bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymology of H2S signaling (227.2)
Authors: Banerjee Ruma
Published: June 2021

Despite the excitement over the discovery of a third gaseous signaling molecule and the consequent profusion of literature on H2S biology, progress in the field has been impeded by the lack of sulfide‐specific sensors for detection/imaging, and an understanding of how cells maintain very low steady‐state levels of H2S, amplifying the signal as needed. We are studying the enzymology of mitochondrial sulfide oxidation, which links it to the electron transport chain and to ATP production. The enzymes of the transsulfuration pathway, cystathionine β‐synthase (CBS) and cystathionine γ‐lyase (CSE) are important for the endogenous production of H2S in addition to the mitochondrial enzyme, mercaptopyruvate sulfurtransferase (MST). We have recently obtained structures of CBS and MST captured with reactive intermediates in the active sites. We have also quantified MST, CBS and CSE levels in murine liver, brain and kidney and assessed the relative contributions of these enzymes to H2S production. We will discuss new data on estimation of hepatic sulfur flux both through H2S biogenesis and O2‐dependent H2S catabolism, which are very high and comparable to the flux of sulfur into the antioxidant pool, glutathione. These data implicate regulation of H2S clearance as a potentially important “off‐switch” in H2S signaling.
RNA editing in trypanosomal parasites (232.1)
Authors: Stuart Ken
Published: June 2021

RNA editing in Trypanosomes is a posttranscriptional process that recodes mitochondrial mRNAs by inserting and deleting uridylates, as specified by gRNAs. The polygenic pre‐mRNA and pre‐gRNA transcripts are processed by several multiprotein complexes; 20S editosomes that serially catalyze RNA cleavage, U addition or removal and ligation and other complexes that add U tails to gRNAs, associate cognate gRNAs and mRNAs and add mRNA poly (A/U) tails. The complexes and accessory factors discriminate among thousands of similar editing sites (ESs) and edit precisely producing the translated mRNAs. The discrimination employs three editosome variants that each have one of three related endonuclease/partner‐protein pairs and different editing site cleavage specificities. The edited mRNAs encode oxidative phosphorylation system components. However, the mRNAs are differentially edited between lifecycle stages by a mechanism that is not yet known. Consequently insect stage T. brucei has a complete oxidative phosphorylation system while the animal stage does not and generates energy by glycolysis. Regulation of RNA editing is integrated within the network of cellular processes that recalibrate metabolic processes and cell surface composition. The animal forms serially express variant surface glycoproteins (VSGs) resulting in antigenic variation while Procyclin surface proteins, not VSGs, are expressed in the insect stage. A future challenge is to elucidate how these multiple cellular processes are integrated. The discovery of editing led to the realization that genomic information can be recoded and the findings that multiple mechanisms of editing exist and are widespread. The exploration of its integration within cellular processes may provide other novel insights.
Mitochondrial dynamics in the heart (234.1)
Authors: Dorn Gerald
Published: June 2021

Although the mammalian heart is the most mitochondrial‐rich organ, adult cardiac myocytes are notable for absence of interconnected mitochondrial networks and paucity of mitochondrial dynamism that typically remodels such networks in other cell types. Nevertheless, proteins whose conventional function is to remodel mitochondrial structure through organelle fission and fusion are highly expressed in mammalian hearts. Using conditional gene manipulation of pro‐fusion mitofusins and pro‐fission Drp‐1 in cultured cells, Drosophila heart tubes, and mouse hearts, our laboratory identified non‐canonical functions for mitochondrial dynamics proteins central to maintenance of metabolic cardiac homeostasis through mitochondrial quality control. In this talk I will review recent work that uncovered mechanisms of interactive molecular cross‐talk between mitochondrial fission/fusion and mitophagy pathways, thus establishing the interdependence of these two processes. Using experimental cardiac‐specific Parkin dysfunction as a model system of mitophagic dysfunction, the therapeutic potential of approaches aimed at restoring the balance between fission/fusion on the one hand and mitophagy on the other will be examined.
Impact of a human missense UCP3 polymorphism on the plasma metabolomic profile: support for a mitochondrial fuel‐partitioning role for UCP3 (248.4)
Authors: Piccolo Brian , Grapov Dmitry , Garvey W. Timothy , Harper Mary‐Ellen , Fiehn Oliver , Adams Sean , Newman John
Published: June 2021

Skeletal muscle uncoupling protein 3 (UCP3) is thought to facilitate fatty acid oxidation (FAO) and protect against excess mitochondrial (MITO) reactive oxygen species (ROS); however, this has yet to be conclusively proven in human studies. We hypothesized that a comprehensive plasma metabolomic characterization of a human UCP3 polymorphism would identify metabolites associated with decreased skeletal muscle FAO and oxidative stress. We measured 490 metabolites using three independent metabolomics platforms in obese African‐American women with or without type 2 diabetes (T2D) containing a missense G304A (G/A) UCP3 polymorphism. Partial least squares‐discriminant analysis (PLS‐DA) revealed altered amino acid profiles and metabolites consistent with enhanced glycolytic oxidation in the context of reduced UCP3 function. UCP3 G/A genotype also revealed decreases in plasma amino acids involved in glutathione metabolism and markers of glutathione synthesis. Stearoylethanolamide (SEA), a putative modulator of MITO oxidative phosphorylation and biogenesis was also significantly elevated in T2D UCP3 G/A individuals. Our results support the idea that UCP3 is involved in regulating muscle MITO fuel selection by promoting fatty acid combustion, perhaps via mechanisms involving ROS signaling.

Grant Funding Source: Supported by: NIH‐NIDDK R01DK078328‐01, UAB‐CCTS UL1 RR025777, WCMC NIH 1 U24 DK097154, USDA‐ARS
Metabolomic profiles of childhood obesity and cardiometabolic risk (248.6)
Authors: Perng Wei , Gillman Matthew , Watkins Steve , Patti Mary‐Elizabeth , Fleisch Abby , Oken Emily
Published: June 2021

Objective To identify metabolite patterns associated with childhood obesity, and to examine their relations with cardiometabolic risk biomarkers.

Methods We quantified 350 metabolites in serum of 262 children 7‐10 y with an untargeted mass spectrophotometry approach. Using principal component analysis, we characterized 18 metabolite patterns. Next, we compared factor scores for each pattern between obese (BMI 蠅 95 %tile; n = 84) and lean children (BMI < 85 %tile; n = 150) to identify patterns related to obesity. Using multivariable linear regression we examined relations of the metabolite factor scores with ln transformed HOMA‐IR, leptin, adiponectin, triglycerides, CRP, and IL‐6.

Results Approximately half (50.6%) of the sample were boys; 58.9% were white. Compared to lean children, obese children had higher factor scores for a branched chain amino acid (BCAA) and an androstene steroid (AS) pattern. After adjustment for age, sex, and race, every 1 unit increment in the BCAA score corresponded to 7% (1%, 14%) higher HOMA‐IR, 15% (6%, 25%) higher leptin, and 31% (14%, 49%) higher CRP. Each unit of the AS score was associated with 4% (1%, 7%) higher triglyceride levels, 7% (2%, 11%) lower adiponectin, and 12% (3%, 20%) higher IL‐6.

Conclusions: BCAA and steroid metabolite profiles associated with obesity are also related to established biomarkers of cardiometabolic risk during mid‐childhood.

Grant Funding Source: National Institutes of Health
Effect of delivering a comprehensive package of services on nutritional status of people living with HIV and AIDS referred for HIV treatment in Rwanda (251.5)
Authors: Lopez Melanie , Muhinda Bosco , Dhlamini Russell
Published: June 2021

Objective: To improve nutritional status for PLWHA referred to HIV treatment by providing an integrated package of clinical nutrition, food security (FS), and economic strengthening services.

Methods: Integrated nutrition services and monitoring of body mass index (BMI); agricultural services such as Farmers Field Schools (FFS) and use of Bio‐Intensive Agricultural Techniques (BIAT); and access to savings and lending schemes (SILC) were provided for 7000 participants referred for HIV treatment across six districts in Rwanda. Pre‐ and post‐test surveys were conducted to assess change in BMI.

Results: Severe malnutrition levels (BMI <16kg/m2) of beneficiaries decreased from 4.6% in 2009 to 0.9% in 2011 and 0.1% in 2013. 92% of beneficiaries adopted at least three BIAT techniques and they reported 3 to 5 times increased production of vegetables. 80% of SILC groups increased their monthly subscription from 500 RWF (Rwandan Francs) to between 1000 to 1500 RWF.

Conclusion: Integrating nutrition, agriculture and economic empowerment interventions for PLWHA appears to have positive effects on BMI, as well as FS and socioeconomic status, for PLWHA.

Grant Funding Source: Catholic Relief Services through the United States President’s Emergency Plan for AIDS Relief (PEPFA
Food neophobia and food preference concordance among parent‐child dyads and parents' offering of new foods (252.1)
Authors: Kaar Jill , Buti Allison , Johnson Susan
Published: June 2021

Our aim was to describe parent/child neophobia, parent offering of new foods, and concordance in food preferences between parent‐child dyads. University email listservs were used to recruit parents of preschoolers (2‐5 y) to complete an online REDCap survey about picky eating. Scales were selected from The Child Feeding Questionnaire+ (offering new foods and pressure to eat), the Child Food Neophobia Scale and 80 items to determine parent‐child food preferences. Concordance (% agreement) and Pearson correlations were calculated in SAS 9.4. The significance level was set at alpha = .05. Respondents (n=258) included mothers (86%), were white (83%), and college educated (65%). Most children (mean age = 40.6 mo ±13.8), were perceived to be of healthy weight (80%) and “good eaters” (60%). Parent (22.0 ± 7.3) and child neophobia (30.0 ± 9.2) correlated significantly (r=.41,p<.01). Offering new foods to children was inversely associated with child (r=‐.52, p<.01) and parent neophobia (r=‐.40, p<.01) and pressure to eat (r=‐.24, p<.01). Parent‐child dyads had >75% concordance in preferences for whole grains, fruit, starchy vegetables and dairy. Lowest concordance (39‐66%) was noted for other vegetables, sweetened beverages, and specific entrees. Discordance occurred when parents had never offered their children a food (e.g. vegetables & proteins), and was uninfluenced by demographic factors. Similarities in parent/child food preferences may be related to the trait of neophobia and, consequently, the specific foods parents offer their children. Educating parents on the potential impact their own neophobia has on their child’s food preferences may be important for early intervention efforts to improve children’s food acceptance.
Intermittent bolus feeding enhances vital organs growth compared with continuous feeding in neonates (258.7)
Authors: El‐Kadi Samer , Boutry Claire , Suryawan Agus , Gazzaneo Maria , Orellana Renan , Srivastava Neeraj , Nguyen Hanh , Kimball Scot , Fiorotto Marta , Davis Teresa
Published: June 2021

Orogastric tube feeding provides enteral nutrition to critically ill neonates unable to ingest normally, and can be administered by intermittent bolus (INT) or continuous (CON) infusion. Recently, we showed that INT compared to CON feeding enhances lean tissue accretion by increasing muscle protein synthesis in neonates. The aim of this study was to determine if these feeding modalities affect vital organ growth. Neonatal pigs (n=6, 6‐d‐old) were fed the same diet in equivalent amounts continuously or intermittently (meal every 4h) for 21d. Arterial branched‐chain amino acid and insulin concentrations measured on the last day of feeding were greater for INT after the meal than for CON pigs (P<0.05). Protein synthesis increased by 14% in jejunum, 48% in ileum, and 22% in liver of INT compared to CON pigs (P<0.05), while for the kidneys the increase was only modest. Jejunum, ileum, liver and kidneys were 41, 36, 73 and 55% heavier for pigs in the INT as compared to the CON group (P<0.05). Phosphorylation of ribosomal protein S6 kinase were higher in ileum and liver in INT compared to CON fed pigs indicating increased translation initiation (P<0.05). The proportion of rpS8 mRNA associated with polysomes in liver was greater in the INT compared to CON fed group (P<0.05). These results suggest that intermittent feeding enhances vital organ growth by up‐regulating protein synthesis.

Grant Funding Source: Supported by NIH AR444474 and USDA/ARS 6250‐51000‐055
Effect of milk fat on LDL cholesterol and other cardiovascular risk markers in healthy humans: the INNOVALAIT project (269.3)
Authors: Bard Jean‐Marie , Drouet Ludovic , Lairon Denis , Cazaubiel Murielle , Marmonier Corinne , Bal dit Sollier Claire
Published: June 2021

The atherogenicity of milk fat is under debate. Milk fatty acid content may depend on the animal breed, its feeding and the season of collection. Dairy calcium may change the kinetics of fat absorption. This study aims to compare the effect on LDL Cholesterol (LDLC) and other risk markers of 4 isolipidic regimen (R) which differ by the fat sources: spring milk fat (R2), winter milk fat (R3), winter milk fat supplemented with calcium (R4) and vegetable fat (R1).

This double blind clinical study included 174 hypercholesterolemic adults (130 mg/dL<LDLC<220 mg/dL, TG <300 mg/dL) randomized in the 4 regimen groups for 8 weeks. R1 and R2 have the same saturated fat (SF) content (12.7% Energy vs 15% for R3, R4). R1, R3 and R4 have similar palmitic acid content (7% vs 5.2% for R2). For 2250 Kcal, the experimental dairy food brings 1040 mg Ca daily (R4) vs 415 mg (R1, R2, R3). 154 subjects (64% women) constitute the per protocol population. LDLC (R1 : 158±29 mg/dL, R2: 154±25 mg/dL, R3: 158±19 mg/dL, R4: 166±37 mg/dL) and TG are comparable at inclusion. After 8 weeks, there is no diet effect on LDLC, HDLC and TG, after adjustment for BMI, sex and baseline values. A diet effect is observed for apo B, apo C3 and CRP but the inter‐group comparison (Dunett) was not significant.

It is concluded that high levels of SF (12.7 to 15%) from milk does not alter conventional cardiovascular risk markers after 8 weeks. In our trial, the origin of milk fat had no influence.

Grant Funding Source: Supported by French Government, BBA, CNIEL and local fundings (Pays de Loire and Bretagne)
Choline status modulates the development of atherosclerosis in LDL receptor knockout mice (272.7)
Authors: Al Rajabi Ala , Leonard Kelly‐Ann , Vine Donna , Proctor Spencer , Jacobs René
Published: June 2021

Background: Phosphatidylethanolamine N‐methyltransferase (Pemt) is the only de novo pathway for phosphatidylcholine (PC) synthesis in mammals; as such, Pemt ‐/‐ mice are chronically choline‐deficient. PEMT deficiency dramatically reduces atherosclerosis development in LDL receptor (LDLR) knockout mice. This atheroprotective effect is believed to be mediated by reduction in circulating lipid levels.

Purpose: to investigate the effect of altering choline status on plasma lipid profiles and atherosclerosis development in LDLR‐/‐ mice, and to test the hypothesis that choline supplementations reverse the atheroprotective effect of PEMT deletion via normalization plasma lipid profiles.

Methods: LDLR‐/‐ and PEMT‐/‐/ LDLR‐/‐ male mice were given free access to high fat high cholesterol diet. Choline in diet (g/Kg) ranged from 0‐10. Atherosclerosis development was assessed by measuring plaque area in cross sectional aortic root.

Results: In LDLR‐/‐ mice, choline‐deficient group gained less weight, presented higher liver TG, CE and PL compared to other groups. Excessive choline group presented significantly higher liver PC and plasma TG.

Choline supplementation caused step‐wise increase in atherosclerosis development in LDLR‐/‐ mice. Significant correlations were observed between choline in diet and plasma PL (r=0.8075, P<0.0001), plasma total cholesterol (r=0.8369, P<0.0001), plasma TG (r=0.7551, P<0.0001), and aortic root atherosclerotic plaque area (r=0.6985, P<0.0001).

As previously reported, PEMT‐/‐/ LDLR‐/‐ mice were protected from atherosclerosis. Increasing dietary choline from 3‐10 (g/Kg) decreased liver weight, TG, and PL. Surprisingly; it didn’t promote atherogenic plasma lipid profiles nor increased plaque formation.

Conclusion: There is a positive correlation between dietary choline and the development of atherosclerosis in the LDLR‐/‐ mice. Choline deficiency cannot solely explain the atheroprotective effect of PEMT deletion in the LDLR‐/‐ mice.

Grant Funding Source: Supported by Canadian Institutes of Health Research (CIHR) & Alberta Innovates Bio Solutions (AI B)
Pkr‐like endoplasmic reticulum kinase (PERK) and glycogen synthase kinase‐3α/β signaling regulate atherosclerosis development (278.5)
Authors: McAlpine Cameron , Werstuck Geoff
Published: June 2021

Recently, a role for endoplasmic reticulum (ER) stress signaling through glycogen synthase kinase (GSK)‐3α/β has been proposed in atherogenesis; this study sought to delineate the underlying molecular mechanisms.

Cultured THP1‐derived macrophages were treated with the ER stress‐inducing agents, glucosamine, thapsigargin or palmitate in the presence or absence of inhibitors of the unfolded protein response (UPR) pathways (PERK, IRE, ATF6) or GSK3α/β (CT99021). ER stress enhanced GSK3α/β activity while inhibition of the PERK pathway, but not the IRE or ATF6 pathways, attenuated GSK3α/β activity. GSK3α/β inhibition did not affect adaptive components of the UPR but did repress the expression of factors downstream of PERK (ATF4 and CHOP). ER stress enhanced the mRNA expression of genes controlling lipid biosynthesis and uptake; while GSK3α/β inhibition attenuated this effect. GSK3α/β inhibition blocked ER stress‐induced cholesterol accumulation in macrophages. To investigate the roles of macrophage GSK3α and GSK3β in a mouse model of atherosclerosis, LDLR‐/‐ myeloid cell GSK3α knockout (LDLR‐/‐GSK3αflox/floxLyzMCre+/‐) and GSK3β knockout (LDLR‐/‐GSK3βflox/floxLyzMCre+/‐) mice were placed on a high fat diet. Myeloid cell GSK3α deficiency, but not GSK3β deficiency, attenuated atherogenesis.

Our findings support a role for macrophage PERK ‐ GSK3α signaling in atherosclerosis development.
Tmod3 participates in platelet formation and sizing in mouse fetal liver (278.9)
Authors: Sui Zhenhua , Robeta Nowak , Sanada Chad , Halene Stephanie , Krause Diane , Fowler Velia
Published: June 2021

Tropomodulins (Tmod) bind tropomyosin and cap the pointed ends of actin filaments. Tmod3 is the only Tmod isoform detected in human and mouse platelets. To study its function in platelets we have examined platelets from Tmod3‐/‐ embryos, which are embryonic lethal (Sui Z et al. 2013. Blood in press), and occasionally display hemorrhage. The platelets in the peripheral blood from E14.5 Tmod3‐/‐ embryos are larger than normal, indicating that platelet formation and sizing might be impaired without Tmod3. Both X‐gal staining and immunostaining with β‐gal antibodies show that Tmod3 is expressed at relatively high levels in megakaryocytes (MKs) of fetal livers. However, MK numbers are not significantly reduced in Tmod3‐/‐ fetal livers, and ploidy analysis of Tmod3‐/‐ MKs shows only a slight increase in the 8N MK population, suggesting that MK differentiation is not affected without Tmod3. Immunostaining of Tmod3 shows that Tmod3 is highly expressed in MK pseudopods and is partially co‐localized with F‐actin in both the MK cell bodies and proplatelets. In MK cultures from Tmod3‐/‐ fetal livers, there are fewer proplatelet‐forming MKs and larger proplatelets, and released proplatelets show abnormal shapes and disrupted F‐actin structures, but no changes in microtubules. In conclusion, our studies show that Tmod3 participates in mouse embryonic platelet formation and sizing, likely through regulating actin dynamics.
Textpresso for oro‐pharyngeal anatomy: a system for searching the full text of anatomy literatures (338.3)
Authors: Van Auken Kimberly , Druzinsky Robert , Done James , Sternberg Paul , Mueller Hans‐Michael
Published: June 2021

Biological databases are a useful resource for parlaying existing data into new experimental hypotheses. Populating such databases with information from the published literature, however, can be labor intensive and thus is aided greatly by text mining tools capable of retrieving key facts from full text.

The Textpresso information retrieval system (http://www.textpresso.org) is used by several Model Organism Database (MOD) communities to assist in curation [Müller HM, Kenny EE, Sternberg PW. 2004. PLoS Biol. Nov;2(11):e309; Van Auken KM, Fey P, Berardini TZ, et al. 2012. Database (Oxford). Nov 17;2012:bas040.] Textpresso searches the full text of literature using keywords and/or categories, groups of semantically related words, to identify sentences within a paper that describe specific aspects of biology.

Using the anatomy ontology of oro‐pharyngeal structures developed in the FEED project [Druzinsky R, et al., in prep ], we are constructing a Textpresso site to aid in curation of the oro‐pharyngeal muscle literature. At this time, we have amassed a database of over 2000 searchable references. We will present a demonstration of Textpresso for Oro‐Pharyngeal Anatomy and show how it can be used to construct meaningful queries on the full text of the literature.
The modern character synthesis: Using semantic tools to aggregate morphological characters across studies (338.5)
Authors: Dececchi Alex , Mabee Paula , Balhoff Jim
Published: June 2021

The growth of morphological character matrices has not addressed the problem of how to synthesize character data. Comparative descriptions in the literature make the information embedded within a character statement opaque to computational approaches. This makes it extremely difficult to integrate across studies or with developmental and/or genetic phenotypic data. The Phenoscape project has developed the use of ontologies to decompose free‐text descriptions into computer‐friendly annotations using an Entity‐Quality approach. We annotated to the Phenoscape Knowledgebase (KB) approximately 15,000 character statements from 114 comparative studies describing over 3,700 extinct and extant vertebrate taxa for 550,000 individual phenotypes. In a pilot study, we queried the KB for all presence/absence data relating to fin, limb, and girdle and parts for all sarcopterygian taxa. These data were automatically aggregated in a supermatrix export through the use of semantic web technologies. Supplementing the original data, were inferred assertions, which greatly broadened character coverage. The results were mapped onto an initial default phylogeny from the taxonomy that we modified to incorporate recent phylogenetic data. Standardized E‐Q terminology allows for the integration of genetic data with this morphology, from the major vertebrate model organism databases for morphology of interest.
TGFβR3 is required for normal vascularization and ossification of the palate (342.5)
Authors: Hill Cynthia , Barnett Joey , Goudy Steven
Published: June 2021

Cleft palate occurs in up to 1:500 live births associated with mutations in multiple genes. Palatogenesis involves a complex choreography of palate shelf elongation, elevation, and fusion. Transforming Growth Factor β (TGFβ) and Bone Morphogenic Protein 2 (BMP2) are known regulators of palatal development, but the contributions of the specific receptors remain elusive. The Type III TGFβ Receptor (TGFβR3) binds all three TGFβ ligands, inhibin, and BMP2. To address the role of TGFβR3 during palatal development we examined Tgfbr3‐/‐ mice. These mice do not survive to E15.5 when palate fusion occurs, but at E14.5, palatal elongation and elevation had failed. Tgfbr3‐/‐ embryos display unorganized and reduced vascularization of the palate at E13.5 and E14.5 which was accompanied by aberrant expression of arterial, venous, and lymphatic markers. Analysis of osteoblast differentiation demonstrated reduced expression of osteoblast determinants at E13.5 and E14.5. In vitro bone mineralization assays revealed reduced capacity of Tgfbr3‐/‐ mouse embryonic palatal mesenchymal cells to undergo osteoblast differentiation and to form bone which was rescued by overexpression of TGFβR3. These data demonstrate that TGFβR3 contributes to palatal development by regulating vascularization and osteoblast differentiation.

Grant Funding Source: 5K08DE17953‐5
β‐Catenin in the renal stroma regulates apoptosis via Bcl2l1 during kidney development (344.4)
Authors: Boivin Felix , Bridgewater Darren
Published: June 2021

Kidney development is dependent upon reciprocal inductive interactions between epithelium, mesenchyme, and stroma cells. β‐catenin is essential for the formation of mesenchyme derived nephrons and epithelial derived collecting duct system. β‐catenin protein is expressed in medullary, cortical, and capsular stroma in embryonic and mature kidneys. However, its role in the renal stroma is not well understood. We generated mice with ß‐catenin deficiency exclusively in the renal stroma (termed ß‐catLOF‐S). Kidneys from ß‐catLOF‐S demonstrated a paucity of renal capsule and a significant reduction in the stromal cell population (4.55 +/‐ 0.19 stromal cells/mm2 WT vs 2.52 +/‐ 0.26 in ß‐catLOF‐S). Analysis of apoptosis in ß‐catLOF‐S revealed a significant increase in apoptotic stromal cells by E13.5 (0.54 +/‐ 0.03 WT apoptotic cells/mm2 vs 1.19 +/‐ 0.14 in ß‐catLOF‐S). We investigated β‐catenin targets that regulate apoptosis and observed significant reductions in Bcl2l1 mRNA and protein in ß‐catLOF‐S kidneys. We demonstrate β‐catenin forms a novel molecular complex with transcriptional co‐activators TBX5, YAP1, and YES1, and binds to the TBX5 consensus site within the Bcl2l1 promoter. These results suggest β‐catenin controls stromal cell survival via regulation of Bcl2l1 by forming a novel transcriptional complex during kidney development.

Grant Funding Source: Supported by NSERC (Natural Sciences and Engineering Research Council of Canada) and KFOC (The Kidne
Growth of a community: ASBMB project to create resources to support the biochemistry and molecular biology community and education (348.1)
Authors: Provost Joseph
Published: June 2021

A consortium of federal and private foundations with biological scientific community resulted in the creation of the strategic document/plan “Vision and Change in Undergraduate Education, a Call to Action”. Principle findings of the plan include a need for core concepts and competencies, building communities for cultural change to develop educational scholars. In support of the key points of “Vision”, the National Science Foundation established the Research Coordination Networks‐Undergraduate Biology Education program to support such collaborations. The ASBMB has been a participant in both programs and in 2010 was funded an NSF RCN‐UBE to engage the BMB community to create core concepts in the field, generate a central resource to support the concepts and their assessment. The initial phase of the project involved starting the BMB community to ask key questions about core concepts and unpack the important components of each concept. Using surveys, small, regional and national meetings a series of core concepts have been developed resulting in a series of publications disseminating the results in the Biochemistry and Molecular Biology Education Journal. The current phase of the project is to transition to build assessment toolkits for faculty to use as they align the foundational concepts. The first of several meeting have already begun to create specific learning objectives, goals, assessments and teaching stratagies that will be used by and for the BMB scientific community. This presentation will provide the history and evolution of the ASBMB project. NSF RCN‐UBE 0957205.
Mass spectrometry‐based metabolomics as a unique biochemical approach for understanding disease pathogenesis (350.3)
Authors: Siuzdak Gary
Published: June 2021

Metabolomics, the quantitative global analysis of endogenous metabolites from cells, tissues, fluids and whole organisms, is becoming an integral part of functional genomics efforts as well as a tool for understanding fundamental biochemistry. While the genome and proteome represent upstream biochemical events, metabolites correlate with the most downstream biochemistry and therefore most closely represent the phenotype. This has been proven by the broad success of metabolite analysis in clinical diagnostics. The experimental aim in our studies is to obtain a comprehensive quantitative view of the metabolome to expand our understanding of what pathways are altered in specific diseases. We have developed a novel mass spectrometry platform for metabolomics including XCMS Online data analysis combined with METLIN, a comprehensive MS/MS metabolite database. As well as nanostructure‐initiator mass spectrometry (NIMS) imaging. These technologies will be presented in the context of their application to discovering new disease therapies/pathways for chronic pain and cancer.
Building asymmetric membranes with P4‐ATPases (351.1)
Authors: Graham Todd , Baldridge Ryan , Xu Peng , Takar Mehmet
Published: June 2021

Type‐IV P‐type ATPases (P4‐ATPases) catalyze translocation of aminophospholipids across the membrane to establish an asymmetric bilayer structure with phosphatidylserine and phosphatidylethanolamine restricted to the cytosolic leaflet, while sphingolipids and phosphatidylcholine are enriched in the extracellular leaflet. Phospholipid asymmetry is a fundamental feature of the eukaryotic plasma membrane that greatly impacts the localization and function of other membrane proteins. The P4‐ATPase “flippases” also play critical roles in vesicle‐mediated protein transport within the secretory and endocytic pathways. The mechanism for how P4‐ATPases recognize and flip specific phospholipid species is poorly understood. This is often described as the “giant substrate problem” because the canonical substrate binding pockets of homologous cation pumps seem too small to accommodate a bulky phospholipid. We have mapped residues that determine the substrate specificity of budding yeast P4‐ATPases to the first four transmembrane segments. These results imply a novel, two‐gate transport pathway at the protein/lipid interface and a potential solution to the giant substrate problem. In addition, we provide insight into how the P4‐ATPases may have evolved the ability to recognize and flip their phospholipid substrate.
Architecture and dynamics of protein complexes containing proliferating cell nuclear antigen (352.3)
Authors: Washington M. Todd
Published: June 2021

Many fundamental biological processes, such as signal transduction and cell cycle regulation, are carried out by dynamic, multi‐protein complexes. Understanding the high‐resolution structures of these complexes is one of the major challenges facing molecular and cellular biologists. Our laboratory is studying protein complexes containing proliferating cell nuclear antigen (PCNA). PCNA is a key replication accessory factor, which is best known for its ability to confer high processivity to DNA polymerases. PCNA interacts with a wide range of proteins that function in replication, repair, and recombination thereby regulating their access to replication forks. In addition, PCNA is post‐translationally modified by ubiquitylation and SUMOylation, which promote the replication of damaged DNA and inhibit homologous recombination, respectively. We are using a combination of approaches including X‐ray crystallography, small‐angle X‐ray scattering, single‐molecule fluorescence, and Brownian dynamics simulations to study the structure and dynamics of ubiquitin‐modified and SUMO‐modified PCNA and the multi‐protein complexes in which they function.
Ubiquitin and intracellular signaling by G protein‐coupled receptors (354.2)
Authors: Trejo JoAnn
Published: June 2021

GPCR signaling is precisely regulated and critical for appropriate cellular responses. Our goal is to understand the mechanisms by which ubiquitin regulates GPCR signaling. Ubiquitin is best known to function in GPCR lysosomal sorting. However, we recently discovered that ubiquitin is not required for lysosomal sorting of a subset of GPCRs including protease‐activated receptor‐1 (PAR1) and the P2Y1 receptor. The adaptor protein ALIX directly links GPCRs to ESCRT‐III to facilitate lysosomal degradation independent of receptor ubiquitination and ubiquitin‐binding ESCRTs. Remarkably, however, PAR1 and P2Y1 receptor are ubiquitinated following agonist stimulation. These findings raise the question: What is the function of GPCR ubiquitination? We found that ubiquitin regulates a non‐canonical GPCR‐stimulated p38 MAPK signaling pathway. We showed that ubiquitination of PAR1 functions as a signal for the recruitment of TAB2 (transforming growth factor‐β‐activated kinase binding protein 2), an adaptor that binds to TAB1, which induces p38 autophosphorylation and activation. The P2Y1 receptor activates p38 in a similar manner. Importantly, we further discovered that the PAR1‐stimulated TAB‐induced p38 signaling is important for regulation of endothelial barrier permeability. These studies extend and challenge our knowledge of existing paradigms for the function of ubiquitination in GPCR regulation.
O‐GlcNAc regulated arginine methylation: a novel paradigm in survival signaling (359.3)
Authors: Zachara Natasha , Fahie Kamau , Henry Roger , Miller Devin , Lee Albert
Published: June 2021

The modification of intracellular proteins by monosaccharides of O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) has emerged as a regulator of cytoprotection. In response to cellular stress and injury, O‐GlcNAc levels are elevated on numerous proteins. Notably, elevating O‐GlcNAc levels before or after the induction of injury is protective in both in vivo and in vitro models. However, the molecular mechanisms by which O‐GlcNAc protects injured cells/tissues remain elusive. In this study, we report that protein arginine methyltransferase 1 (PRMT1) is O‐GlcNAc modified and associates with the O‐GlcNAc transferase (OGT). Our data demonstrates that the association of OGT with PRMT1 inhibits methyltransferase activity. As a consequence, lowering OGT expression leads to an elevation of arginine assymetric dimethylation in vivo. Enhanced arginine assymetric dimethylation is associated with the development of cardiovascular disease, reduced lifespan, and sensitivity to apoptotic stimuli. Consistent with a model in which OGT inhibits PRMT1 leading to a stress‐tolerant phenotype, the sensitivity of cells with reduced O‐GlcNAcylation to oxidative stress can be ablated by PRMT1 knockdown or inhibition. Together, these data highlight one mechanism by which O‐GlcNAc/OGT regulates cell survival and demonstrates that OGT may be as important as its catalytic activity in promoting cell survival during injury.
Catalyzing and proofreading pre‐mRNA splicing (361.2)
Authors: Staley Jonathan , Fica Sebastian , Semlow Daniel , Mefford Melissa , Koodathingal Prakash , Tuttle Nicole , Novak Thad , Li Nan‐Sheng , Dai Qing , Piccirilli Joseph
Published: June 2021

Pre‐mRNA splicing is catalyzed and proofread by the spliceosome, an RNP machine composed of proteins and snRNAs. Decades ago, after the discovery of self‐splicing group II introns, the snRNAs were proposed to catalyze splicing. Definitive evidence, however, has remained elusive. Hindering studies, proofreading mechanisms discriminate against compromised spliceosomes. By disabling proofreading and by exploiting metal rescue strategies, we found that U6 snRNA catalyzes both splicing reactions, positioning two divalent metals that interact with the scissile phosphates. Notably, all of the U6 catalytic metals ligands correspond stereospecifically to the catalytic metal ligands observed in group II intron structures. In group II introns, the metal binding configuration hinges on three RNA base‐triple interactions. We found by genetics and crosslinking similar base‐triple interactions form between the catalytic and 5’ splice site‐binding domains of U6, implying a mechanism for docking. Finally, we investigated fidelity mechanisms by single molecule FRET and found that proofreading DEAH‐box ATPases discriminate against catalytically deficient spliceosomes by undocking the splicing substrate from the catalytic core. Overall, our findings indicate that group II introns and the spliceosome share common RNA‐based catalytic mechanisms, and likely common evolutionary origins.
Structural basis of membrane protein insertion: regulation of the SRP and GET systems (362.2)
Authors: Sinning Irmgard
Published: June 2021

More than 25% of the cellular proteome comprise membrane proteins that have to be inserted into the correct target membrane. Most membrane proteins are delivered to the membrane by the signal recognition particle (SRP) pathway which relies on the recognition of an N‐terminal signal sequence. In contrast to this co‐translational mechanism, which avoids problems due to the hydrophobic nature of the cargo proteins, tail‐anchored (TA) membrane proteins utilize a post‐translational mechanism for membrane insertion ‐ the GET pathway (guided entry of tail‐anchored membrane proteins). Both, the SRP and GET pathways are regulated by GTP and ATP binding proteins of the SIMIBI family. Insights into the molecular mechanisms of SIMIBI proteins, their unifying principles and specific adaptations have been obtained by an integrated structural biology approach and will be discussed.
Investigation of SecY protein‐translocation channel in action using a novel in vivo tool (362.3)
Authors: Park Eunyong , Akey Christopher , Rapoport Tom
Published: June 2021

Many proteins are targeted by signal sequences to a conserved protein‐conducting channel, formed by the prokaryotic SecY or eukaryotic Sec61 complex, and are translocated across the membrane. The channel forms an hourglass‐shaped pore with a constriction in the middle across the membrane. The channel also has a lateral gate that can open toward the lipid phase. During initiation of translocation, a ribosome‐nascent chain complex binds to the SecY/Sec61 channel, resulting in insertion of the nascent chain. To study the SecY channel engaged in translocation, we developed a novel method generating defined translocation intermediate complexes in E. coli cells. Using this tool, we addressed how the channel is opened by a ribosome‐bound nascent chain and transports large polypeptides and yet prevents passage of small molecules. We determined structures of inactive and active ribosome‐channel complexes with cryo‐electron microscopy. These structures demonstrate that the nascent chain opens the channel through mostly rigid body movements of the two halves of SecY. The polypeptide inserts as a loop into the channel, with the hydrophobic region of the signal sequence intercalated into the open lateral gate. Biochemical analysis shows that the pore residues of SecY form a gasket‐like seal around the translocating polypeptide chain, preventing the permeation of small molecules. This work was supported by HHMI and NIH.
Exploring and exploiting aminoglycoside resistance enzymes (363.1)
Authors: Tsodikova Sylvie
Published: June 2021

Aminoglycosides are broad‐spectrum antibiotics use against serious bacterial infections. The main mechanism of resistance against these drugs consist of aminoglycoside‐modifying enzymes, which chemically modify the antibiotic scaffold for deactivation. In this presentation, the mechanism of a novel aminoglycoside‐resistance enzyme will be revealed and application of these enzymes as tools for chemical biology will be discussed.
Design of a novel redox biocatalyst to specification (363.3)
Authors: Bommarius Andreas , Abrahamson Michael , Schuermann Martin , Au Samantha , Bommarius Bettina
Published: June 2021

Recently, we obtained the first amine dehydrogenases, termed L‐AmDH and F‐AmDH, from leucine DH [1] and phenylalanine DH [2], respectively, allowing direct reductive amination of ketones to chiral amines.

Starting from a scaffold devoid of such activity, a robust AmDH has been evolved with a single two‐site library allowing for the direct production of (R)‐1‐(4‐fluorophenyl)‐propyl‐2‐amine from para‐fluoro phenyl acetone with a kcat value of 6.85 s‐1 at 25oC. Only (R)‐amine was formed (> 99.8% e.e.), showing that perfect enantioselectivity of the template was conserved.

Successful domain shuffling created a chimeric amine DH with enhanced substrate specificity that allows to accommodate sterically very demanding side chains, such as adamantly groups. We will elucidate the impact of restricted libraries in the evolution towards higher activity and broader specificity.

Many interesting substrates feature low solubility in aqueous solution. We describe how utilizing a two‐phase system can also lead to pinpointing broadened substrate specificity, as substrate candidates that were previously missed, can register as hits in the search.
Seafood consumption and blood mercury concentrations in youth ages 1‐19 years, 2007‐2010 (369.4)
Authors: Nielsen Samara Joy , Kit Brian , Aoki Yutaka , Ogden Cynthia
Published: June 2021

Background: Consuming seafood has health benefits, but seafood can also contain methyl mercury, a neurotoxin.

Objective: To examine seafood consumption and blood mercury concentrations in US youth.

Design: In the 2007‐2010 National Health and Nutrition Examination Survey, blood mercury and seafood consumption in the past thirty days were collected from 5,744 youth, ages 1‐19 years. Seafood consumption was categorized as fish or shellfish and by frequency of consumption: none, 1‐2, 3 or more times per month. Association between seafood consumption and blood mercury was examined using multiple linear regression adjusted for sex, age and race/Hispanic origin.

Results: In 2007‐2010, 60.5% (SE,1.0), of youth reported consuming seafood, 35.9%, (SE,1.2), shellfish, and 47.6% (SE,1.0) fish in the preceding month. Less than 1% of youth had blood mercury concentrations at or above 5.8 µg/L. The geometric mean blood mercury concentrations were 0.31 (SE, 0.01) for non‐seafood consumers and 0.54 (SE, 0.02) for seafood consumers. Higher frequency of seafood consumption was associated with higher log10(blood mercury concentrations) in the adjusted model (fish: (β=0.70 (95% CI, 0.62,0.78), shellfish:(β=0.43 (95% CI, 0.31,0.54)).

Conclusions: Very few youth have blood mercury concentrations at or above 5.8 µg/L but among US youth, blood mercury concentration is associated with both fish and shellfish consumption.
Central adiposity influences the relationship between 25(OH)D and indices of plasma insulin (37.7)
Authors: Wright Christian , Weinheimer‐Haus Eileen , Fleet James , Peacock Munro , Campbell Wayne
Published: June 2021

Low serum 25‐hydroxyvitamin D3 (25(OH)D) is inversely associated and central adiposity is positively associated with plasma insulin (INS) concentrations. We assessed whether the relationship between 25(OH)D and INS is confounded by central adiposity. 336 overweight and obese adults (age: 48 ± 8 y, BMI: 30 ± 3 kg/m2) from West Lafayette, IN (40.4°N) were studied. Among all subjects, vitamin D status was as follows: 2% deficient (8‐10 ng/ml); 26% insufficient (10‐20 ng/ml); 53% adequate (20‐30 ng/ml); and 19% proposed optimal (>30 ng/ml). After controlling for age, race, and sex, 25(OH)D was inversely correlated with indices of INS (fasting INS, r= ‐0.12; oral glucose challenge INS area under the curve (AUC), r= ‐0.12; HOMA‐IR, r= ‐0.12; all p<0.05). Android fat mass index was also correlated with indices of INS (fasting INS, r= 0.32; INS AUC, r= 0.32; HOMA IR, r= 0.31; p<0.001) and 25(OH)D (r= ‐0.11, p<0.05). Similar correlations were observed for trunk fat mass and trunk body fat percent. These correlations (p<0.001) between central adiposity and INS persisted after controlling for 25(OH)D. However, after adjusting for individual markers of central adiposity, the significant correlations between 25(OH)D and indices of plasma INS did not persist. Our data do not support an independent effect of 25(OH)D on INS, but suggest that 25(OH)D is a marker for the relationship between central adiposity and insulin sensitivity.

Grant Funding Source: U.S. Whey Protein Research Consortium; NIH T32AG025671 and UL1 TR000006
Metabolites of polyphenols preserve indices of endothelial cell nitric oxide bioavailability under glucotoxic conditions (372.6)
Authors: Qian Ying , Panneerseelan Leenalitha , Babu P.V.A. , Symons J. , Jalili Thunder
Published: June 2021

We hypothesized that biological metabolites of quercetin, resveratrol, and grape seed extract previously identified in human plasma can prevent impairment of nitric oxide (NO) bioavailability due to glucotoxic conditions (e.g. Type 1 or 2 diabetes).

Human aortic endothelial cells were treated for 24 h with 2μΜ Quercetin‐3‐O‐glucoronide, 5μΜ Piceatannol, or 1μΜ 3‐Hydroproponoic acid. Cells were next exposed to normal (5mM) or high (25mM) glucose for 48h, then treated with insulin (100nM, 10 min) to stimulate NO production.

In the absence of polyphenols, insulin stimulation increased (P<0.05) indices of NO production, phosphorylated to total Akt (p‐AktSer473:Akt), and endothelial nitric oxide synthase (p‐eNOSser1177:eNOS) in cells grown in 5mM but not 25mM glucose. Pretreatment of cells with polyphenol metabolites prior to 25mM glucose exposure preserved insulin stimulated increases (P<0.05) in NO, p‐AktSer473:Akt and p‐eNOSser1177:eNOS. These effects may be secondary to oxidative stress as elevations (P<0.05) in reactive oxygen and nitrogen species in cells treated with 25mM glucose were completely prevented by all polyphenol metabolites.

These data indicate that biological metabolites of quercetin, resveratrol, and grape seed extract protected against gluocotoxic impairment of insulin dependent NO bioavailability, and preserved the insulin ‐ Akt ‐ eNOS signaling axis.
DNA methylation in an enhancer region of the FADS cluster is associated with FADS activity in human liver (373.8)
Authors: Chilton Floyd , Mathias Rasika , Seeds Michael , Herrington David , Hixson James , Hawkins Greg , Sergeant Susan , Miller Leslie , Howard Timothy
Published: June 2021

Omega‐6 (n‐6) and omega‐3 (n‐3), long chain polyunsaturated fatty acids (LcPUFAs) impact a wide range of biological activities ranging from immune signaling to brain function. Two desaturase steps (Δ6, encoded by FADS2 and Δ5, encoded by FADS1) are rate‐limiting in the conversion of dietary essential 18 carbon PUFAs such as linoleic acid (n‐6) to arachidonic acid and α‐linolenic acid (n‐3) to eicosapentaenoic and docosahexaenoic acids. Genetic variants in and around FADS1 and FADS2 are highly associated with levels of LcPUFAs as well as cardiovascular disease risk factors. Particularly, rs174537 is associated with both FADS activity and circulating AA levels (p = 5.95 x 10‐46). FADS1 and FADS2 sit adjacent (5’ to 5’) on chromosome 11 (11q12.2). We hypothesized that SNPs such as rs174537 were associated with DNA methylation variation that impacted LcPUFA levels and FADS activities. To test this hypothesis, we performed a genome‐wide allele‐specific methylation study with rs174537 in 144 human liver samples. This approach identified a strong association of rs174537 with CpG sites in an enhancer region between FADS1 and FADS2 (p=2.69x10‐29). These data suggested that rs174537 was serving as a genetic proxy for methylation variability in a critical enhancer region sitting between two proximal promoters for FADS1 and FADS2. Importantly, methylation levels of the significant probes were shown to be strongly associated with FADS1 and FADS2 activities within the liver.

Grant Funding Source: NIH ‐ P50 AT002782