bluuebunny/crossref_metadata_2025_split huggingface dataset:
AGPAT4 is a Mitochondrial Lysophosphatidic Acid Acyltransferase that Regulates Learning and Memory in Mice
Authors: Bradley Ryan , Mardian Emily B , Mitchell Andrew S , Bloemberg Darin , Marvyn Phillip , Bombardier Eric , Moes Katherine , Tupling A. Russell , Quadrilatero Joe , Duncan Robin E 
Published: November 2023

We have previously characterized acylglycerophosphate acyltransferase 4 (AGPAT4) as a mitochondrial lysophosphatidic acid acyltransferase. We have also determined that AGPAT4 is important in the regulation of brain levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI), since levels of these glycerophospholipids were decreased significantly by 38%, 32%, and 52%, respectively, in the brains of Agpat4−/− mice. We investigated whether there were any impairments in cognition that may occur due to the significant loss of these three major glycerophospholipid species in the brains of Agpat4−/− mice. We tested the behaviour of Agpat4−/− mice and found that they have impaired spatial learning and memory as compared to wildtype littermates when assessed using the Morris Water Maze (MWM). Mechanisms underlying this effect were studied. No differences in whole‐body physiological parameters including respiratory capacity, food intake, motion and activity, or respiratory exchange ratio were evident. Similarly, we did not detect any changes with loss of Agpat4 in the brain mitochondrial content, or in the function of mitochondrial complexes as assessed in isolated brain mitochondria using high‐resolution respirometry. And, finally, we found that downstream targets of polyphosphorylated PI‐mediated signaling were also unchanged. However, Agpat4−/− mice did have a significantly lower brain content of the NMDA receptor subunits NR2A and NR2B, as well as the AMPA receptor subunit GluR1. Our findings indicate that neuronal membrane phospholipid content and composition are important in regulating NMDA and AMPA receptor content with implications for learning and memory.Support or Funding InformationThis work was supported by grants to RED from the Canada Foundation for Innovation – Leader's Opportunity Fund and Ontario Research Fund (Project#30259), and a Discovery Grant (#418213‐2012) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. EBM is the recipient of an NSERC Master's Scholarship (PGS‐M). RMB, JJAH, ASM, DB, PMM are the recipients of an NSERC Doctoral Scholarship (PGS‐D).
Investigation of a Role for AGPAT4 in Mouse Skeletal Muscle
Authors: Bradley Ryan M , Bloemberg Darin , Mitchell Andrew S , Fajardo Val A. , Henao Juan Jose Aristizabal , Bellissimo Catherine , Mardian Emily B , Paré Marie‐France , Bombardier Eric , Moes Katherine , Tupling A. Russell , Quadrilatero Joe , Stark Ken D , Duncan Robin E 
Published: November 2023

Acylglycerophosphate acyltransferases (AGPATs) are a group of homologous proteins that function in the de novo synthesis and remodeling of glycerophospholipids, catalyzing the acyl‐CoA‐dependent acylation of lysophospholipids. We have recently determined that AGPAT4 is mitochondrial, and is a canonical AGPAT, preferentially utilizing lysophosphatidic acid as an acyl acceptor. However, we found that it functions in brain to regulate levels of phosphatidylinositol, phosphatidylcholine (PC), and phosphatdylethanolamine (PE), rather than phosphatidic acid per se. Whether this effect is tissue specific has not yet been investigated. The presence of multiple AGPATs suggests that different homologues may feed phosphatidic acid into unique phospholipid species synthesis pathways, although this remains poorly characterized. Phosphatidic acid (PA) metabolism is of particular interest in skeletal muscle, where levels of this phospholipid are involved in mediating muscle force contractility, muscle size, and initiation of mTOR signaling, among other effects. We have therefore investigated a role for AGPAT4 in skeletal muscle biology. We performed immunohistochemistry on AGPAT4 in cross sections from C57Bl/6J mouse soleus and extensor digitorum longus (EDL) muscle, and found that it is predominantly visualized in type IIa muscle fibres, as compared to type I, IIb, or IIx muscle fibres. We next investigated the impact of Agpat4 gene ablation on muscle physiology and function. Force contraction was significantly reduced in soleus muscle but not EDL from Agpat4−/− mice, in agreement with the higher proportion of type IIa fibres in the former muscle type. Interestingly, however, our studies indicate no difference between Agpat4−/− mice and wildtype littermates in measures of time to exhaustion during treadmill running tests. We found a significantly higher content of PA and PE in the soleus muscles of Agpat4−/− mice, with no difference in TAG content. As well, there were no apparent differences in mitochondrial content or function, suggesting that mitochondrial function is not impaired by Agpat4 deficiency in skeletal muscle. This work is the first to characterize the role of a bona fide AGPAT in skeletal muscle function.Support or Funding InformationThis work was supported by grants to RED from the Canada Foundation for Innovation – Leader's Opportunity Fund and Ontario Research Fund (Project#30259), and a Discovery Grant (#418213‐2012) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. RMB, JJAH, DB are supported by an NSERC PGS‐D, VAF was supported by a CIHR Doctoral Scholarship, ASM was supported by an NSERC CGS‐D award, EBM was supported by an PGS‐M scholarship.
LplT‐Aas system: The primary mechanism for lysophospholipid remodeling in E. coli
Authors: Zheng Lei , Lin Yibin , Tong Shuilong , Guan Ziqiang , Bogdanov Mikhail 
Published: November 2023

Lysophospholipids (LPLs) are metabolic intermediates in phospholipid turnover and are derived from multiple endogenous and exogenous sources in bacterial membrane envelope. Distinct from their diacyl counterparts, these detergent‐like lipid molecules alter the local membrane properties and their accumulation may lead to severe membrane disruption and eventually cell lysis. LplT and Aas form a novel lysophospholipid remodeling system found exclusively in Gram‐negative bacteria. LplT is a member of Major Facilitator Superfamily and facilitates energy‐independent lipid flipping across the bacterial inner membrane. Aas catalyzes acyl transfer to LPL from acyl‐ACP on the cytoplasmic surface. The biological function and molecular mechanism of this lipid remodeling system remain largely unknown. We found that LplT‐Aas effectively facilitate remodeling of all three major bacterial phospholipids, PE, PG and cardiolipin, in E.coli. LplT imports lyso‐PE, lyso‐PG into the cells, which are subsequently acylated by the action of Aas to form their respective diacyl forms. We also identified a novel cardiolipin hydrolysis reaction by venom phospholipase A2 to form diacyl cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives are both translocated by LplT and then remodeled by Aas to form a triacyl form of cardiolipin. These results reveal the first cardiolipin remodeling mechanism in bacteria. LplT cannot transport lyso‐PC or lyso‐PA, suggesting its primary role in phospholipid repairing mechanism in the bacterial membrane envelope. To understand this novel LPL transport mechanism, we found that the substrate fatty acid chain is not required for LplT transport and its substrate binding was not inhibited by either orthophosphate or glycerol 3‐phosphate, suggesting that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Membrane phospholipids could not serve as a competitive inhibitor in vitro. This strict selectivity for lyso lipids allows LplT‐Aas to fulfill their efficient lipid repairing function in a membrane environment. Based on a structural model, we propose a “sideway sliding” mechanism to explain how a conserved membrane‐embedded α‐helical interface excludes membrane from the LplT binding site to facilitate efficient flipping of LPL across the cell membrane. Very recently, we found that LplT‐Aas play a potent role in maintaining membrane stability and integrity in the bacterial envelope and may be involved in the bacterial resistance against secretory phospholipase A2‐mediated host immune attack. These data demonstrate the first evidence for the biological function of this novel LPL remodeling system in E.coli.Support or Funding InformationNIH grant R01GM098572
Structure‐function Analysis of Yeast Pah1 Phosphatidate Phosphatase
Authors: Park Yeonhee , Han Gil‐Soo , Carman George M 
Published: November 2023

Mg2+‐dependent phosphatidate phosphatase (PAP), encoded by PAH1 in Saccharomyces cerevisiae, catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. The diacylglycerol produced in the reaction is utilized for the synthesis of triacylglycerol or the major membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. The PAP enzyme from yeast contains two conserved domains; the NLIP domain is found at the N‐terminus and the haloacid dehalogenase (HAD)‐like domain is found in the middle region of Pah1. The G80R mutation in the NLIP domain of Pah1 causes a 98% reduction in PAP activity along with other phenotypes characteristic of the pah1Δ mutation. The HAD‐like domain of Pah1 contains the DXDX (T/V) motif that is essential for catalytic activity. In this study, we carried out a structure‐function analysis of the conserved and non‐conserved regions of yeast Pah1. For constructs expressed and purified from Escherichia coli, G80R and G80A mutations, as well as deletion of the NLIP domain caused > 99% loss of PAP activity; the deletion of the non‐conserved region at the N‐terminus had little effect on PAP activity; and the deletion of the non‐conserved region at the C‐terminus caused a 3‐fold increase in PAP activity. The effects of the mutations on the in vivo function of Pah1 were examined by the ability to complement cold or warm sensitivities exhibited by the pah1Δ mutant. Cells expressing the deletion of the non‐conserved region at the N‐terminus complemented both the cold and warm sensitive phenotypes of the pah1Δ mutant, whereas the mutations in the conserved NLIP domain and the deletion of the non‐conserved region at the C‐terminus did not rescue the temperature sensitive phenotypes. The effects of the mutations on Pah1 PAP functions such as the synthesis of triacylglycerol and lipid droplet formation are being investigated.Support or Funding InformationSupported by NIH grant GM028140
Lantibiotic synthetase C‐like protein 2 (LanCL2) is necessary for 3T3‐L1 Differentiation to Adipocytes
Authors: Dutta Debapriya , Chen Jie , van der Donk Wilfred 
Published: November 2023

Adipocyte differentiation is a highly regulated process, necessary for metabolic homeostasis and nutrient sensing. The expression of peroxisome proliferator‐activated receptor gamma (PPARγ) and the subsequent transactivation of adipogenic genes is critical for adipogenesis, and is controlled through the coordinated efforts of CCAAT‐enhancer‐binding (C/EBP) proteins and a PPARγ ligand. In this study, we identified lanthionine synthetase C‐like protein 2 (LanCL2), as a positive regulator of adipogenesis in 3T3‐L1 cells. Knockdown of LanCL2, but not LanCL1, inhibited adipocyte differentiation, and this effect was not mediated through cAMP or Akt signaling pathways. The expression of early adipogenic markers C/EBPβ and C/EBPδ remained intact in LanCL2 knockdown cells, and an arrest in differentiation was observed at the stage of PPARγ activation. The addition of a PPARγ ligand, troglitazone rescued differentiation in LanCL2 knockdown cells and restored expression of late adipogenic markers. Furthermore, addition of troglitazone partially restored the transactivation potential of PPARγ. Taken together, our study reveals a novel role of LanCL2 in adipogenesis, acting through the ligand‐mediated activation and/or propagation of PPARγ signaling.Support or Funding InformationThe work was supported by the Howard Hughes Medical Institute (HHMI)
Use of the auxin‐induced degradation system to dissect factors important for exocyst assembly and localization
Authors: Heard Rachael D , Lepore Dante , Munson Mary 
Published: November 2023

Exocytosis is the delivery of cargo molecules from the Golgi to the plasma membrane. In the budding yeast, S. cerevisiae, the proteins regulating the process of exocytosis are found predominantly at the bud tips in G1 phase and mother‐bud necks in G2/M phase. The exocyst complex regulates localization and fusion of secretory vesicles at these sites. Exocyst is a multi‐subunit tethering complex that is required for post‐Golgi vesicle tethering at the plasma membrane. This hetero‐octameric protein complex is composed of the following subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. Our lab recently showed that each subunit has equal stoichiometry within the complex which is predominately soluble, and all but Sec3 are essential in maintaining the structure of the complex. Using electron microscopy, the assembly and architecture of exocyst shows that this complex forms an elongated conformation consisting of two modules of four helical rods each.We were interested in understanding the roles of different subunits in controlling the polarized localization of exocyst. Specifically, we asked what happens to the structure, stability and localization of exocyst when other subunits are degraded. An auxin induced degradation system (AID) will be used with the subunits Sec6, Sec3, and Exo70 to determine if the structure of the complex is affected by this degradation. The AID system allows for fast depletion of targeted proteins in response to the hormone auxin. We take advantage of the GFP tag on Sec8 in order to determine what subunits are still attached to Sec8 when degradation occurs, by pulling down the complex onto GFP‐bound nanobody beads. Next, we are determining where Sec8‐GFP is localized in the cell after Sec6, Sec3, and Exo70 degradation. These results will help elucidate the requirements for exocyst complex assembly, localization and secretory vesicle trafficking.Support or Funding InformationThis work supported by NIH R01GM068803 parent grant (to MM) and an R01GM068803‐11S1 Diversity Supplement (to RDH).
Mapping the Atg9 Binding Site on Atg11, a Central Organizer of the Selective Autophagy Initiation Complex
Authors: Meyer Mitchell , Winzeler Jasmine , Sheppard Damien , Backues Steven K 
Published: November 2023

Autophagy is a dynamic process by which cytosolic material is sequestered into double membrane vesicles called autophagosomes and then sent to the vacuole / lysosome for degradation. Selective autophagy, which targets specific unwanted components such as a damaged mitochondria, protein aggregates and pathogens, plays central roles in innate and adaptive immunity, regulation of inflammatory responses, pathogen clearance and protection against Parkinson's and Alzheimer's diseases. Autophagy is performed by dozens of autophagy‐related proteins (Atg) that assemble in a hierarchal fashion to form the pre‐autophagosomal structure (PAS). Atg11, a protein crucial for selective autophagy in the yeast Saccharomyces Cerevisiae, contains two coiled‐coil domains, CC2 and CC3, which are the primary binding sites used to organize the autophagy initiation complex and recruit Atg9‐containing vesicles to the PAS. It is not known whether Atg11 performs both of these processes simultaneously, or whether there is competition between its binding partners that would suggest a sequential model of action. Here we use a random mutagenesis / yeast‐2‐hybrid screen to determine the specific amino acid residues in the Atg11 CC2 region that are necessary for the interaction with Atg9 in order to more precisely map its binding site. Knowledge of the specific binding sites of all of Atg11's partners will help us to determine how Atg11 organizes these proteins to promote the creation of the autophagosome and accomplish selective autophagy.Support or Funding InformationNSF RUI Grant #1613653 “Scaffold or Assembly Line: How Does Atg11 Organize its Binding Partners for the Initiation of Selective Autophagy?” to S. K. Backues
Understanding the Effect of Community‐Based Health and Nutrition Program on Uptake of Iron and Folic Acid Supplementation and Breastfeeding Practices in a Pastoralist Community in Rural Ethiopia
Authors: Kulkarni Shibani , Frongillo Edward A , Girma Mamo 
Published: November 2023

Maternal and child health and nutrition are important issues to address in Ethiopia. Poor socioeconomic context and inadequate healthcare affects availability, access, and utilization of health services. The Community‐Based Maternal and Neonatal Health and Nutrition (CBMNH‐N) project by the Micronutrient Initiative aimed at strengthening the health system capacity to improve access and quality of maternal and newborn health and nutrition services by increasing the knowledge and skill of community health workers, traditional birth attendants, and health care providers. We undertook a qualitative inquiry from the endline data from four intervention and six comparison woredas to understand the effect of CBMNH‐N project on iron and folic acid supplementation and breastfeeding practices in the Afar community in Ethiopia. We used open coding to identifying themes and analyzed 46 key informant interviews from fathers, health extension workers, midwives, traditional birth attendants, and clan leaders, and 11 focus‐group discussions with pregnant and lactating mothers. Overall, mothers, health workers, and community members in the intervention woredas were more knowledgeable about the use of iron and folic acid supplementation and showed improvement in early initiation and exclusively breastfeeding practices as compared to the comparison woredas. Mothers noted that health extension workers were instrumental in increasing knowledge and awareness of iron and folic acid supplements, and trained traditional birth attendants helped with follow‐up and compliance in taking the supplements. Program efforts helped the community, including clan leaders to be more aware and supportive of the importance of iron and folic acid supplementation during pregnancy and the post‐partum period. Community members and clan leaders also noted the importance of early initiation and exclusive breastfeeding. Most respondents also discussed the change from traditional feeding practices such as providing the baby with goat milk to putting the baby to the breast immediately after birth and exclusively breastfeeding. Mothers, health extension workers, and traditional birth attendants were aware of the health benefits of exclusively breastfeeding. Several respondents attributed their knowledge of infant feeding to health education provided by the health extension workers and traditional birth attendants. Our findings suggest that community‐based programs can build capacity, increase knowledge, and improve health practices that can ultimately promote maternal and child health and nutrition.Support or Funding InformationFunding: Micronutrient Initiative
An Integrated Approach to Harmonize Nutrition Knowledge across Sectors in the Upper Manya Krobo District of Ghana
Authors: Birks Katherine , Marquis Grace , Matey Stephen , Owusu Raymond , Aidam Bridget , Aryeetey Richmond , Thompson‐Colón Theresa 
Published: November 2023

Determinants of nutrition are addressed by multiple service delivery platforms, including government sectors such as agriculture, education, finance, and health, in addition to community members and volunteers. Consistent messages must be integrated across these platforms to make education efforts more effective, particularly among vulnerable communities. As part of the Building Capacity for Sustainable Livelihoods and Health in Ghana (Nutrition Links) project, a needs assessment was conducted to identify knowledge gaps among service providers in the Upper Manya Krobo District. An integrated, multi‐level approach was subsequently developed to address these needs: (i) training workshops (e.g., Community‐Based Growth Promotion, Mother‐to‐Mother Support Group) for health sector staff and community volunteers; (ii) workshops to harmonize nutrition promotion messages (e.g., Essential Nutrition Actions) for non‐health sector (agriculture and finance institutions) staff; and (iii) Lots Quality Assurance Sampling and data analysis workshops for staff from all government sectors (health, agriculture, education, social development, environmental health, and governance) and finance institutions. These training sessions allowed participants to interact with multiple sectors and use local data to examine specific district‐wide nutrition concerns. Each session had on average 26 people in attendance and ranged from 4 to 7 days in duration. A questionnaire was administered before and after each training session and the results were compared to determine if participant knowledge had improved. All pre‐ and post‐training comparisons demonstrated an increase in participants' knowledge (p<0.0001). Training sessions with the greatest improvement included Mother‐to‐Mother Support Group for community members (mean increase = 70 ± 11 %), Essential Nutrition Actions for staff from the agriculture sector and finance institutions (mean increase = 60 ± 15 %), and Lots Quality Assurance Sampling (mean increase = 50 ± 14 %). Integrated, multi‐sector training sessions can be useful in harmonizing nutrition knowledge and may provide opportunities for improving communication and coordination of services across sectors.Support or Funding InformationGlobal Affairs Canada, McGill University, World Vision
Integrated DHS and National Micronutrient Survey: A Case Study from Malawi
Authors: Rhodes Elizabeth , Tripp Katie , Mshali Glory , Nyirenda Eunice , Suchdev Parminder 
Published: November 2023

ObjectiveTo document lessons learned from an integrated Micronutrient Survey (MNS) and Demographic and Health Survey (DHS) in Malawi from 2015–2016.MethodsWe conducted semi‐structured, in‐depth interviews with 26 individuals who planned and implemented the integrated MNS‐DHS. We used purposive recruitment to ensure that we captured a diversity of views and interviewed individuals from each of the partner organizations. Interviews were conducted in‐person or by telephone and audio recorded. We analyzed the data using thematic analysis to identify key themes related to the benefits, drivers of success, and challenges of an integrated MNS‐DHS.ResultsPreliminary findings indicate that an integrated approach increased data collection efficiency, reduced time burden for respondents, and enabled research inquiries into the socio‐demographic determinants of micronutrient deficiencies using DHS data. The perceived drivers of success for this integration included investment in coordination activities and regular communication among the sub‐teams carrying out the DHS and MNS. A primary challenge was overcoming implementation obstacles (e.g., delays in procuring equipment, lengthy decision‐making processes due to the command chain structure) and ensuring the DHS and MNS began and ended simultaneously.ConclusionsAn integrated MNS‐DHS was feasible, efficient, and acceptable to respondents in Malawi. Further, the integration of the surveys can help from a research perspective; it provides the opportunity to examine the determinants of micronutrient deficiencies using DHS data. The lessons learned in Malawi can inform other countries on how to best integrate MNS and DHS, which can provide nationally representative nutrition and inflammation biomarkers in the same cycle as DHS.Support or Funding InformationEmory University