bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quality Control Of Nuclear Pore Complex Biogenesis
Authors: Lusk Patrick , Webster Brant , Jaeger Jens , Thaller David 
Published: November 2023

Nuclear pore complexes (NPCs) are massive protein assemblies that are inserted into the nuclear envelope and control the bidirectional exchange of molecules between the nucleus and cytoplasm. Each NPC is made up of ~30 proteins termed nups, which are found in multiple copies such that hundreds of nups ultimately build an individual NPC. Understanding the molecular mechanisms that underlie the spatial and temporal assembly of NPCs remains a central challenge in the field, particularly during interphase when NPC assembly occurs into intact nuclear membranes. Moreover, given that a single defective NPC could compromise the barrier function of the nuclear envelope, it is essential to understand how NPC quality is ensured and whether there are mechanisms capable of recognizing and removing defective NPCs. Consistent with this idea, our recent work showed that the endosomal sorting complexes required for transport (ESCRT) machinery helps ensure the formation of functional NPCs in budding yeast. Moreover, we identified the storage of improperly assembled NPCs compartment (SINC) that sequesters defective NPCs in mother cells to prevent their inheritance by progeny. We continue to investigate the mechanism by which the ESCRTs contribute to NPC quality control and are investigating the physical interactions between ESCRT‐III, nucleoporins and the Lap2‐emerin‐MAN1 (LEM) domain containing integral inner nuclear membrane proteins, Heh1 and Heh2, using several in vivo and in vitro approaches. We have also tested interactions with the mammalian orthologue of the ESCRT‐III‐like protein, CHMP7, which was recently implicated in recruiting ESCRT‐III to the nuclear envelope in a mammalian tissue culture model. Our data are consistent with the interpretation that Heh1 recruits the CHMP7 orthologue to the nuclear envelope during nuclear pore biogenesis.Support or Funding InformationNIH GM105672
Identifying vulnerable steps in the coenzyme A biosynthesis pathway of M. tuberculosis
Authors: Mizrahi Valerie , Evans Joanna , Trujillo Carolina , Eoh Hyungjin , Ehrt Sabine , Schnappinger Dirk , Boshoff Helena , Rhee Kyu , Barry Clifton E. 
Published: November 2023

Enzymes in the pantothenate and coenzyme A (CoA) biosynthesis pathways have attracted considerable interest as potential targets for the development of drugs against a number of human pathogens, including M. tuberculosis. While potent inhibitors have been developed against M. tuberculosis pantothenate synthase and pantothenate kinase, these have failed to translate into compounds with significant whole‐cell activity. These findings illustrate the challenges inherent in target‐led approaches to tuberculosis drug discovery, which is confounded by issues of permeability, metabolism and efflux, by a lack of understanding of target vulnerability.To identify vulnerable steps in the pantothenate and CoA biosynthesis pathway in M. tuberculosis, a set of anhydrotetracycline (ATc)‐regulated mutants of M. tuberculosis was constructed using a transcriptional silencing system. The mutants were used to assess the impact of depletion of specific targets in the pantothenate and CoA pathway on growth and survival of the organism. Mutants with inducer‐dependent growth phenotypes were obtained for panB, panC, coaBC, coaD and coaE whereas those in panB, panE and panK were refractory to ATc‐regulated growth. While transcriptional silencing of panB, panC or coaE was bacteriostatic, coaBC silencing was apparently bactericidal in vitro, as determined by CFU enumeration. CoaBC was likewise shown to be required for growth and persistence of M. tuberculosis in vivo based on enumeration of bacillary loads in the lungs of mice in which coaBC was silenced immediately post‐infection, during acute infection, or during chronic infection. However, by taking advantage of the fact that M. tuberculosis is capable of CoaBC bypass via CoA salvage, coaBC silencing was shown to result in a transient, ‘non‐growing but metabolically active’ state from which non‐culturable bacilli can be rescued by uptake and assimilation of pantethine. These findings highlight the limitations of colony‐forming ability as a proxy for viability in M. tuberculosis. In further studies, the impact of silencing of these genes is being investigated by analysing changes in the metabolomic profile of M. tuberculosis with the aim of identifying similarities and differences in the metabolic response to target depletion at different steps of the pathway.Support or Funding InformationThis work was supported by grants from the TB Drug Accelerator program of the Bill and Melinda Gates Foundation (to C.E.B. III, D.S., K.R. and V.M.), the South African Medical Research Council, the National Research Foundation of South Africa and an International Research Scholar's grant from the HHMI (to V.M.) and, in part, by the Intramural Research Program of NIAID (H.B. and C.E.B. III).
Alkaloid Biosynthesis In Plants
Authors: O'Connor Sarah 
Published: November 2023

Plants are– arguably– the world's best chemists. All plants synthesize thousands of complicated molecules that they use to protect themselves from predators, attract pollinators and communicate with other plants. Thousands of years ago, humans realized that many of these plant‐derived molecules also have a powerful impact on human health and well‐being. Advances in genomic and transcriptomic sequencing have rapidly advanced our understanding of the complex metabolic pathways that produce these high‐value chemicals. Here we show how transcriptomic and genomic data can be used to elucidate the alkaloid pathways of Madagascar Periwinkle, a medicinal plant that produces compounds that are used to treat a variety of cancers.Support or Funding InformationBBSRCERC
Altered fatty acid metabolism remodels the hypothalamic metabolome to stimulate feeding behavior in BCO2 knockout mice
Authors: Guo Xin , Wu Lei , Soh Traces , Chowanadisai Winyoo , Clarke Stephen , Lucas Edralin , Smith Brenda , Wang Weiqun , Medeiros Denis , Lin Dingbo 
Published: November 2023

Objectiveβ,β‐carotene‐9′,10′‐oxygenase2 (BCO2) is a carotenoid cleavage enzyme localized to the inner membrane of mitochondria and is broadly expressed in many tissues, such as liver and hypothalamus. We recently found that systemic depletion of BCO2 impaired lipid metabolism in a mouse model of diet‐induced obesity. The aim of this study is to explore the mechanism by which BCO2‐regulated lipid metabolism impacts hypothalamic metabolome and subsequent stimulation of feeding behavior.MethodsAfter weaning, male BCO2 knockout (KO) and wild type (WT, 129S6) mice were fed a standard chow for 3 weeks. Food intake, body weight, and fasting blood glucose level were monitored on a weekly basis. Gluconeogenesis was examined via a pyruvate tolerance test. At the end of study, mice were sacrificed after a 3 hour fast. Blood and hypothalamic tissues were collected. Plasma biomarkers of lipid metabolism were tested, including cholesterol, lipoproteins, triglycerides (TG), and nonesterified fatty acids (NEFA). Fresh hypothalamic mitochondria were isolated and subjected to mitochondrial respiratory activity assays. Metabolomics profiling assay on hypothalamic tissues was conducted. Western blot was employed to determine altered lipid metabolism and energy homeostasis.ResultsAverage daily food intake was significantly increased (14 %, p<0.01) in the KO mice compared to the WT controls. The KO mice also exhibited a higher (p<0.05) fasting NEFA and blood glucose compared to the WT mice. However, the body weight of the KO mice was reduced (p<0.05) compared to age and gender‐matched WT mice. The KO mice developed pyruvate intolerance. Enhanced expression of hypothalamic acetyl‐CoA carboxylases, peroxisome proliferator‐activated receptor α, and forkhead box O1, and suppression of AMP‐activated protein kinase confirmed impaired lipid metabolism and disturbed energy homeostasis in KO mice. Hypothalamic metabolomics results showed decreases in oleate and acyl‐carnitine contents, suppression of long chain fatty acid transport into mitochondria, and inhibition of bile acid biosynthesis in KO mice. The mitochondrial respiratory capacity of complex II was significantly decreased in the hypothalamus of KO mice.ConclusionsBCO2 may exert a role in regulation of hypothalamic mitochondrial metabolism. Ablation of BCO2 results in decreased fatty acid oxidation, impaired lipid sensing, and potentiated gluconeogenesis
Dietary Glycine Alters One‐Carbon Metabolic kinetics in vivo
Authors: Huang Yu‐Hsuan , Chiang En‐Pei Isabel 
Published: November 2023

Folate‐dependent one‐carbon metabolism is required for nucleotide biosynthesis and for the remethylation of homocysteine to methionine. Glycine‐N methyltransferase (GNMT) is a major hepatic enzyme that converts S‐adenosylmethionine to S‐adenosylhomocysteine while generating sarcosine from glycine, hence it can regulate mediating methyl group availability in mammalian cells. The metabolic role of GNMT in folate‐mediated one‐carbon metabolism and how glycine supply may affect one carbon metabolic kinetics are not fully understood. In the present study, we investigated how GNMT expression may affect cellular one carbon flow under various glycine conditions. Hepatocyte‐derived cell‐lines with and without GNMT expression were cultured in minimum essential medium (MEM) with and without high glycine supply. Methylene‐tetrahydrofolate (mTHF) utilization and endogenous formate biosynthesis were carefully investigated using stable isotopic tracers and GC‐MS. We discovered that high glycine inhibits the M+1 specie enrichments in deoxythymidine (dT+1) from 2,3,3 D3‐serine, suggesting that high glycine may inhibit endogenous formate production in these in vitro models. In a parallel experiment that exogenous formate was used as the 1C source, enrichments in dT+1 significantly increased from exogenous formate, supporting our hypothesis that high glycine inhibits endogenous format production for thymidine synthesis. On the other hand, high glycine promotes M+1 specie enrichments in methionine from 2,3,3 D3‐serine, indicating that high glycine supply may favor the utilization of methyleneTHF derived one carbon for homocysteine remethylation. High glycine supply can promote folate dependent methionine synthesis in our cell models. Furthermore, our preliminary in vivo experiments demonstrated that high dietary glycine can promote folate dependent methionine synthesis, consistent with our in vitro findings. These results showed that high dietary glycine promotes methionine remethylation in vitro and in vivo.Support or Funding Informationsupported by MOST 104‐2320‐B005‐010‐MY3; MOST104‐2911‐I005‐301; and Ministry of Education, Taiwan, R.O.C. under the ATU plan
Vitamin B12 Deficiency Defined Using Total Cobalamin, Holo‐Transcobalamin II, and an Algorithm of Both Total Cobalamin and Holo‐Transcobalamin II in Vegetarians, Vegans, and Omnivores
Authors: Arnold Taylor , Knurick Jessica , Johnston Carol 
Published: November 2023

Vitamin B12 (cobalamin) is a water‐soluble vitamin essential for purine and pyrimidine synthesis, DNA methylation, and the myelination of nerve cells. As vitamin B12 is only found in animal products, vegetarians and vegans are at an inherently higher risk of developing a deficiency than the omnivorous population. The standard measures used to evaluate vitamin B12 nutriture in the clinical setting include total cobalamin, as well as homocysteine and methylmalonic acid (MMA). Each of these markers have flaws, however, as total cobalamin includes biologically inactive cobalamin, homocysteine can be influenced by folate status, and MMA is not present in significant amounts when vitamin B12 nutriture is adequate or excessive. Because of this, holo‐transcobalamin II (TCII), which is the carrier of biologically active vitamin B12, is becoming increasingly popular in research settings to accurately detect cobalamin status. Consequently, there continues to be a discrepancy between the standard analysis methods used in the clinical and research settings. In this study, we sought to compare deficiency prevalence in omnivores, lacto‐ovo vegetarians, and vegans using three different definitions of vitamin B12 deficiency: serum B12 < 148 pmol/L, serum TCII < 35 pmol/L, and serum B12 < 243 pmol/L + TCII < 35 pmol/L. To accomplish this, we analyzed blood samples of 82 subjects between the ages of 19 and 50 years old (27 omnivores, 27 lacto‐ovo vegetarians, and 28 vegans). Chi squared tests revealed significant differences in deficiency detection across the three deficiency definitions (p=0.046). In particular, there was a significant difference in the frequency of deficiency detected using serum B12 (4.9%) and serum TCII (17.1%) (p=0.025) in the total study population. The prevalence of deficiency detected when using serum TCII, rather than serum total cobalamin, appeared to increase more in lacto‐ovo vegetarians (from 7.4% to 25.9%) and vegans (from 3.6% to 17.9%) than in omnivores (from 2.7% to 7.4%), although this difference was not significant. Changing the clinical standard to detect vitamin B12 deficiency may improve deficiency detection, particularly at early onset. In addition, using TCII to detect deficiency cases may improve detection of marginal cases, which can allow practitioners to treat individuals who are deficient before symptoms progress. Future research is needed to examine the feasibility of using TCII in a clinical setting.Support or Funding InformationGraduate and Professional Student Association, Arizona State University
Food Insecurity is Associated with Depression and Stress Among a Cohort of Pregnant Kenyan Women of Mixed HIV Status
Authors: Tsai Irene , Krumdieck Natalie R. , Collins Shalean , Widen Elizabeth M. , Wekesa Pauline , Onono Maricianah , Young Sera L. 
Published: November 2023

Food insecurity (FI) is well established to be harmful among children, adolescents, and adults, but little is known about either its prevalence or covariates during pregnancy. Given the importance of maternal health for the lifelong well‐being of children, a greater understanding of FI may provide insight into potentially modifiable determinants of maternal and child health. Therefore, we examined (1) the population prevalence of food insecurity among all pregnant women attending 7 urban and rural antenatal clinics in Nyanza province, Kenya, and (2) assessed the predictors of FI at baseline among women enrolled in Pith Moromo, an ongoing observational pregnancy cohort study in this region.Cohort participants (n=363) were enrolled at <30 weeks gestational age, and were purposively sampled to achieve equal proportions of HIV‐infected and –uninfected women across low, moderate, and severe tertiles of food insecurity. FI was measured by clinic‐based study staff using an individually focused food insecurity access scale (IFIAS, Natamba et al., 2014). Staff assessed depression using the Center for Epidemiologic Studies Depression Scale (CES‐D), and assessed stress using the Perceived Stress Scale (PSS). An asset score was created by assessing patterns of asset ownership, and weighted using principal component analysis. Linear regression was used to evaluate predictors of FI at enrollment into the cohort, including maternal age, gestational age, HIV status, marital status, perceived stress, and urban/peri‐urban/rural locality. In the population‐based sample (n=1,188), the mean score for FI was 12.9 (SD 7.4), 39.44%, 34.54%, and 26.01% of women were in the low, moderate, and severe FI groups. Among participants in the cohort, the mean score for FI was 13.6 (SD 7.1). In multivariate linear models among cohort participants, CES‐D score was positively associated with FI (β 0.11, 95% confidence interval 0.00 to 0.21, p<0.05), and greater perceived stress was also associated with higher FI (β 0.19 score, 95% confidence interval 0.00 to 0.39, p<0.05). Greater asset index scores were associated with lower FI (β −1.43, 95% confidence interval −2.02 to −0.85, p<0.001). Maternal HIV and marital status were not significantly associated with FI.In sum, FI is experienced by many pregnant women in this region. In our cohort, we observed significant positive correlations between FI and psychosocial indicators, including probable maternal depression and perceived stress. Understanding the relationships between mental health and food insecurity may help inform the development of psychosocial health interventions to improve mental health and/or food security among pregnant women.Support or Funding InformationSupport: SLY NIH K01 MH098902
ZnT2‐Mediated Zn Transport Is Required for Vesicle Acidification And Milk Secretion during Lactation
Authors: Lee Sooyeon , Rivera Olivia C , Kelleher Shannon L 
Published: November 2023

Successful lactation requires optimal regulation of mammary gland differentiation, milk secretion and milk composition. Recently, we determined that the loss of the vesicular zinc (Zn) transporter ZnT2 impairs mammary gland differentiation, reduces milk secretion and leads to suboptimal milk composition in lactating mice. In this study, we aimed to determine how the loss of ZnT2 compromises milk secretion during lactation. We hypothesized that ZnT2‐mediated Zn transport regulates vesicle acidification, which is critical for vesicle trafficking and secretion. First, we studied the role of ZnT2 in vesicle trafficking in mammary epithelial cells (MECs) in vitro. We found that while ZnT2‐expressing cells successfully trafficked beta‐casein‐containing vesicles toward the membrane in response to the lactogenic hormone prolactin, beta‐casein‐containing vesicles were retained in the perinuclear region in ZnT2‐attenuated MECs, suggesting that ZnT2 is important for vesicle trafficking and secretion. Vesicle trafficking and secretion in neuronal and pancreatic cells requires vesicle acidification through the vacuolar ATPase (V‐ATPase) proton pump, which generates a vesicular pH gradient that permits trafficking and exocytosis of secretory components. To test the hypothesis that ZnT2 regulates vesicle acidification, we first determined if ZnT2 and V‐ATPase interact, and determined the localization of V‐ATPase using immunofluorescence in mouse mammary glands. We found that the cytoplasmic subunit V1 domain was localized to the apical membrane of MECs, indicating active vesicular secretion. In contrast, the V1 domain remained cytoplasmic in the MECs of ZnT2‐null mice. We next measured vesicular pH using the fluorescent ratiometric probe Lysosensor DND‐160 in isolated MECs. We found that ZnT2‐attenuated MECs failed to acidify vesicles (pH 6.0) while acidification was augmented in ZnT2‐overexpressing cells (pH 4.9) compared to mock‐transfected cells (pH 5.5). Importantly, we found that ZnT2 is directly associated with both the cytoplasmic V1 and the membrane‐bound V0 domains of V‐ATPase, suggesting a role for ZnT2 in V‐ATPase complex assembly. Taken together, these results establish that ZnT2 is critical for V‐ATPase complex assembly, which is coupled to proton gradient formation to acidify vesicles for milk secretion.Support or Funding InformationIntramural funds to SLK
Acidic Human Milk Oligosaccharides Vary Across Populations and Over Lactation
Authors: Newburg David S , Chen Ceng , Cline Allison , Morrow Ardythe L 
Published: November 2023

BackgroundHuman milk oligosaccharides (HMOS) inhibit pathogens, promote colonization through a prebiotic effect, and inhibit inflammation of the intestinal mucosa. The content of HMOS can vary qualitatively and quantitatively among individuals and over the course of lactation.ObjectiveTo compare the acidic HMOS composition of milk from mothers in Cincinnati, OH, Shanghai, China, and Mexico City, Mexico.MethodsA total of 120 mothers per site were included, and 760 milk samples were analyzed by LC‐MS as part of the Global Exploration of Human Milk (GEHM) study. Eligibility required healthy, singleton, term delivery and > 75% of feedings from breast milk for at least 3 months. Milk samples were collected between 9AM–1PM at 2, 4, 13 and 26 wks postpartum using Medela Symphony breast pumps, and represented the contents of one breast that was not used for feeding for two hours prior to pumping.ResultsTotal oligosaccharides decreased over lactation, from 5.8 (week 2) to 2.8 g/L (week 26, p<0.001). However, the most rapid decline in HMOS occurred in the acidic oligosaccharide fraction, from 2.4 (week 2) to 0.4 g/L (week 26, p<0.001). Milk from Shanghai was significantly lower than the other two sites in total HMOS (at 1 month: 4.1 vs 5.1 g/L, p<0.001). The most abundant individual acidic HMOS across the populations were 3′‐sialyllactose (3′SL), 6′‐sialyllactose (6′SL), and disialyllacto‐N‐tetraose (DSLNT). Comparing populations, 3′SL was higher (p<0.001), 6′SL was lower (p<0.001), and DSLNT was lower (p<0.001) in Mexican than Cincinnati or Shanghai mothers. The concentrations of 6′SL and DSLNT, but not of 3′SL, declined over the first 26 weeks of lactation.ConclusionsConcentrations of acidic HMOS differ among populations, and change over the course of lactation. The biological ramifications of these differences warrant investigation.Support or Funding InformationSupported in part by HD013021, HD059140, and Mead‐Johnson