time, the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH‐induced locomotion without altering basal locomotion. We present the first demonstration of how PIP2 interactions with a membrane protein can regulate organismal behaviors, such as locomotion.

Grant Funding Source: Supported by DGE0909667 & F31 DA 035535‐01(PJH), P22893(HHS), DA13975(AG), P01 DA12408(AG, HW, JAJ)
Food security and access to water in Mexican households (805.1)
Authors: Monroy‐Torres Rebeca , Naves‐ Sánchez Jaime , Melgar‐Quiñonez Hugo
Published: June 2021

Introduction: Food security is the state in which all persons always have access to the food needed according their biological requirements, but since 2002, the FAO has mentioned that food security cannot be achieved without considering the access to water. Objective: To identify the state of food security and access to water in Mexican households. Methods: Food Security was assessed using a validated scale applied in 352 households in rural and urban communities, as well as a pilot scale to assess access to water. Results: 73 % of households were classified as food secure (level 1), 15 % as being mildly food insecure (level 2), 7% were considered food insecure at a moderate level (level 3) and only 4 % were households with severe food insecurity (level 4). At all food security levels interviewees were worried about not having enough access to water, and in the last three months 50% of households experiences water scarcity. Most of the households used tap water to prepare the milk for the children, as well as for personal hygiene. 25% of the interviews reported that water availability has declined in their households, while the cost of water has increased. Conclusions: Food security cannot be conceived without taking into account the water situation. The majority of the households report lack of access to enough water, which usually does not meet the conditions of safety for human consumption and food preparation.

Grant Funding Source: None funding
Serving vegetables in multiple locations in school lunchrooms can lead to increased vegetable selection (808.10)
Authors: Hanks Andrew , Just David , Wansink Brian
Published: June 2021

Objective: To determine the impact on selection and consumption of fruits and vegetables in school cafeterias when fruits and vegetables are served both on the hot lunch line and in a separate location, such as a salad bar.

Methods: Twenty‐five schools of all age levels agreed to participate in the study. Of these schools, 5 served fruits and vegetables both on the hot lunch line. Three of these schools were elementary schools, and two were middle schools. Fruit and vegetable selection and consumption data were collected on four different days in all the schools. Standard t‐tests were used to compare differences in fruit and vegetable selection and consumption.

Results: In cafeterias where fruits and vegetables were served in two separate locations, students took 20% more hot vegetables (p=0.015) and 41% more cold vegetables (p<0.001), such as salad. Fresh fruit selection, on the other hand, decreased by 9% (p=0.023).

Conclusions: More is not always better. In school lunchrooms, serving vegetables in more than one location tends to increase the number of servings of vegetables, both hot and cold, that students take. Yet, when fruits are served in two locations, students take fewer servings of fresh fruits, possibly because they gravitate towards vegetables. Yet, there are methods available for gently nudging students to take more of the healthy items they are offered, regardless of the number of locations in which they are served.
Environmental cues have the greatest impact on eating in the absence of hunger in college students (811.19)
Authors: Goett Taylor , Johnston Carol , Lee Chong
Published: June 2021

Hunger regulation involves genetics and learned behaviors. The behavior constructs for hunger are grouped into emotional, environmental, or physical factors, factors that also influence disordered eating, such as eating in the absence of hunger (EAH). Continuing EAH is defined as continuing to eat immediately after satiation at mealtime, and beginning EAH is defined as beginning to eat while not hungry several hours after being satiated. College students are at high risk for weight gain due to stress, social pressures, and the constant availability of energy dense food options. The purpose of this study was to validate a modified EAH survey developed for children in a college population and to identify the behavior factors that best predicted continuing and beginning EAH in this population. The modified survey displayed good internal consistency reliability for both continuing and beginning EAH (Cronbach's alphas = 0.72,0.84) with emotion the strongest predictor of EAH. However, the environmental factor (e.g., “food looks, tastes, or smells good”, “others are still eating”, “time of day”, and “the food is available”) had the greatest influence on the likelihood to continue eating in the absence of hunger. These results can be utilized to develop educational tools aimed at reducing EAH in college students, and ultimately reducing the likelihood for unhealthy weight gain and health complications related to obesity.
Dietary behaviors are rapidly changing in China, 1991 ‐ 2011 (811.24)
Authors: Du Shufa , Du Wenwen , Zhang Bing , Wang Huijun , Popkin Barry
Published: June 2021

In low and middle income countries, few studies on away‐from‐home consumption exist. Our study examines the patterns and trends using China Health and Nutrition Survey data (1991– 2011) for 32,024 individuals aged 2 years old and older. Detailed dietary data was collected by three consecutive 24–hour recalls with weighing of all foods and condiments. Meal preparation locations were categorized into home, restaurants, work/school cafeteria, and their combinations. Random effects regression models were used to examine the patterns and trends. Over the 20 years of study period, age‐standardized energy from food prepared at home significantly decreased, while restaurant food increased from 6.6% to 15.9%, from 7.9% to 21.9%, and from 8.6% to 15.0% among participants aged 2‐18 years, 19‐59 years, and 蠅60 years, respectively. About 50.0% of energy was from away‐from‐home‐eating in 3 autonomous mega cities (Beijing, Shanghai, and Chongqing) in 2011. Modernization (both urban and rural areas measured by an urbanicity index) and increased income are driving these dietary patterns shifts. China is rapidly shifting away from home‐cooking rapidly, although overall energy intake from away‐from‐home‐eating remains lower than that in higher income countries.

This study was supported by the NIH (R01‐HD30880) and the Fogarty International Center, NIH (5D43TW007709).

Grant Funding Source: NIH
Alternative transcripts in the human milk fat globule proteinogenic RNA transcriptome with emphasis on desaturases (818.8)
Authors: Kothapalli Kumar , Guo Xiaoxian , Sun Xuepeng , Hyon Stephanie , Ran‐Ressler Rinat , Zou James , Gu Zhenglong , Brenna James Thomas
Published: June 2021

Introduction. Human breastmilk carry fat mostly in milk fat globules (MFG). Our aim was to investigate proteinogenic RNA with emphasis on PUFA synthesis in MFG.

Methods. Fresh human breastmilk sample from a single donor was centrifuged and MFG collected for mRNA isolation. An Illumina HiSeq implementing RNASeq detected >15,000 transcripts.

Results. The most highly expressed transcripts were associated with protein translation and elongation, milk protein, MFG formation, fatty acid metabolic process and transport, iron ion transport, regulation of apoptosis, and cellular component size. Six transcripts (CSN2, LALBA, CSN3, FTH1, CSN1S1 and CSN1S2AP) accounted for the bulk of the MFG transcriptome. Alternative splice sites were identified for >3500 genes in MFG. A novel FADS2 alternative transcript (FADS2AT2) with 386 amino acids was detected. FADS2, FADS2AT1 and 5 out of 8 known FADS3AT were expressed in MFG; FADS1, FADS3AT3, and FADS3AT5 are undetectable. Transcripts involved in cellular process of translation, respiratory chain and ATP synthesis were the enriched functional gene groups identified in MFG. A lipid metabolism network using Ingenuity Pathway Analysis identified UBC as the outstanding interaction partner.

Conclusion. To our knowledge this is the first report to show existence of evolutionarily conserved AT in MFG. We conclude that MFG may be a unique mRNA vector for the breast fed infant.

Grant Funding Source: NIH R01AT007003
PGC‐1α‐mediated branched‐chain amino acid metabolism in the skeletal muscle (820.1)
Authors: Hatazawa Yukino , Tadaishi Miki , Nagaike Yuta , Morita Akihito , Ogawa Yoshihiro , Ezaki Osamu , Kitaura Yasuyuki , Shimomura Yoshiharu , Kamei Yasutomi , Miura Shinji
Published: June 2021

PGC‐1α is a co‐activator of various nuclear receptors and other transcription factors. In skeletal muscles, the role of PGC‐1α in fibre type specificity, mitochondrial biogenesis, angiogenesis and improved exercise performance has been demonstrated. Branched‐chain amino acid (BCAA) metabolism primarily occurs in the skeletal muscles. In this study, microarray analysis revealed that the BCAA catabolic pathway was co‐ordinately activated in the skeletal muscles of transgenic mice overexpressing PGC‐1α. Using murine skeletal muscle and cultured cells overexpressing PGC‐1α, we investigated whether PGC‐1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice specifically overexpressing PGC‐1α in the skeletal muscles increased the expression of branched‐chain aminotransferase (BCAT) 2 and branched‐chain α‐keto acid dehydrogenase (BCKDH), which catabolize BCAA. The amount of BCAA in the skeletal muscles was significantly decreased in the transgenic mice compared with that in the wild‐type mice, suggesting the activation of muscle BCAA metabolism by PGC‐1α. In C2C12 myoblast cells, the overexpression of PGC‐1α significantly increased the expression of BCAT2 and BCKDH. In conclusion, PGC‐1α in the skeletal muscles is likely to contribute significantly to BCAA metabolism.
Selenium contents in Korean snack and dairy products (826.1)
Authors: Lee Okhee , Moon Jongwha , Chung Yongsam
Published: June 2021

Se is a trace micro‐mineral and antioxidant protecting human body from the free radical induced lipid peroxidation. However, there are little reports on the Se intake status of Koreans because of deficient Se database on foods. Furthermore, the accuracy of existing data is questionable because they were usually analysed using methods as pre‐treatment process such as wet digestion or dry digestion, which can cause selenium loss. This study was aimed to evaluate Se contents in edible portion of snack and dairy products by instrumental activation analysis(=INAA) method using freeze‐drying as a pre‐treatment process. The most frequently consumed snacks and dairy products by Korean were selected for the analysis of Se contents. To minimize contamination, all the instruments and storage tools were used after washing with HNO3. Each food samples were individually mixed using homogenizer equipped with titanium blade. The homogenized food samples of 10~20g were freeze‐dried at temperature of ‐60 C for 24~96 h and subsequently, used to analyze Se contents by IINAA‐method. Analytical quality control was conducted using NIST SRMs. The analysis limit of Se in foods by INAA was 0.01ppm. Se contents in processed foods were in the range of 0.22ug~14.9ug in 100g foods. The Se contents were various according to the type of processed foods. The average Se contents in 100g sandwich, snack, milk, soy milk, joghurt were presented. This result will be useful in establishing the food Se database for Koreans.

Grant Funding Source: Korean Research Foundation
Dietary uridine supplementation decreases tumor number in Apc(Min/+) mice (829.11)
Authors: Field Martha , Stover Patrick
Published: June 2021

Colorectal cancer affects 1.23 million people and accounts for more than 600,000 deaths each year worldwide. Deficiency of methyl donors including folate and methionine has been linked to increased colon cancer risk in experimental and epidemiological studies. The Apc(Min/+) mouse model is a powerful tool for studying the genetic and dietary mechanisms which contribute to intestinal cancer phenotypes. A previous study showed that Apc(Min/+) mice also lacking one copy of the Shmt1 gene, which results in perturbed de novo thymidylate (dTMP) synthesis, displayed increased intestinal tumor number that was exacerbated by folate deficiency. dTMP is the only nucleotide that is synthesized at the replication fork as needed for DNA replication and repair. dTMP can either be synthesized de novo from the folate‐dependent reductive methylation of deoxyuridylate (dUMP) to dTMP through a multi‐enzyme complex or can be generated by phosphorylating thymidine to dTMP by the salvage pathway enzyme thymidine kinase (TK1). In this study, we sought to determine whether dietary supplementation of dTMP precursors (thymidine, deoxyuridine or uridine) could modify tumor number in the Apc(Min/+) model. Surprisingly, dietary uridine supplementation reduced intestinal tumor number and tumor burden by 40% (p<0.05) relative to the control diet; neither thymidine nor deoxyuridine affected tumor number.

Grant Funding Source: Supported by NIH R37DK58144
Date seed (Phoenix dactylifera) extract reduces the proliferation of pancreatic cancer cells, DNA damage and superoxide‐dependent iron release from ferritin in vitro (829.20)
Authors: Habib Hosam , Platat Carine , AlMaqbali Fatma , Ibrahim Wissam
Published: June 2021

It has been well established that date seeds contain high amounts of polyphenols (50.2 mg/g), mainly epicatechin and catechin, and exert antioxidant activities in vitro and in vivo. These polyphenolic compounds have been shown to inhibit or attenuate the initiation, progression and spread of cancers in vitro and in animals in vivo. The objective of the present study was to investigate the effects of date seed extract on the proliferation of Mia‐Pa‐Ca‐2 pancreatic cancer cells, DNA damage and superoxide‐dependent iron release from ferritin in vitro. Interestingly, date seed extract inhibited the proliferation of pancreatic cancer cells in a dose‐dependent manner, reaching about 90% inhibition at a concentration of 0.1 mg/ml after 48‐hour exposure. Moreover, DNA damage and superoxide‐dependent iron release from ferritin were also reduced in a dose‐dependent manner by date seed extract. Additionally, date seed extract dose‐dependently exerted free radical scavenging and antioxidant activities in vitro, as evidenced by results obtained from using the DPPH, ABTS, Nitric Oxide, FRAP, and Total Antioxidant Capacity assays. Results obtained support a very promising anticancer potential of date seed extract. Studies are underway to determine the antitumor property of date seed extract on various cancer cell lines, determine the underlying mechanisms, and investigate its in vivo anti‐tumorigenic properties.
The effects of freeze‐dried watermelon on bone parameters of ovariectomized mice (829.3)
Authors: Mahmood Maryam , Castleberry Kayla , Meister Maureen , Eldoumi Heba , Peterson Sandra , Ketz‐Riley Connie , Perkins‐Veazie Penelope , Clarke Stephen , Smith Brenda , Lucas Edralin
Published: June 2021

The role of fruits and vegetables in the prevention and treatment of chronic diseases such as osteoporosis are continuously being explored. The purpose of this study was to investigate the dose‐dependent effects of freeze‐dried watermelon in the prevention of bone loss in ovariectomized (OVX) mice, a model of post‐menopausal osteoporosis. We hypothesized that compounds in watermelon such as lycopene and citrulline would help prevent bone loss in this animal model. Three month old C57BL/6 female mice (n=68) were sham‐operated or OVX and randomly assigned to treatment groups for 12 weeks: sham‐control (SHAM), OVX‐control, OVX + 1%, 10% or 25% (w/w) freeze‐dried watermelon (WM), or OVX‐control with alendronate (OVX‐ALN) injection (100 μg/kg body weight). All diets were isocaloric, isonitrogenous and had the same calcium and phosphorus concentrations. The 25% WM group had significantly higher final body weight and % body fat compared to the OVX‐control group. The DXA scans using PIXImus showed watermelon was not able to prevent the decrease in whole body, tibial, and lumbar bone mineral content and density due to estrogen deficiency. Micro‐computed tomography (μCT) analyses showed no similarities between WM‐fed groups and SHAM for tibial trabecular and cortical bone microarchitecture. However, the lumbar trabecular micro‐architecture analyses revealed that the WM 10% group had similar connectivity density to the SHAM group and higher than the other two doses of WM and OVX‐control groups. Bone volume, structure model index and trabecular separation of the WM 10% group is similar to the SHAM and OVX‐control groups. Watermelon supplementation increased liver catalase mRNA and tended to increase plasma glutathione peroxidase activity. Our data indicates that watermelon, in a moderate amount, might be a suitable dietary option for preventing post‐menopausal osteoporosis.
Taurine may affect redox homeostasis via regulation of thioredoxin interacting protein in endothelial cells (830.29)
Authors: Vincent Andrew , Scheers Nathalie , Gabrielsson Britt , Sandberg Ann‐Sofie
Published: June 2021

Taurine, a bioactive compound abundant in herring, has been shown to be active against endothelial dysfunction, through the alleviation of high‐glucose induced apoptosis and increased proliferation in endothelial cells. The nutrient‐sensitive inhibitor of anti‐oxidant defence, TXNIP, has been shown to be up‐regulated during endothelial dysfunction. Through ELISA and western blot analyses we have found that TXNIP levels are affected by taurine treatment in endothelial cells. The results indicate that dietary taurine may be important for maintaining redox homeostasis of endothelial cells.

Grant Funding Source: Supported by The Swedish Council for Environment, Agricultural Sciences and Spatial Planning
Streptococcal collagen‐like protein (Scl‐1) promotes resistance to phagocytic killing of group A Streptococcus (836.2)
Authors: Etesami Neelou , Doehrmann Simon , Anik Sabina , Nizet Victor , Okumura Cheryl
Published: June 2021

The up‐regulation of the gene encoding Streptococcal collagen‐like protein (scl‐1) in invasive Group A Streptococcus has been hypothesized to promote bacterial resistance to host defenses. Our previous data demonstrated that when incubated with phagocytic cells, an scl‐1 mutant strain exhibits significantly decreased survival compared to wild‐type and plasmid‐complemented strains. In our current study, we have collected results that support two possible molecular mechanisms by which Scl‐1 contributes to bacterial evasion of phagocytic killing. First, the surface protein Scl‐1 may serve as a physical barrier to mediate resistance to antimicrobial molecules contained in phagocytic cell granules. Scl‐1 directly contributes to bacterial resistance to the antimicrobial peptide human cathelicidin LL‐37, granule proteases and reactive oxygen species, though Scl‐1 does not affect peroxide production by phagocytic cells per se. Secondly, Scl‐1 contributes to bacterial survival by suppression of degranulation and release of antimicrobial molecules, and consequently the formation of neutrophil extracellular traps (NETs). Thus, the results show that Scl‐1 is a multi‐faceted virulence factor that mediates bacterial evasion to phagocytic killing by both suppressing the host response and by mediating direct resistance to antimicrobial molecules.
Lubiprostone prevents NSAID‐induced small intestinal damage via prostaglandin EP4 receptors (840.7)
Authors: Takeuchi Koji , Hayashi Shusaku , Amagase Kikuko , Kurata Naoto
Published: June 2021

We examined the effect of lubiprostone, a bicyclic fatty acid derived from PGE1 and a possible chloride channel type‐2 opener, on indomethacin (IM)‐induced small enteropathy and investigated the mechanisms involved, including the relationship to EP4 receptor. Methods: Male SD rats without fasting were given IM (10 mg/kg) s.c and killed 24 h later to examine hemorrhagic lesions developed in the small intestine. Lubiprostone (0.1 mg/kg) was given p.o. 30 min before IM treatment. Results: IM caused hemorrhagic lesions in the small intestine, accompanied by the upregulation of iNOS and TNFα mRNA expressions as well as enterobacterial invasion. Lubiprostone prevented the ulcerogenic response to IM, with concomitant inhibition of the upregulation of iNOS and TNFα expressions and bacterial invasion. These effects of lubiprostone were significantly abrogated by pretreatment of AE3‐208, an EP4 antagonist, but not CFTR(inh)‐172, a CFTR inhibitor. Conclusion: Lubiprostone prevents IM‐induced enteropathy via the EP4 receptor‐dependent mechanism, and this effect may be associated with suppression of the expression of inflammatory mediators such as iNOS and TNFα. In addition, it is unlikely that CFTR chloride channels contribute to the protective effect of lubiprostone against NSAID‐induced enteropathy.
Enigmatic chloride channels ő how to contribute to cerebrovascular remodeling (841.10)
Authors: Guan Yong‐Yuan , Wang Guan‐Lei
Published: June 2021

TMEM16A and ClC‐3 are responsible for Ca2+ activated (CaCC) and volume‐regulated (VRCC) Cl‐ channels respectively in rat basilar smooth muscle cells (BASMCs). The activity of CaCC and TMEM16A expression in BASMCs isolated from 2‐kidney, 2‐clip (2K2C) hypertensive rats were decreased , and CaCC activity negatively correlated with the blood pressure and the medial cross sectional area in basilar artery during hypertension. Knockdown of TMEM16A facilitated, whereas, overexpression of TMEM16A inhibited, angiotensin II induced cell cycle transition and cell proliferation. TMEM16A affected cell cycle progression mainly through regulating the expression of cyclin D1 and cyclin E. These results indicated that TMEM16A CaCC is a negative regulator of cell proliferation. ClC‐3 expression and volume‐regulated Cl‐ movement were enhanced in BASMCs from 2K2C hypertensive rats. The Cl‐ movement was positively correlated with increase of blood pressure. Enhanced ClC‐3 channel activity in hypertension was closely associated with cerebrovascular structural remodelling. ClC‐3 evoked up‐regulation of cyclin D1 and cyclin E, and down‐regulation of p27KIP and p21CIP through Akt pathway, leading to cell cycle G1/S transition and cell proliferation. These results suggest that both VRCC and CACC mediate cerebrovascular remodeling by different mechanisms. It is still enigmatic that the interaction between TMEM16A and ClC‐3 Cl‐ channel in the development of cerebrovaascular remodeling.

Grant Funding Source: Supported by National Natural Science Foundation of China (No.81230082,81370897,813111115)
The 2.7‐Å crystal structure of Gα12 in complex with a regulator of G protein signaling homology (RH) domain (843.1)
Authors: Kreutz Barry , Chanthavong Dara , Santarsiero Bernard , Kozasa Tohru
Published: June 2021

Heterotrimeric G proteins Gα12 and Gα13 parse signals from G protein‐coupled receptors at the cell surface to activation of RhoA through a subset of RhoA‐specific exchange factors (RhoGEFs), including human proteins p115 RhoGEF, PDZ‐RhoGEF, and leukemia‐associated RhoGEF/LARG. Each of these proteins share a well‐conserved regulator of G protein signaling homology (RH) domain which binds with high affinity and specificity to active forms of Gα12 and Gα13. Concurrent with their activation as effectors, p115 RhoGEF and LARG were shown to function as GTPase‐activating proteins (GAPs) to downregulate Gα13, and to a lesser extent, Gα12, through direct stimulation of their rates of GTP hydrolysis. To identify the structural determinants of interaction between Gα12 and RH domain‐containing RhoGEFs, we purified and crystallized a 1:1 complex of an activated Gα i/12 chimera and the RH domain of p115 RhoGEF. The 2.7‐Å crystal structure of this complex was solved by molecular replacement. Detailed comparisons were made between the refined model and previously‐reported crystal structures of Gα13 in complex with the RH domain of either p115 RhoGEF or PDZ‐RhoGEF. The interaction surface of Gα12 and the p115 RhoGEF RH domain is bipartite and highly similar to that seen in Gα13:p115 RH domain complex structures. We are currently testing the hypothesis that the engagement of p115 RhoGEF by either Gα12 or Gα13 is differentially transmitted to residues involved in the mechanism of GTP hydrolysis, and this effect is primarily responsible for measured differences in GAP activity toward Gα12 and Gα13.

Grant Funding Source: Supported by a grant from the National Institutes of Health GM061454 (to T.K.).