bluuebunny/crossref_metadata_2025_split huggingface dataset:
Computational Modeling of Human Fatty Acid Transport Protein 2
Authors: Perez Vincent Michael , Puniya Bhanwar Lal , Helikar Tomáš , DiRusso Concetta C , Black Paul N 
Published: November 2023

Fatty acid transport protein 2 (FATP2) functions in the uptake and trafficking of fatty acids through vectorial acylation and belongs to the family of the solute carrier 27A proteins (SLC27A), which have been classified based on their ability to transport exogenous fatty acids into the cell. The FATP2 knockout mouse has depressed serum triglyceride levels and a reduced ability to assimilate dietary fatty acids suggesting a key role in fatty acid transport across the intestinal epithelium. To more fully understand the role of FATP2, we have developed in silico studies to inform the activity of this protein in vivo. Two techniques were employed in these studies: [1] Genome‐scale metabolic modeling, and [2] Ligand docking using a predicted three‐dimensional structure of FATP2. The human genome scale metabolic network model, Recon 2.2, was used to test systematic impacts of FATP2 perturbations on metabolism. Preliminary predictions using Recon 2.2 revealed that knocking out FATP2 is predicted to alter the metabolic flux of 436 reactions (215 up and 221 down). These reactions are encoded by 265 genes. Of interest, 22 of these genes are involved in fatty acid metabolism, 5 of which had increased function and 17 had decreased function after FATP2 perturbation. In addition, we completed preliminary ligand docking studies using the predicted structure of FATP2 retrieved from the Phyre2 server. Using this approach, the substrate binding sites within FATP2 (ATP, long chain fatty acid, and CoA) were predicted to be distinct, yet adjacent to core of the enzyme that may facilitate both substrate binding and product release. Collectively, predicted metabolic flux and ligand binding sites results within FATP2 provide key information that can be tested in vivo to better understand protein‐mediated transport of exogenous fatty acids.Support or Funding InformationThis work is funded by the Agricultural Research Division and Office of Research and Economic Development at the University of Nebraska.
The Ebola Virus Matrix Protein and Membrane Fluidity
Authors: Johnson Kristen A , Stahelin Robert V 
Published: November 2023

IntroductionWhile the Ebola virus is considered a rare and neglected disease, it received worldwide attention during the 2014–15 epidemic. This recent outbreak was eleven times larger than all previous Ebola virus outbreaks combined, taking a large toll on the lives and economies of Western Africa. Unfortunately, there are still no FDA approved therapeutics or vaccines for Ebola virus treatment. The most abundant protein of the virus, which was also the most conserved in amino acid sequence during the course of the outbreak, is the matrix protein VP40. VP40 is sufficient to form virus like particles from human cells independent of other viral proteins and has been suggested to be viable therapeutic target. We aim to better understand how VP40 is able to interact with lipid membranes consisting of different glycerophospholipids and sphingolipids and bend them to form the virus particle. The Stahelin lab has found that VP40 requires the anionic lipids phosphatidylserine (PS) and phosphatidylinositol (4,5)bisphosphate (PI(4,5)P2) at the plasma membrane for proper assembly. We are now investigating if the physical property of membrane fluidity plays a roll in VP40 function.ObjectiveWe are implementing several assays to investigate the role of membrane fluidity in VP40 membrane binding, oligomerization, and membrane bending.MethodsConfocal Laser Scanning Microscopy (CLSM), Number and Brightness analysis (N&B), Liposome Pelleting Assays, and Transmission Electron Microscopy (TEM).ResultsVP40 assembly is sensitive to membrane fluidity and has greater binding to ordered membranes than disordered membranes. When membrane fluidity is changed in cells, VP40 localization and large oligomers at the plasma membrane disassemble. We have identified FDA approved drugs that modulate membrane fluidity and are testing their efficacy against VP40 function.ConclusionsThis project aims to better understand VP40 membrane binding and bending functions in relation to membrane fluidity. We have observed that VP40 function is sensitive to changes in membrane fluidity in vitro and in live cells. We hope that a deeper understanding of VP40 dependency on membrane fluidity will allow for expedited drug discovery or reapplication of FDA Approved drugs.Support or Funding InformationFinancial Support: NIH AI081077
The Yeast Homolog of the Mammalian Oncogene, Bax Inhibitor‐1, Regulates the Unfolded Protein Response by Altering the ER Microenvironment.
Authors: Benko Savannah , Eagan David , Alisch Joseph , Brown Melissa , McCarthy Morgan , Cavedon William , O'Brien James , Ritzer Lukas , Berry B. Michael , Austriaco Nicanor 
Published: November 2023

Bax inhibitor‐1 (BI‐1) is an anti‐apoptotic gene whose expression is upregulated in a wide range of human cancers. The yeast homolog of BI‐1, yeast Bax inhibitor‐1 (BXI1), encodes a protein that belongs to the Bax Inhibitor (TMBIM) family of proteins. The crystal structure of a prokaryotic member of the family, BsYetJ, has revealed that the Bax inhibitor proteins are pH sensitive calcium leaks. Our studies have shown that yeast Bxi1p is localized to the ER and is involved in the unfolded protein response (UPR) that is triggered by ER stress. We have recently found that Bxi1p regulates the UPR downstream of endonuclease Ire1p clustering but upstream of HAC1 mRNA splicing. Studies with an ER localized calcium reporter and an ER redox reporter suggest that Bxi1p accomplishes this by altering the ER microenvironment.Support or Funding InformationOur laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.
ER stress and molecular targets of platinum anticancer compounds
Authors: Cunningham Rachael , DeRose Victoria 
Published: November 2023

Platinum anticancer compounds have been reported to induce ER stress responses in yeast and mammalian cancer cell lines. In this work, using modified ‘click’‐enabled Pt reagents, localization of Pt compounds in treated cells has been followed using post‐treatment fluorescent labeling. Platinum localization to both nucleoli and the ER are observed in Pt‐treated cancer cell lines. The ER stress response has been followed using Xbox1 and/or HAC1 splicing in response to platinum treatment. Finally, platinum association with proteins has been assessed using post‐treatment conjugation to biotin followed by pulldown and MS analysis. Taken together, the results of these studies show that treatment with platinum compounds induces an ER stress response, and support the hypothesis that direct targeting of ER proteins plays a role in this response.Support or Funding InformationSupport form the American Heart Association (predoctoral to R.C.) and the National Science Foundation (CHE‐CLP to VJD) is gratefully acknowledged.
Regulation of the mitochondrial protein quality control factor VMS1
Authors: Fredrickson Eric K. , Nielson Jason R. , Hill Christopher P. , Rutter Jared 
Published: November 2023

Mitochondrial function is constantly threatened by damaging agents, including mitochondria‐derived reactive oxygen species. This stochastic burden damages mitochondria, which left unchecked leads to mitochondrial dysfunction. The detrimental consequences of mitochondrial dysfunction are highlighted by its presence in many human diseases including cancer, Parkinson's Disease, and diabetes. To prevent mitochondrial dysfunction, cells have evolved mitochondrial quality control mechanisms, which include protein degradation. A critical component in mitochondrial protein quality control degradation is the protein VCP/Cdc48‐associated Mitochondrial Stress‐responsive (Vms1), which together with Cdc48, translocates to mitochondria under stress. Through the action of Cdc48, an ATPase critical for ER‐associated degradation, Vms1 promotes the proteasomal degradation of proteins embedded in the mitochondrial outer membrane. Deletion of VMS1 results in oxidative stress that leads to a shortened lifespan in yeast and nematodes, which is highly suggestive of mitochondrial dysfunction. Mitochondrial localization of the Cdc48/Vms1 complex depends on a highly conserved Mitochondrial Targeting Domain (MTD), which is inhibited by an intramolecular interaction with the N‐terminal Leucine Rich Sequence (LRS) of Vms1 in unstressed conditions. We aimed to further our understanding of the stress‐responsive localization of Vms1 to mitochondria through structural and biochemical studies. Toward this goal we have: (1) identified a modified mitochondrial sphingolipid that is necessary and sufficient for Vms1 mitochondrial localization, and (2) determined the structure of Vms11–417 (2.7Å, 25% R‐free), which includes the MTD/LRS interaction. These data suggest a model in which the novel MTD fold binds the sphingolipid receptor head group in a pocket that is masked in the auto inhibited state by the LRS and becomes accessible following a conformational change. This model is being tested by structure‐based mutagenic, biochemical, and genetic experiments.Support or Funding InformationNIH GM115129AHA Postdoctoral Fellowship 14POST20380216
Effects of Deceased Mitochondrial Ca2+ Uptake in an in vitro Model of Parkinson's Disease
Authors: de la Rosa‐González Nicole , Lee Amy , Joiner Mei‐ling 
Published: November 2023

Parkinson's disease is characterized by a progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) of the midbrain. All cells use Ca2+ among other ions to produce signals that regulate cell function. Ca2+ signals in neurons alter electrical activity and stimulate release of neurotransmitters. One of the roles of mitochondria is to help maintain appropriate concentrations of Ca2+ within the cell. The mitochondrial calcium uniporter (MCU) allows Ca2+ into the mitochondria matrix. Under stress conditions, more cytosolic Ca2+ is present in the cell, which can cause excessive Ca2+ uptake by the MCU. It is thought that this excess mitochondrial Ca2+ uptake causes mitochondrial dysfunction which may promote Parkinson's disease. A mechanism that regulates excess mitochondrial Ca2+ uptake in dopaminergic cells might reveal a new target for therapeutic strategies to halt the progressive degeneration of SNc neurons in Parkinson's disease. One such mechanism would be to inhibit MCU, then test if this inhibition protects the cell from high levels of cytosolic Ca2+. For this experiment, we inhibited the activity of the MCU by expressing a dominant‐negative (DN) MCU variant, restricting Ca2+ uptake into the mitochondria matrix of SH‐SY5Y cells, a dopaminergic cell line, that are treated with the dopaminergic neurotoxin, 6‐hydroxydopamine (6‐OHDA). Resulst showed that the expression of DN‐MCU does not reduce cell death, and in this case, is promoting cell death. The expression of DN‐MCU may generate persistent excessive levels of cytosolic Ca2+, generating stress conditions that ultimately lead to cell death and the promotion of Parkinson's disease.Support or Funding InformationThanks to Amy Lee's lab for the intensive training during the summer of 2016. Funding for this project was provided by the Summer Research Opportunities Program, a Carver Trust Collaborative Pilot Grant and, the Ponce Research Initiative for Scientific Enhancement (RISE) under the grant # 2R25GM096955.
Cardiac Mitochondrial Phospholipid Acyl Chains are Remodeled in Murine Obesity but do not Impair Supercomplex Formation, Respiratory Enzyme Activity, or Respiration
Authors: Sullivan Elizabeth M. , Fix Amy , Crouch Miranda J. , Sparagna Genevieve C. , Zeczycki Tonya N. , Brown David A. , Shaikh Saame Raza 
Published: November 2023

Cardiac phospholipids, notably cardiolipin, undergo acyl chain remodeling and/or loss of content in diseases such as aging, diabetes and cardiovascular disease. Acyl chain remodeling and loss of content are postulated to mediate mitochondrial dysfunction in these disease states. Surprisingly, less is known about how diet‐induced obesity influences phospholipid acyl chain composition and thereby mitochondrial responses. Here we tested if a high fat diet remodeled murine cardiac phospholipid acyl chain composition and the mitochondrial response. Several mitochondrial endpoints were assayed in response to a high fat diet. We found that mice consuming a high fat diet displayed 0.2–3.3 fold changes in acyl chain remodeling of cardiolipin, phosphatidylcholine, and phosphatidylethanolamine, measured via LC/MS, but no change in total phospholipid mass. Analysis of monolayers constructed using a Langmuir trough from the mitochondrial phospholipids of obese mice revealed an accompanying impairment in the packing properties of the membrane. Although there were alterations in the mitochondrial membrane composition and organization, the high fat diet, relative to the lean controls, had no influence on cardiac mitochondrial supercomplex formation, respiratory enzyme activity, or respiration. These effects were tissue specific however, since complex I to III liver respiratory enzyme activity was diminished with the high fat diet compared to the control diet. These results suggest that diet‐induced obesity leads to modest acyl chain remodeling of mitochondrial phospholipids but does not disrupt mitochondrial supercomplexes, respiratory enzyme activity, or respiration in the heart. Altogether, our findings support an emerging view suggesting that mitochondrial acyl chain remodeling is not a major driver of disrupting mitochondrial function.Support or Funding InformationFunding supported by NIH R15HL12292201 (SRS and DAB) and NIH R01HL123647 (SRS and DAB).
Single, long molecule PCR for the detection of rare mutations in Mitochondrial DNA
Authors: Li Housaiyin , Annis Sofia , Woods Dori , Tilly Jonathan , Khrapko Konstantin 
Published: November 2023

IntroductionOocytes contain the whole mitochondrial genomes of mammals, and the inheritance pattern of mitochondrial DNA in oocytes do not follow Mendel's rules of heredity. Mitochondrial DNA is maternally inherited in the oocytes, and only mother's mitochondrial genome can be passed to the next generation. The mitochondrial genome is a closed, circular piece of DNA which has a length of about 16kb, and there are hundreds to hundreds of thousands mitochondrial DNA in a single cell. Mitochondrial DNA mutation rate is generally difficult to detect and is often over‐estimated. This research is focusing on developing a sequencing strategy to accurately get mitochondrial DNA mutation rate of single, long molecules from a wild type mouse germline.Research ObjectivesThe research goal is to develop an efficient and accurate way to assess the mutation rate of mitochondrial DNA from wild type mouse oocytes.MethodsWe isolated and individually lysed single oocytes from a wild type female mouse. A serial dilution of the DNA sample was made to find its working concentration for generating single, long molecules, and prepared a PCR reaction by using the specific DNA sample concentration. A high‐fidelity DNA polymerase was used to generate 16kb PCR products originating from single template molecules. Sanger sequencing was used to sequence these DNA molecules.ResultsWe used single molecule PCR to amplify the mitochondrial DNA from three wild type mouse oocytes and sequenced all the PCR products, yielding a total coverage about 330,392bp. Twenty‐two mitochondrial genomes from three oocytes were sequenced, with an average length of about 15,017bp. Mouse mitochondrial DNA length is 16,299kb, so each of our mitochondrial DNA molecule has an average coverage about 92.13% of the whole mitochondrial DNA genome. Only two mutations were found in two separate mitochondrial genomes, and the resulting mutation rate of mitochondrial DNA in the oocytes was 6.05×10^‐6. This is a very low mutation rate across over 300,000bp.ConclusionThe mutation rate of mitochondrial DNA in wild type mouse germline is remarkable low in our research by using single, long molecule PCR. This PCR method allows for the accurate detection of mutations in the mouse mitochondrial DNA in oocytes. Our data indicates that, by using single, long molecule PCR combined with Sanger sequencing, we can accurately get informations from a molecule that has the most coverage of its genome. Additionally, we used a high‐fidelity enzyme to serve as DNA polymerase in the PCR reaction, thus preventing the chance of artificial mutations from the beginning of the PCR reaction. With the right procedure of our sequencing strategy, the rare mutations and the mutation rate in single germline cells can be accurately determined.Support or Funding InformationEllison Medical Foundation; NIH R37‐AG012279
Fis1 activity in pre‐ and post‐assembly of the yeast mitochondrial fission machinery.
Authors: Harwig Megan Cleland , Hill R. Blake 
Published: November 2023

Far from static organelles, mitochondria frequently undergo fusion and fission events to help maintain a healthy mitochondrial network. These processes are vital for a variety of cellular tasks including maintenance of the mitochondrial genome as well as the mitochondrial membrane potential. In yeast, a mitochondrial outer membrane fission protein 1 (Fis1) is essential for mitochondrial fission; however, the precise role of Fis1 has been a matter of debate. Fis1 is highly conserved and interacts with two cytosolic fission proteins, Mdv1 and Dnm1 to possibly recruit them to sites of scission, however, the details of these interactions are not well understood. We recently demonstrated that mutating a key residue (E78) of Fis1 helped demonstrate a post‐recruitment role for Fis1. Here, our analyses with additional point mutations of Fis1 points to a revised model for Fis1 activity in yeast mitochondrial fission.Support or Funding InformationNIH R01 GM067180
A plasma proteome is associated with carotenoid status in young Nepalese children
Authors: Eroglu Abdulkerim , Schulze Kerry Jean , Cole Robert N. , Christian Parul , Nonyane Bareng Aletta Sanny , Lee Sun Eun , Wu Lee Shu Fune , Khatry Subarna , West Keith P. 
Published: November 2023

BackgroundDietary carotenoids are naturally occurring pigments that may function in the body as vitamin A precursors, antioxidants, anti‐inflammatory agents or biomarkers of recent intake of vegetables and fruits, and thus be important for population assessments of nutritional status. Yet, carotenoid status is rarely assessed due to cost and difficulty. Quantitative plasma proteomics may offer an indirect approach to assess carotenoid status by identifying potentially predictive plasma proteins.ObjectiveTo detect and quantify a set of plasma proteins associated with major dietary carotenoids including β‐carotene, α‐carotene, β‐cryptoxanthin, lycopene, lutein and zeaxanthin in a cohort of 500 6–8 year old rural Nepalese children.DesignStudy children were born to mothers enrolled in an earlier antenatal micronutrient trial in Sarlahi, Nepal. Plasma carotenoid concentrations were measured by conventional HPLC analytical methods. For analysis of the plasma proteome, plasma aliquots were immunoaffinity‐depleted of six high abundance proteins, digested with trypsin, labeled with isobaric mass tags (iTRAQ 8‐plex reagents), and analyzed for protein relative abundance on tandem mass spectrometry (MS/MS). Linear mixed effects (LME) models were used to derive estimates of relative protein abundance, determine the direction and strength of correlation between individual proteins and plasma log2 concentration of each carotenoid, accepting a false discovery rate of q <0.10, and predicting, when feasible (AIC ≥ 30, q<0.10, p<0.0004), plasma carotenoid distributions.ResultsWe quantified 982 plasma proteins in >10% of all child samples. Fifty‐one proteins were correlated with log2 β‐cryptoxanthin, primarily involved in lipid transport, oxidative stress, immune system regulation, and lipid peroxidation. Three modeled plasma proteins (APO‐A1, protein phosphatase, Mg2+/Mn2+ dependent, 1M and leucine rich repeat containing 47) explained 50% of the variance in log2 β‐cryptoxanthin (model R2=0.50). Plasma β‐carotene was associated with 4 proteins (orosomucoid 1, APO‐A1, pyruvate kinase, muscle [PKM] and TNFAIP3 interacting protein 1[TNIP1]). Two (APO‐A1 and PKM) were predictive of plasma β‐carotene (R2=0.66). Eleven plasma proteins were correlated with lutein/zeaxanthin, including APO‐A1, TNIP1 and 3 were modeled to be predictive of plasma concentration (model R2=0.55) (all q<0.10). No predictive models proteomes were identified for α‐carotene and lycopene.ConclusionsWe identified plasma proteomes that varied in size and were moderately predictive of plasma concentrations of β‐cryptoxanthin, β‐carotene and lutein/zeaxanthin. Plasma proteomics may offer an approach to assess carotenoid status in undernourished populations.Support or Funding InformationSupported by the Bill & Melinda Gates Foundation (OPP5241).
Carotenoids and apo‐carotenoids in human plasma after continued consumption of high β‐carotene or high lycopene tomato juice
Authors: Cooperstone Jessica L , Riedl Ken M , Cichon Morgan J , Francis David M , Curley Robert W , Schwartz Steven J , Novotny Janet A , Harrison Earl H 
Published: November 2023

Many studies have found associations between diets rich in carotenoids and a decreased risk for chronic diseases. Recent work has suggested that putative carotenoid metabolites resulting from the cleavage of β‐carotene (β‐C) and lycopene (LYC) by β‐C 15,15′‐oxygenase or β‐C 9,10‐oxygenase may be responsible for bioactivity attributed to the parent carotenoids. As a result, significant efforts have attempted to quantify these elusive carotenoid metabolites in vivo, with limited success. The objective of our study was to quantify both carotenoids and apo‐carotenoids in the blood of individuals consuming β‐C and LYC derived from whole foods. Healthy non‐smokers (n=35) consumed a low carotenoid diet for two weeks, and then consumed the same basal diet for 4 weeks with one of three juices: high β‐C tomato juice (360 mL, 30 mg β‐C, 35 μg apo‐carotenoids/day) or high LYC tomato juice (360 mL, 43 mg LYC, 120 μg apo‐lycopenoids/day) or a calorie matched cucumber juice control (360 mL, no detectable carotenoids). Targeted HPLC‐MS/MS methods were developed to quantitate apo‐carotenoids (apo‐8′, 10′, 12′, 14′, 15′‐carotenals, 13‐carotenone) and apo‐lycopenoids (apo‐6′, 8′, 10′, 12′, 14′, 15′‐lycopenals, 13‐lycopenone) in blood plasma with a limit of quantitation (LOQ) of ~50 pM. Plasma carotenoids were determined by HPLC. After four weeks, plasma β‐C levels reached 0.36, 0.62 and 3.18 μM in groups fed control diet, high‐LYC and high β‐C tomato juice, respectively, significantly increasing in the high‐β‐C group (P<0.001). Plasma lycopene reached 0.25, 1.40 and 0.58 μM in the control, high‐LYC and high β‐C groups, respectively, significantly increasing in the high‐LYC group (P<0.001). Plasma retinol was unchanged during this intervention, and not different among groups. Apo‐10‐ and 12‐carotenal were the only carotenals detected in plasma but not above our LOQ. Apo‐6′‐lycopenal was the only lycopenoid detected. Apo‐13‐carotenone was quantifiable in all subjects, measuring 0.46 ± 0.11 nM in control and 0.53 ± 0.12 in high‐LYC groups but higher (P<0.05) in those on the high β‐C juice at 1.01 ±0.27 nM at the end of study. These data suggest blood apo‐13‐carotenone increases with supplemental β‐C intake, but its occurrence is not dependent on it. New data also suggests that apo‐13‐carotenone can be derived from vitamin A.Support or Funding InformationThis study was supported by NCI‐USDA‐IAA #ACN15005‐001‐00000. Carotenoid and apocarotenoid analyses were conducted within the Nutrient and Phytochemical Analytical Shared Resource (NPASR) at the OSU Comprehensive Cancer Center (NIH P30 CA016058).