bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lysosome enlargement in PIKfyve inhibited cells proceeds through homotypic lysosome fusion rather than growth of individual lysosomes
Authors: Saffi Golam 
Published: November 2023

Phosphoinositides are important lipids that modulate various functions including signal transduction, membrane trafficking, and organelle identity. Phosphatidylinositol‐3,5‐bisphosphate [PI(3,5)P2] is a phosphoinositide found on lysosomes where it controls a wide array of functions such as stress‐induced signalling, autophagic flux, ion‐channel activity, trafficking and endolysosome morphology. Lysosomes are degradative organelles where macromolecules, foreign pathogens, and intracellular organelles arrive through endocytosis, phagocytosis and autophagy for degradation, respectively. PI(3,5)P2 is synthesized from phosphatidylinositol‐3‐phosphate [PI(3)P] via the lipid kinase PIKfyve. One of the most dramatic phenotypes in cells deficient for PI(3,5)P2 synthesis is the enlargement of lysosomes. However, the dynamics and mechanism of lysosome enlargement due to PIKfyve inhibition remains mostly unexplored. We aimed to distinguish whether lysosome swelling proceeds through growth of individual lysosomes or if multiple lysosomes fuse together culminating in enlarged lysosomes in cells treated with apilimod, a specific inhibitor of PIKfyve. Using a combination of spinning disc microscopy and volumetric/particle counting analysis, we show that lysosome enlargement in PIKfyve‐inhibited cells results from homotypic lysosome fusion rather than by growth of individual lysosomes. This is supported by a decrease in lysosome number and increase in volume of individual lysosomes, while total lysosome volume remained constant. These effects are reversed upon washing of apilimod. Consistent with this, disruption of microtubules and microtubule‐associated motor complexes, dynein and kinesin‐1, reduced the rate of lysosome enlargement in PIKfyve‐inhibited cells, while accelerating shrinkage of lysosomes during apilimod removal. We propose a model whereby lysosomes undergo constant kiss‐and‐run (fusion and fission) events. In the absence of PIKfyve, lysosomes “kiss” but fail to “run”, leading to a shift towards homotypic fusion, which increases the size of individual lysosomes and a reduction in total lysosome number.Support or Funding InformationThis work was made possible by NSERC and Canada Research Chair Program.
Stereochemical characterization of acyl‐phosphatidylglycerol and bis‐(monoacylglycerol)phosphate using nuclear magnetic resonance
Authors: Peros Colin S , Garrett Teresa A 
Published: November 2023

Gram‐negative bacteria such as Escherichia coli contain mainly glycerophospholipids (GPLs), with phosphatidylethanolamine (PA), phosphatidic acid (PA) and phosphatidylglycerol (PG) comprising about 95% of the total lipids in the cell. Low level (~0.1% total GLPs) lipids are formed when an additional acyl chain is added to the headgroup of PG or lyso PG to form acyl PG and bis(monoacylglycero)phosphate (BMP), respectively. Two enzymes that affect headgroup‐acylated GPL levels are E. coli PldB and the Arabidopsis thaliana lysophospholipid acyltransferase At1g78690. It was previously assumed that the stereochemistry of the sn‐2 and sn‐2′ positions of the substrates involved in the lipids' biosynthesis set the configuration of the glycerols but given that there is precedence for inversion of the glycerol stereochemistry by the enzymes involved in BMP formation in eukaryotes, we wanted to directly investigate the stereochemistry of BMP and acyl PG formed in E. coli. Furthermore, other works suggest that during extraction the acyl chain can migrate from the sn‐2′ position to the sn‐3′ position. Understanding the exact structure, including the stereochemistry of the sn‐2 and sn‐2′ position, will allow us to fully understand the function and biosynthetic origin of the acyl‐PG and BMP isoforms. We are structurally characterizing acyl‐PG and BMP using nuclear magnetic resonance (NMR) following covalent modification of the glycerol hydroxyls of the polyglycerolphosphate backbone. The enantiomers of α‐methoxyphenylacetic acid (MPA) will be added to the hydroxyl of the base deacylated isolated acyl PG or BMP molecules, followed by 1H‐NMR on the resulting conjugated lipid. The covalently‐bonded MPA leads to shielding and deshielding on the proton of the chiral glycerol carbons that allow discrimination of the stereochemical configuration by comparing the chemical shifts of the (R)‐ and (S)‐MPA modified molecules. Determination of the stereochemistry will give insight into the mechanism of biosynthesis, origin, and function of these low abundance lipids.Support or Funding InformationNational Science Foundation Research at Undergraduate Institutions Grant #1516805 and #1152463, and National Science Foundation Major Instrumentations Grant #1039659 and #1526982
Structure and Organization of Mitochondrial Cristae Membranes
Authors: Ryan Robert O. , Ikon Nikita 
Published: November 2023

A fundamental question in biology, under investigation for over six decades, is the structural organization of mitochondrial cristae (singular = crista) membranes. Long known to harbor electron transport chain proteins (ETC), crista membrane integrity is key to establishment of the proton gradient that drives oxidative phosphorylation. Visualization of cristae morphology by electron microscopy/high voltage electron tomography has provided evidence that cristae are tube‐like extensions of the mitochondrial inner membrane (IM) that project into the matrix space. The signature lipid component of mitochondria, cardiolipin, comprises 18% of IM phospholipid mass. Unlike other glycerophospholipids, cardiolipin possesses two phosphatidyl moieties that share a glycerol head group. This combination of compact anionic polar head group and four esterified fatty acyl chains results in a distinctly cone‐shaped molecule. Based on its properties, cardiolipin has been classified as a “non‐bilayer” phospholipid. Interestingly, phosphatidylethanolamine (PE), which comprises 34% of IM phospholipid mass, is also a cone‐shaped, non‐bilayer phospholipid. How does a membrane that contains ~50% non‐bilayer phospholipids maintain a bilayer structure? Whereas a planar bilayer would not be able accommodate such high percentages of PE and cardiolipin, these phospholipids are well suited to reside in, and stabilize, a curved membrane. Taken together, ultrastructural and lipid compositional data provides support for a model of cristae as continuously curved cylinder‐shaped bilayer membranes capped by a dome‐shaped membrane tip. In this model of crista membrane structure cardiolipin and PE partition into the negatively curved monolayer leaflet facing the crista lumen while the opposing, positively curved, matrix facing monolayer leaflet contains predominantly phosphatidylcholine (~80%), with lesser amounts of phosphatidylinositol and phosphatidylserine. In addition to housing ETC proteins, cristae are home to the dynamin‐like GTPase, Opa1, the transacylase, tafazzin, the scaffold protein, prohibitin and the molecular chaperone, DNAJC19. These proteins function in distinctive ways to establish and maintain crista membrane lipid (i.e. cardiolipin) composition and structure. The accumulation of negative curvature lipids along with the presence of protein factors that establish and maintain crista structure, have a direct impact on mitochondrial functions including oxidative phosphorylation, fusion/fission and apoptosis.Support or Funding InformationNIH R37 HL64159NIH T32 DK061918
Characterizing Lipid Production in Chlorella Vulgaris Exposed to Sethoxydim, an Acetyl‐CoA Carboxylase Inhibitor
Authors: Smythers Amanda L. , Holland Aaron T. , Roberts Aaron , Kolling Derrick R.J. 
Published: November 2023

Microalgae, with their ability to grow in adverse environments using a variety of water sources, can be a lucrative energy source that will not compete with agriculture (Sharma et al. 2012). In order to realistically produce fuel on an industrial scale, microalgal lipid production must be increased. Microalgae with 70% oil by biomass would satisfy 50% of the United States' transport fuel needs while using less than 2% of the existing cropping area used for fuel production (Chisti 2007).The objective of this work is to characterize the process through which Chlorella vulgaris, a unicellular green alga, produced elevated lipid levels under acetyl‐CoA carboxylase (ACCase) inhibition by sethoxydim, a common herbicide. Previous work aimed to use applied natural selection, or directed evolution, to create a modified C. vulgaris culture with increased ACCase activity that would increase the overall lipid yield for use in biofuels. In the preliminary experiments, a puzzling phenomenon emerged: cells exposed to sethoxydim produced over 3X more lipids than unexposed cells. Furthermore, there were metabolic changes in sethoxydim‐exposed cells: rapid chlorosis indicated a decrease in photosystem II activity that was absent in the control. Further experimentation confirmed that this paradox is not an artifact, but rather a novel response due to the presence of sethoxydim.The current experimental approach is twofold: to identify the chemical compounds in sethoxydim‐exposed lipid samples and to characterize the mechanism through which this lipid accumulation occurs. Gas chromatography‐mass spectrometry will be used for identification. This is particularly useful in determining the presence of non‐membrane fatty acids, including triacylglycerides, the lipid most desired for biodiesel. Since the herbicide inhibits ACCase, based on currently known biological information, there should be few fatty acids in the experimental sample as compared to the control. If that is the case, the next step will be to look for the presence of other lipids, such as isoprenoids.Isoprenoids represent a variety of lipids formed from isopentenyl diphosphate (IPP), a 5‐C building block. Currently, two pathways are known for the biosynthesis of IPP, the mevalonate pathway and the methylerythritol phosphate pathway (Lohr et al. 2012). The mevalonate pathway is of particular interest to this experiment as it begins with the condensation of 3 acetyl‐CoA molecules. If acetyl‐CoA is building up in the cell and cannot be catalyzed by ACCase, it would make sense to follow this pathway toward the synthesis of isoprenoids.The increased lipid mass under sethoxydim stress was highly unexpected and the mechanism through which it occurs is not yet found in literature. Identifying the chemical makeup of these lipid samples and characterizing this mechanism could lead to fundamental science that could potentially be used in industrial applications.
Stronger Together: The role of cysteine residues in Ebola viral matrix protein VP40
Authors: Baker Sarah Catherine Briann , Johnson Kristen , Stahelin Robert 
Published: November 2023

IntroductionOne hallmark of an intracellular Ebola infection is the formation of virus‐like particles, or VLPs, on the extracellular surface of infected cells. The formation of VLPs is known to be facilitated by oligomerization of the viral matrix protein VP40. The tendency of VP40 to oligomerize into dimers, hexamers, and octamers is well‐established, yet the mechanism of this oligomerization is not fully understood. An examination of the secondary structure of VP40 reveals the presence of only two cysteine residues, the positions of which are highly conserved across all five sub‐species of Ebola virus. In the protein's fully‐folded conformation, these cysteine residues would be solvent exposed in the dimer conformation. In the 4LDB VP40 crystal structure, these residues are shown to be at the interface of the VP40 tetramer.ObjectiveWe aim to better understand the role of the cysteine residues on VP40.MethodsSite directed mutagenesis, protein purification, lipid binding assays, confocal imaging.ResultsWhen VP40 cysteines are reduced with DTT, lipid binding in‐vitro is changed. We aim to understand how this affects VP40 function in cells my comparing WT VP40 and VP40 with cysteine to alanine mutations. Our preliminary data suggests that VP40 cysteine residues are important for protein function. Future work in cellular and lipid‐binding assays with WT and mutant VP40 may shed further light on how the cysteine residues interact at the membrane, and analysis of wild‐type proteins exposed to reducing agents may elucidate the importance of disulfide bonds specifically.Support or Funding InformationFinancial Support: NIH AI081077
Ceramide Induced Regulated Alternative Translocation of TM4SF20 and Beyond
Authors: Denard Bray , Chen Qiuyue , Lee Ching‐En , Han Sungwon , Ye James , Ye Jin 
Published: November 2023

TM4SF20 (transmembrane 4 L6 family 20) is a polytopic membrane protein that normally inhibits proteolytic processing of CREB3L1 (cAMP response element‐binding protein 3‐like 1), a membrane‐bound transcription factor that inhibits cell proliferation and activates collagen synthesis. Recently, we have discovered that ceramide stimulates CREB3L1 cleavage by inverting the topology of TM4SF20 in membranes. In the absence of ceramide, the N‐terminus of the first transmembrane helix of TM4SF20 is inserted into the endoplasmic reticulum lumen. In the presence of ceramide, the N‐terminus of the first transmembrane domain of TM4SF20 is exposed to cytosol. Consequently, the membrane topology of TM4SF20 is inverted, and this form of TM4SF20 stimulates CREB3L1 cleavage. We designate the mechanism causing regulated inversion of the membrane topology as “Regulated Alternative Translocation”.We identified a GXXXN motif in the first transmembrane helix of TM4SF20 that is critical for Regulated Alternative Translocation of TM4SF20. Bioinformatics analysis revealed hundreds of membrane proteins containing this motif. Our result suggests that Regulated Alternative Translocation could be a general mechanism regulating a large family of transmembrane proteins.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (GM‐116106, AI‐70116, and HL‐20948)
Effectiveness of sub‐therapeutic staurosporine on inhibition of budding and replication of lipid‐enveloped viruses
Authors: Fraser Mark , Stahelin Robert 
Published: November 2023

Infectious Diseases are a major cause of morbidity and mortality internationally, disproportionately affecting developing countries. Many of the deficits caused by infectious diseases are due to viruses, specifically lipid‐enveloped viruses. By identifying a common infectious pathway via viral phosphatidylserine (PS) to host TIM protein receptor and a class of drug that was recently found to potentially interfere with this common pathway, it was hypothesized that staurosporines, specifically UCN‐01, can inhibit viral infection and replication of lipid‐enveloped viruses dependent on viral PS and host TIM receptor interactions, including but not limited to Ebola virus, Marburg virus, HIV‐1, Dengue virus, Chikungunya virus, and Zika virus. The effects of UCN‐01 on the effects of PS localization in mammalian cells, the effects of UCN‐01 on GFP tagged viral matrix proteins in cell culture, and the efficacy of UCN‐01 on live virus infected cells are being studied. It was shown using confocal microscopy that UCN‐01 is able to significantly reduce plasma membrane localization of both ebola VP40 and HIV‐gag proteins in HEK cells. Flow cytometry was also performed on samples under the same experimental conditions showing alterations in external cell membrane PS expression and drug toxicity over a range of concentrations. This pathway was further supported by previous work done in this lab by showing that Ebola VP40 protein induces PS expression in a cell culture model by using confocal microscopy and flow cytometry. By testing UCN‐01 to determine cell toxicity using Caspase 3 and MTT assays, the dosing used in these experiments were shown to affect cellular metabolism, but at sub‐lethal levels to the cells. By investigating how UCN‐01 affects cell lipidomics using mass spectrometry, more can be understood about the mechanism of action of these drugs interfering with PS and their appropriateness for pursuing as broad class antivirals. In addition, UCN‐01 will be studied in how it can interfere with virus like particle production. After showing efficacy at sub‐toxic levels in the matrix protein model, live virus studies were initiated and have already shown complete viral infectivity inhibition using low MOIs of Dengue virus using TCID‐50 assays. These studies, if successful at decreasing infectivity or viral replication, could lead into future animal model studies that may be taken into clinical trials, producing a broad‐spectrum anti‐viral therapy that would have the potential to eliminate a large portion of the global health burden.Support or Funding InformationFellowship provided by University of Notre Dame Eck Institute for Global Health
Neuroprotective effects of lutein in neonatal hypoxic‐ischemic brain injury
Authors: Foster Chase H , Sambalingam Devaraj , Gong Xiaoming , Rubin Lewis P 
Published: November 2023

BackgroundHypoxic‐ischemic (HI) brain injury is a leading cause of neonatal mortality and, in survivors, of severe neurological disability and it is a major source of developmental and cognitive impairments in children and adults. In HI, injury pathways include excitotoxicity, inflammation, and oxidative stress. The carotenoid lutein (and its isomer zeaxanthin) are selectively taken up and accumulated in the brain. Based on the lutein effects in improving later life cognition, we postulated lutein may have neuroprotective effects in HI.MethodsWe used a validated neonatal rat model of HI brain injury induced by unilateral carotid artery ligation (ischemia) on postnatal day 7 (P7) and subsequent exposure to 8% oxygen (hypoxia) for 2.5 h. Dams were fed a lutein‐rich or control chow diet throughout pregnancy and lactation. From P7 (HI) to P10 (sacrifice), nursing pups also received lutein or vehicle by intra‐peritoneal injection. Brain sections were examined with H&E, Nissl, and immunohistochemical staining and apoptotic assays. Protein expression (e.g., HIF‐1α, nNOS) was evaluated by western blot. RT‐qPCR expression arrays were used to evaluate changes in expression of targeted genes involved in hypoxic, apoptotic, and neuroinflammatory pathways.ResultsLutein supplementation resulted in lutein accumulation in milk and neonatal brain tissues. Lutein treatment, in this model, reduced histological brain injury scores and suppressed: (a) RNA and protein expression of key neuroinflammatory mediators, (b) expression of hypoxia‐inducible proteins (including HIF‐1α), and (c) microglial (inflammatory) activation in several brain regions particularly susceptible to HI injury. Additionally, lutein exerted anti‐apoptotic effects including altered expression of several apoptosis‐related genes: BIK, Birc5, Bnic2, and Casp2–6.ConclusionThese results demonstrate: (1) there is maternal/fetal transfer and uptake of dietary lutein into milk and brain in the rat; (2) this rodent model is informative for testing the effects of lutein on HI brain injury and neurodevelopment; and (3) pre‐ and peri‐injury lutein administration suppresses multiple deleterious response pathways in HI. These findings suggest lutein supplementation is a promising preventive and therapeutic strategy for limiting pathological consequences of HI brain injury. This model including manipulation of lutein intake is applicable to other brain disorders including traumatic brain injury, intracranial hemorrhage, and stroke.Support or Funding InformationSeed grant from Texas Tech University Health Sciences Center El Paso
High fat diet induces gender‐specific differential gene expression in Drosophila melanogaster brain
Authors: Stobdan Tsering , Azad Priti , Heinrichsen Erilynn , Sahoo Debashis , Hartley Iain , Zhou Dan , Haddad Gabriel G 
Published: November 2023

BackgroundThe high‐fat diet is ranked as the second most lethal habit after smoking. Beside obesity, which counts for more than one‐third (34.9% or 78.6 million) of US adults, high‐fat diets also accounts for most of the leading causes of death e.g., heart disease, diabetes, and cancer. Recent finding also suggests that regularly eating high‐fat diet could damage brain cells, however the knowledge on its causes and effects are limited.ObjectiveIn this study we hypothesize that the whole transcriptome analysis of Drosophila melanogaster brain kept on high‐fat diet would provide us with clues pertaining to metabolic disturbances that are potentially organ‐specific.Method & ResultsAdult males and females, 1–3 days old, Drosophila melanogaster collected and kept the experimental group on high‐fat diet (20% w/v coconut oil) and controls on regular diet for one week. Heads and body were collected on liquid N2 for RNA isolation. In this particular study, we compared the transcriptomic profile of the heads of flies kept on high‐fat vs regular diet, both in males and females and used a Boolean method for mining specific genes and pathways. A considerable number of genes were differentially expressed in the head as a result of being on high‐fat diet. Interestingly, these genes were gender specific, with only few that were common between male and female heads. The composite network analysis of these genes reveals that the male specific genes are involved in odorant, pheromone and chemical sensing. In the females the genes were limited to lipid metabolic processing.ConclusionOur results demonstrate, for the first time to our knowledge, that high‐fat diet triggers the differential expression of genes in brain and these were gender‐specific. It is worth speculating that such gender‐specific variability in gene expression, particularly in the brain, could pave the way to understanding gender‐specific metabolic disturbances as a result of high fat diet.Support or Funding InformationNIH, Emerald Foundation
Migraine as a Metabolic Disease
Authors: Stanton Angela A 
Published: November 2023

Migraine is classified as a neurovascular disease and most treatment is aimed at pain prevention or release. However, the cause of migraine is poorly understood and medicinal treatments often fail. Here we identify that genetic metabolic sensitivities are the cause of migraine and demonstrate that migraine treatment and prevention is possible without the use of medicines. We summarize available research and logically lead to key points that show that migraine is a metabolic disease. Although we did not run laboratory experiments, we used surveys and self‐reporting in a migraine group on Facebook with thousands of members of whom several hundred agreed to participate with surveys, reports, and metadata research. The outcome showed that genetic mutation caused metabolic changes make migraineurs sensitive extremely to carbohydrates and to the difficulties associated with the maintenance of electrolyte homeostasis, which is easily disrupted by the consumption of carbohydrates. When migraineurs became aware and reduced their dietary carbohydrates, managed their electrolytes by increasing sodium and potassium rich foods, increased water and fat in their diet, migraines were prevented. The changes were strong enough to encourage migraineurs to quit all their medicines, surgically remove their neuronal stimulators, and remained migraine free—some almost three years by now, the start of our research.Support or Funding InformationNo funding was requested or providedNo financial support was receivedNo conflict of interest