bluuebunny/crossref_metadata_2025 huggingface dataset:
Cancer‐Specific Cell Death in Response to Palmitoylcarnitine is Associated with Increased Mitochondrial Hydrogen Peroxide
Authors: Turnbull Patrick C , Perry Christopher G. R.
Published: November 2023

Rationale and Hypothesis

A benchmark of cancer cells is the reliance on glycolysis characterized by decreased mitochondrial oxidative phosphorylation (eg. Warburg effect). Recent evidence suggests that fatty acids (palmitoylcarnitine; PCarn) may force a shift to mitochondrial oxidative phosphorylation in HT29 colon cancer causing cell death, yet non‐cancer cells appear to be resistant. These results present the intriguing possibility that ‘lipid‐therapy’ might be well tolerated by healthy cells while retaining potency in cancer. However, the mechanism of lipid‐induced cancer cell death is not known. Glutathione is the main antioxidant found within the cell, we hypothesize that cell survival is determined by heterogeneity of basal intracellular glutathione. We theorize that cells with low basal glutathione will be more susceptible to elevated levels of mitochondrial H2O2 from beta‐oxidation of PCarn, whereas cells with higher glutathione will be more resistant to elevated levels of H2O2.

Experimental approach

We developed a live cell kinetic assay to measure H2O2 emission in real time; using this assay we exposed three different cancer lines (HT29 colorectal adenocarcinoma, MCF7 breast adenocarcinoma and HepG2 hepatocellular carcinoma) and one non‐cancerous cell line (CCD841 colon epithelial cells) to increasing amounts of PCarn (25μM, 50μM 100μM) for 2, 24 and 48hrs. As a measure of cell proliferation, we adapted a BCA protein assay to digest adhered cells in‐well as a measure of total cell content.

Results

After 48hr incubation of 100μM PCarn, H2O2 emission increased in MCF7 (20%) and HT29 (170%), which corresponded to decreased protein contents of 20% and 50% respectively, thereby demonstrating a graded relationship between degree of H2O2 emission and reduction in cell content. HepG2 demonstrated increased H2O2 emission after 48hr incubation with 25μM (150% increase) and 50μM PCarn (218% increase) with a 50% reduction in protein content in both cases. In the non‐cancerous CCD841, we noticed an increase in H2O2 (115%) emission, despite no change in protein content. Collectively, this data suggests that increases in H2O2 production through PCarn oxidation is well tolerated by non‐cancerous cells, however cancer cells are unable to withstand the concomitant increase in mitochondrial H2O2 emission albeit in response to different PCarn concentrations. Future work will determine the manner by which these cancer‐specific redox‐cell death relationships are regulated by glutathione buffering.

Support or Funding Information

Funding was provided to C.G.R.P. by National Science and Engineering Research Council (#436138‐2013) with infrastructure supported by Canada Foundation for Innovation, Ontario Research Fund and the James H. Cummings Foundation. P. C. T. was supported by an NSERC CGS‐D scholarship.
Metformin Kills Breast Cancer Cells by Inducing NADH Accumulation, Pyruvate Depletion and Reductive Stress
Authors: Iglesias Clara L. , Ratti Bianca A. , Kang Christie , de Abreu Andre L. , Bonini Marcelo G.
Published: November 2023

AMP‐activated kinase (AMPK) is a central metabolic regulator activated by deficits in nutrient supply. The role of AMPK in cancer has been contentious with both oncogenic and tumor suppressive roles being proposed. Studies by our group indicated that in breast cancer, AMPK activation promotes tumor evolution towards more aggressive phenotypes. These studies, however, were heavily criticized based on the argument that metformin, a biguanidine drug used to treat diabetes, activates AMPK and kills breast cancer cells. Hence, we set up to test the alternative hypothesis that AMPK activation by metformin relieves metformin toxicity by alleviating energy deficits, which are severe enough to cause cell death despite of AMPK activation. To examine this possibility we used AMPK‐competent (MCF7ev) and AMPK‐deficient (MCF7shAMPK) MCF7 cells, which are a estrogen receptor positive breast cancer cell line. Our results showed that MCF7shAMPK are slightly more sensitive to metformin than parental cells particularly at higher doses (20 mM). In addition, we found that pyruvate suppresses metformin toxicity towards parental cells but not MCF7shAMPK. Combined with metabolomics studies that revealed significant AMPK‐dependent distortions in amino acid metabolism caused by metformin our results indicate that complex I inhibition with the accumulation of NADH are the likely causes of metformin toxicity. This is alleviated by pyruvate (a NADH oxidant) as long as AMPK is functional and activates NADH oxidation by lactate dehydrogenase. We also conclude that metformin may be best utilized to treat tumors deficient in AMPK activity.

Support or Funding Information

NIH Grant Number: 3R01HL125356 ‐ 03W1
Succinate Drives Aggressiveness in Breast Cancer
Authors: Kang Christie , Bonini Marcelo
Published: November 2023

Succinate is an intermediate metabolite in the Kreb cycle which accumulates in hypoxia. It is also becoming clear that succinate is active in signaling directly or indirectly affecting nuclear factor activity and gene expression. We hypothesize that the accumulation of succinate in cancer promotes more aggressive phenotypes by either increasing the production of mitochondrial reactive oxygen species (ROS) or driving hypoxia‐inducible factor (HIF) signaling. We further hypothesize that sirtuin3 (sirt3), an established tumor suppressor, counteracts succinate's push towards more malignant phenotypes and that the loss of sirt3 in certain tumors enhances the pro‐carcinogenic effects of succinate accumulation. In support of the hypothesis, we found that MDA‐MB‐231 (triple negative) and MCF7 (estrogen receptor positive, ER+) cell lines had increased levels of hydrogen peroxide (H2O2) when treated with dimethyl succinate for two weeks. The rise in H2O2 levels was accompanied by a significant increase in cancer stem cell population particularly in MDA‐MB‐231. In addition, we found that sirt3 expression and activity are determinant of the effects of succinate in promoting cancer stem cell (more aggressive) phenotypes in MDA‐MB‐231. Together, our results indicate that the accumulation of succinate, which happens in hypoxia, promotes breast cancer phenotypes associated with treatment resistance, poorer patient outcomes and lower survival rates.

Support or Funding Information

1RO1 HL125356 and 67263‐RT‐REP
Environmental Lead (Pb)‐Exposure, when Combined with Diet‐Induced Obesity, Induces Metabolic and Behavioral Abnormalities in Mice
Authors: Lisznyai Eric Anthony , Yamin Danyoul , Lisieski Michael , Stemmer Paul , Leff Todd , Perrine Shane
Published: November 2023

Multiple factors contribute to the well‐documented rise in diabetes and related metabolic disorders in the US population. Nutritional and lifestyle factors together with chemical and psychosocial environmental stressors may act in concert to increase diabetes risk. Poor urban populations display higher diabetes rates compared to the general population, possibly because they are exposed to urban‐specific environmental stressors. Our preliminary findings indicate that exposure to lead (Pb), which is more common in urban environments such as Detroit and Flint, can cause metabolic and gene expression abnormalities in the liver that might contribute to the development of diabetes. We hypothesize that there is an interaction between high fat diet (HFD) induced obesity and Pb‐exposure that alters the feeding behavior and worsens the diabetes‐inducing effect of HFD. In this study we investigated the interaction between Pb and HFD by examining their impact on metabolic and behavioral outcomes, including glycemia, exploratory activity (open field (OF) testing) and reward‐based learning (conditioned place preference (CPP)). Six week old male C57BL/6 mice (n=12 per experimental group) were given 0 ppm (controls) or 20 ppm Pb in drinking water (chosen to generate a blood Pb levels of approximately 20 μg/dl) and either a high fat or low fat diet for 12 weeks. Mice underwent behavioral testing at 12 weeks on study and metabolic phenotyping at 16 weeks on study. Results demonstrated a significant interaction between HFD and Pb‐exposure in both OF and CPP tests, with mice exposed to both stressors exhibiting significant deficits in reward‐based learning and general motor activity. Similar co‐morbid effects of Pb‐exposure and HFD were observed on metabolic phenotypes; animals given HFD and exposed to Pb showed significantly higher blood glucose levels when compared to control HFD mice. Interestingly, Pb exposure did not cause hyperglycemia in lean control mice fed a low fat diet, suggesting that Pb effects vary according to diet and/or adiposity. Molecular analysis of brain and liver tissues from these animals is underway, and current work is utilizing cell culture models to investigate the effects of Pb on gluconeogenesis, insulin signaling and enzymatic regulation. Overall, our findings suggest that Pb‐exposure, in the context of diet‐induced obesity, exacerbates both behavioral impairments and metabolic abnormalities associated with diabetes.
Attenuation of Mitochondrial Dysfunction During Hepatic Steatosis by Avocado Oil Supplementation
Authors: Garcia‐Berumen Claudia isabel , Ortiz‐Avila Omar , Márquez‐Ramírez Cristian Adrián , Calderón‐Cortés Elizabeth , Montoya‐Pérez Rocío , Rodriguez‐Orozco Alain R. , Molina Alfredo Saavedra , Cortés‐Rojo Christian
Published: November 2023

Nonalcoholic Fatty Liver Disease (NFLD) is a clinical syndrome characterized by excessive fat accumulation in the liver and hepatic cellular degeneration even in the absence of alcohol consumption. Mechanisms of alcoholic liver disease have been extensively studied; however, the events that cause progression to hepatitis and fibrosis/cirrhosis are still undefined. It has been recently recognized that mitochondrial dysfunction and mitochondrial oxidant production may play a critical role in the development of NAFLD and progression to nonalcoholic steatohepatitis (NASH). We have recently reported that avocado oil intake has protective effects against mitochondrial dysfunction during diabetes by increasing the resistance of the respiratory chain (RC) to oxidative stress, decreasing ROS production and enhancing glutathione availability Therefore, we aimed to test whether avocado oil protects from the alterations elicited by diabetes on RC function in liver mitochondria from rats with NAFLD. Mitochondria from rats with NAFLD exhibited decreased respiration, impaired activity of complex III and diminished reduction of cytochromes b and c+c1. All these effects were corrected when avocado oil was supplemented after the establishment of NAFLD but not before the induction of the disease. These data suggest that avocado oil could be beneficial to decrease NAFLD progression by improving mitochondrial function.

Support or Funding Information

Acknowledgments: this work was supported by a Coordinación de la Investigación Científica‐UMSNH grant (CCR).
Comparative Effects of Avocado Oil and a Thiol Reductant on Impaired Complex I Activity in Mitochondria from Diabetic Rats
Authors: Hernandez Manjury Jatziry , Flores‐Ledesma Claudia Guadalupe , Sánchez‐Briones Luis Alberto , Alonso‐Velázquez Celeste , Aguilar‐Toral Roxana , Campos‐García Jesús , Saavedra‐Molina Alfredo , Cortés‐Rojo Christian
Published: November 2023

Diabetes is a chief factor for the progression of non‐alcoholic liver disease, which in turn, has been closely linked to mitochondrial dysfunction and excessive ROS production. Complex I may have a prominent role in the development of liver disease because its inhibition may drive to impaired hepatic fatty acid oxidation via decreased reoxidation of the NADH generated during beta‐oxidation. Complex I activity decreases by oxidation of specific thiol groups of the protein. Previously, we have demonstrated that avocado oil intake increases the concentration of reduced glutathione (GSH) in liver mitochondria from diabetic rats. As GSH is essential to maintain thiol groups of complex I in a reduced state, in the present study we aimed to compare the effects of avocado oil and beta‐mercaptoethanol, a thiol reductant, on complex I activity of liver mitochondria from type I‐diabetic rats. Respiration in state 3 was impaired in diabetic rats when a complex I substrate was tested. This was attributed to a decrease in complex I activity in diabetic rats. 90‐days avocado oil intake prevented these defects. When mitochondria from diabetic animals were incubated with beta‐mercaptoethanol, it was observed a full recovery of complex I activity. Besides, upregulated activity of catalase was detected in mitochondria from diabetic animals, indicating an increase in the mitochondrial production of H2O2. All these results suggest that diabetes decreases mitochondrial respiration due to an increment in H2O2 levels, causing the inhibition of complex I due to the oxidation of the free thiol groups of the enzyme, with avocado oil preventing complex I impairment probably by preventing thiol oxidation because enhancement of mitochondrial levels of GSH.

Support or Funding Information

Acknowledgments: this work was supported by a Coordinación de la Investigación Científica ‐ UMSNH grant (CCR).
Effects of 3D culture and aqueous cinnamon extract on 3T3‐L1 adipogenesis
Authors: Wells Emily , Dinsmore Olivia , Aulthouse Amy , Kinder David , Marchiano Joe , Stockert Amy
Published: November 2023

Extracts of cinnamon have long been used medicinally and recent research has solidified its use for controlling blood glucose levels. The correlation between type 2 diabetes and obesity make research in carbohydrate and lipid metabolism essential. We utilized the 3T3‐L1 cells as a model to examine degrees of differentiation to mature adipocytes by examining lipid droplet accumulation at various time points post treatment. Lipids from micro‐droplets were exchanged with oil red O as a marker of adipogenesis, and where appropriate the lipid volume was estimated and assayed. Culture periods were extended up to two weeks to reach mature adipocytes. A reduction in cell size was observed in both monolayer and three‐dimensional culture, but the size changes appear more significant when grown in three‐dimensional culture. A comparison was also made between a monolayer 3T3‐L1 culture and an agarose 3D culture, neither of which was treated to cinnamon. Using western blot analysis, adipogenesis markers were used to determine if growth in 3D culture is inducing differentiation into adipocytes. Cinnamon treated cells appear to have more lipid droplet accumulation, yet the lipid volume did not correlate directly with the reduction in cell size, suggesting a true change in metabolic signaling rather than an osmotic contraction. In three‐dimensional culture, there was an obvious cluster of lipid droplets, yet in monolayer this was not as obvious due to the single layer of cells. Monolayer cells were counted in each quadrant of the culture to determine the percent of cells that had rounded morphology compared to those that remained flat and fibroblast‐like. Of the rounded cells, the portion containing at least 50% space fill with lipid droplets were considered adipogenic. In monolayer, an estimate of lipid droplet volume was not reliable and a quantitation of extracted dye was necessary.

Support or Funding Information

Departments of Pharmaceutical and Biomedical Sciences and Biological and Allied Health Sciences, Ohio Northern University.
The Ebola Virus Matrix Protein VP40 Interacts Selectively with Plasma Membrane Lipids to Promote Viral Egress
Authors: Vecchio Kathryn Del , Shwarz Anna , Saphire Erica Ollmann , Stahelin Robert
Published: November 2023

Filoviruses are filamentous viruses and include Ebola (EBOV) and Marburg (MARV), which are morphologically identical but antigenically distinct. Mortality rates can be has high as 90% and to date there are no FDA approved vaccines or small molecules for treatment outside the realm of emergency situations under the compassionate use clauses of FDA policy.

EBOV harbors a genome of 7 proteins, the most abundantly expressed in mature virions is Viral Protein 40 (VP40) also known as the matrix protein. VP40 is required for the assembly and budding of EBOV and alone VP40 can form virus like particles (VLPs) from the plasma membrane of host cells. Recent work by the Stahelin and Ollmann Saphire labs indicates that VP40 adopts multiple different structures to elicit different functions in the viral life cycle. Cellular data demonstrates that each structure adopts a specific function, one for budding form the plasma membrane of human cells and one for regulation of viral transcription. This work investigates how distinct VP40 structures assemble in the presence of synthetic lipid vesicles and at the inner leaflet of the plasma membrane in live cells. This project aims to determine lipid composition requirements for functionality of VP40 mutants vs. wild‐type VP40, and to elucidate the function of VP40 oligomerization with site‐specific mutants.

The C‐terminal domains of the VP40 homodimer exhibit a highly conserved basic patch which is thought to promote interaction with anionic lipids. Preliminary data confirms that VP40 binds with nanomolar affinity to liposomes containing phosphatidylserine (PS), an anionic lipid found within the inner leaflet of the plasma membrane, and has specificity for this anionic lipid in particular. Cellular data from the Stahelin lab provides precedent that VP40 requires PS in order to bud from the plasma membrane. Lipidomics analysis of mammalian cells transfected with VP40 has aided in the investigation of the species and saturation of lipid populations of in the plasma membrane versus lipid populations in VLPs. Preliminary data suggest that VP40 selects for not only specific species of lipids, but for particular saturation states of lipids within the plasma membrane.

Support or Funding Information

This work has been supported by NIH grant AI081077 and NIH grant T32GM075762.
Optimization of ESI Parameters for Comprehensive Lipid Analysis
Authors: Gathungu Rose M , Larrea Pablo , Sniatynski Matthew J , Kristal Bruce S
Published: November 2023

Untargeted lipidomics is mainly performed using electrospray ionization mass spectrometry (ESI‐MS) either with or without chromatographic separation. ESI‐MS enables structural annotation and quantitation of lipids, which can then be used to determine the biological relevance of lipids. The use of ESI‐MS requires the optimization of the MS method to ensure detection of as many lipids as possible and the correct annotation of these lipids. Here we look at two factors that affect ESI‐MS that, in turn, influence lipid detection and identification. First, we show that in‐source fragmentation at the ESI source can generate artifacts that can have identical masses to other common lipids leading to the misannotation of the in‐source fragment ions as true lipids. In‐source fragmentation can lead to (i) missing the biologically relevant lipids (i.e., a false negative), and/or; (ii) incorrect assignation of a phenotype to an incorrect lipid (i.e., false positive). To mitigate this problem, we have conducted a systematic evaluation of ESI source parameters with the objective of obtaining uniformly appropriate source conditions for a wide range of lipids, while, at the same time, reducing in‐source fragmentation. Second, since analysis in both the positive and the negative ionization modes is required for a complete coverage of the lipidome, we show that the optimal conditions for analysis of lipids in the positive and negative MS ionization modes are different. We have evaluated the additive effect on ionization of lipids in both the positive and negative ionization modes. Our results indicate that the optimal additives for negative ionization of lipids are 1 mM ammonium acetate with 0.1 acetic acid and the optimal additives for positive analysis are 5 mM ammonium formate with 0.1% formic acid.

Support or Funding Information

NIH
Homeostatic Regulation of Serine Palmitoyltransferase (SPT) is Mediated by a Direct Interaction of Ceramide with the SPT/ORMDL Complex
Authors: Davis Deanna , Wattenberg Binks
Published: November 2023

Sphingolipids, the large class of lipids based on the sphingosine backbone, have diverse functions in health and disease. The overall rate of synthesis of the sphingolipid pool is determined by serine palmitoyltransferase (SPT), the initiating enzyme in the de novo biosynthetic pathway. SPT is subject to homeostatic regulation such that elevated cellular sphingolipid results in decreased SPT activity. This regulation is mediated by small proteins of the endoplasmic reticulum: the Orms in yeast and ORMDLs in mammalian cells. The Orms and ORMDLs are negative regulators of SPT. Here we describe the mechanism by which sphingolipids are sensed by the ORMDL/SPT system. The mammalian ORMDLs are not phosphorylated under any conditions, in contrast to the yeast Orms, which are regulated by phosphorylation. To characterize regulation of the ORMDL/SPT complex we devised a cell free system that recapitulates that regulation. Using isolated membranes we demonstrate that the triggering sphingolipid is ceramide. Moreover, regulation does not require ATP or cytosolic proteins, eliminating the role of protein expression or post‐translational modification as potential regulatory mechanisms. A distinct spectrum of ceramide structures activate ORMDL regulation of SPT. Strikingly, only the native stereoisomer of ceramide will trigger this regulation, the hallmark of a specific binding interaction. These data strongly suggest that ORMDL dependent regulation of SPT is not due to a complex signal transduction pathway or sphingolipid‐induced changes in membrane properties but instead is due to direct binding of ceramide to a membrane‐bound ceramide sensor, likely ORMDL.

Support or Funding Information

University of Louisville Bridge Grant National Institutes of Health‐R01HL131340
Single‐Molecule Analysis of PKB/AKT‐Lipid Interactions
Authors: Singh Nilmani , Arauz Edwin , Aggarwal Vasudha , Ha Taekjip , Chen Jie
Published: November 2023

Protein kinase B (PKB)/AKT is a serine/threonine kinase with a wide range of essential functions in cell proliferation, survival, differentiation, and metabolism. Growth factor stimulation of phosphoinositide‐3 kinase (PI3K) results in generation of phosphatidylinositol‐3,4,5‐trisphosphate [PI(3,4,5)P3] or PI(3,4)P2 at the plasma membrane, which recruits AKT. At the membrane AKT is phosphorylated and activated, and subsequently dissociates from the membrane and translocates to cytoplasm or nucleus in order to phosphorylate its targets. Hence, it is postulated that phosphorylation of AKT dampens its affinity for membrane lipids. We have employed a newly developed lipid single‐molecule pulldown (lipid‐SiMPull) assay to study AKT interaction with lipid vesicles and its functional implication. Quantitative and kinetic measurements were made with wild‐type and phosphor‐mimetic or unphosphorylatable mutants of AKT in whole cell lysates for their binding to PIP3 and PIP2 vesicles. Indeed, phosphorylation of AKT drastically decreased its affinity for the lipids. The potential role of phosphotidylserine (PS) in AKT membrane recruitment was also examined. Furthermore, distinct binding behaviors were observed with full‐length AKT and the PH domain alone, suggesting that other domains in AKT modulate its lipid interaction and thus membrane association. Our results provide new insights into the molecular mechanism of AKT activation, and also highlight the unique advantages of the lipid‐SiMPull method in probing protein‐lipid interactions with full‐length proteins in a near‐physiological context.

Support or Funding Information

NIH NIGMS R01GM089771
Sphingosine kinase 1 inhibition modulates crosstalk between p53 and autophagic cell death
Authors: Lima Santiago , Takabe Kazuaki , Leopoldino Andreia Machado , Newton Jason , Dent Paul , Milstien Sheldon , Spiegel Sarah
Published: November 2023

Sphingosine‐1‐phosphate is a highly bioactive lipid that promotes cellular growth and survival important to the etiology of cancer, and levels of the enzyme that produces it, sphingosine kinase 1 (SphK1), are elevated in many human cancers and are associated with higher tumor grade and worse prognosis. The great majority of human cancers have mutations in the tumor suppressor p53, a key regulator of cell cycle arrest and programmed cell death. There are important associations that suggest SphK1 levels are p53‐dependent in tumors and in preclinical cancer models. Therefore, we sought to better understand whether alterations in SphK1 activity might also regulate activation of p53‐dependent processes, such as apoptosis and autophagy. By using an SphK1 isotype specific inhibitor in cancer cell lines with or without p53 expression, we establish that inhibiting SphK1 activity leads to phosphorylation of p53, induction of downstream p53 targets, increased processing of autophagic markers such as LC3 in a p53‐dependent manner, and the formation of autophagic and multi‐vesicular bodies. Moreover, depleting protein levels of master regulators of autophagic flux, Beclin1 and ATG5, dramatically decreased the cytotoxicity of the SphK1 inhibitor only in cells with p53 expression. Hence, our study reveals that SphK1 inhibition is cytotoxic to cancer cells in a p53‐dependent manner via autophagic and apoptotic pathways, suggesting crosstalk between these important players in human cancers.

Support or Funding Information

NCI grant K22 CA187314‐01 to S.L. and R01GM043880 to S.S
Macrophage Cholesterol Efflux and Atherosclerosis in Psoriasis: A role for microRNA‐33
Authors: Karunakaran Denuja , Dwevidi Girish , Rayner Katey
Published: November 2023

Psoriatic patients have an increased risk in developing atherosclerosis. However, the precise mechanism by which the localized skin inflammation drives atherosclerosis is unclear. Macrophages have been shown to promote localized skin inflammation and also drive atherosclerosis by retaining modified excess cholesterol. Macrophage cholesterol efflux depends on 2 elements: (i) functional circulating HDL interacting with (ii) macrophage ABCA1 cholesterol transporter to release the excess cholesterol into HDL particles to be cleared. microRNA‐33 (miR‐33) levels are markedly elevated in CAD and it directly targets various genes regulating cholesterol transport, mitochondrial function and inflammation to inhibit macrophage cholesterol efflux, promoting atherosclerosis. Thus, novel therapeutics aims to inhibit miR‐33 to treat atherosclerosis. Nonetheless, the mechanism by which macrophage miR‐33 drives atherosclerosis in psoriasis and how psoriasiform‐inflammation accelerates atherosclerosis remains to be elucidated. Here, we aim to determine if psoriasiform macrophages have elevated miR‐33 levels and are defective in mediating cholesterol efflux and thus, accelerating atherosclerosis. Preliminary data indicate that human macrophages treated with key cytokines (e.g. IL‐17A or IL‐23 or TNFα) that drive psoriatic inflammation have increased miR‐33a expression and decreased macrophage cholesterol efflux. Ongoing studies are investigating whether human primary macrophages derived from psoriatic patients have decreased cholesterol efflux relative to control. These studies will provide mechanistic insight as to how miR‐33 is regulated in psoriasiform inflammation and whether anti‐miR33 therapy or other novel therapeutic targeting the macrophage efflux pathways can be used to treat cardiovascular disease in psoriatic patients.
NPC1‐mediated cholesterol export from lysosomes
Authors: Pfeffer Suzanne R.
Published: November 2023

Endocytosed, LDL‐derived cholesterol is exported from the interior of lysosomes by a process that requires the action of NPC1 and NPC2 proteins. We have recently shown that LAMP1 and LAMP2 proteins interact with NPC1 and NPC2 and also bind cholesterol (1). LAMPs are highly abundant, ubiquitous, mammalian proteins that line the lysosome limiting membrane, and protect it from lysosomal hydrolase action. LAMP2 deficiency causes Danon's disease, an X‐linked hypertrophic cardiomyopathy. LAMP2 is needed for chaperone‐mediated autophagy, and its expression improves tissue function in models of aging. We have shown that human LAMP2 binds cholesterol in a manner that buries the cholesterol 3β‐hydroxyl group; it also binds tightly to NPC1 and NPC2 (1). Quantitation of cellular LAMP2 and NPC1 protein levels suggest that LAMP proteins represent a significant cholesterol binding site at the lysosome limiting membrane, and may signal cholesterol availability. Functional rescue experiments show that the ability of human LAMP2 to facilitate cholesterol export from lysosomes relies on its ability to bind cholesterol directly (1). Why do LAMP proteins bind cholesterol and what is the significance of their interaction with NPC1 and NPC2 proteins? Our recent determination of the structure of a complex of NPC1 and NPC2 proteins in collaboration with Xiaochun Li and Günter Blobel (2) provides a model for how NPC2 may deliver cholesterol to NPC1. Current work is exploring the subsequent steps involved in exporting that cholesterol across the lysosomal glycocalyx (3) and into the cytoplasm.

Support or Funding Information

This research was funded by the Ara Parseghian Medical Research Foundation and the NIH (DK37332).
HDLs and LDLs in the Brain: Characterizing the Lipid Secretome of Astrocytes
Authors: Nguyen Amanda
Published: November 2023

Comprising up to a hundred‐fold more lipid than a typical cell, a neuron has extensive processes like dendrites and axons. The magnitude of the neuronal membrane surface area suggests the importance of the regulation of the lipids in the membrane to maintain normal neuronal function. While particles like high density lipoproteins (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL) normally circulate throughout the body for lipid regulation, these particles cannot pass the blood brain barrier. The brain must synthesize lipids locally in order to fulfill the biosynthetic/repair needs of the neurons. Lipid synthesis and transportation in the brain are facilitated by astrocytes, the helper glial cells of the nervous system. Astrocytes package and secrete lipids into “HDL/LDL‐like” particles, comprising a lipid core and a shell of proteins, like apolipoproteins, on the exterior. These particles function to regulate the lipid membrane of adjacent neurons, but they also vary in functionality depending on the particles' ratio of lipids to proteins (or how “LDL‐like” or “HDL‐like” the particles are). For example, apolipoprotein E (apoE) is a protein that is synthesized by astrocytes and is essential for cholesterol transportation to neurons. In Alzheimer's Disease (AD) specifically, the apoE4 polymorphism is implicated in higher AD frequency than other apoE polymorphisms because apoE4 is a weaker carrier of cholesterol than the other apoE variants. The apoE variant functionality suggests the imperative role of cholesterol homeostasis to maintain normal function in neurons. ApoE has also been shown to be the main constituent of the HDL‐like particles in the brain. Our initial studies of the lipid secretome of primary cultured mouse astrocytes revealed that the younger astrocytes secreted nearly ten‐fold less cholesterol than the older astrocytes while protein secretions between the different ages were not significantly different. These results suggest the “HDL‐like” particles are secreted more by the younger astrocytes while the “LDL‐like” particles are secreted by older astrocytes, revealing how aging plays a role in lipid homeostasis. We are continuing to isolate the lipid secretome of our primary astrocytes by using the lipid‐binding resin, Cleanascite. In order to understand the composition of these lipid particles, we are using thin layer chromatography to observe the presence of different lipids qualitatively and quantitatively.

Support or Funding Information

Mach Research Fellowship and Cowles Professorship
Lessons from evolutionary medicine for sepsis and critical care
Authors: Alcock Joe
Published: November 2023

Sepsis remains a leading cause of death, causing approximately 60,000 deaths yearly. Despite hundreds of clinical trials and billions of dollars, only one agent aimed specifically at sepsis was ever approved by the FDA. It was subsequently withdrawn from the market after it was shown to be ineffective. Despite this and similar failures, researchers continue to search for therapies aimed at blocking inflammation and other physiologic changes in sepsis. These approaches have failed to improve mortality in human sepsis in part because they overlook functional adaptations and evolved tradeoffs in sepsis physiology. We have followed a series of 160 patients with severe sepsis admitted to the intensive care unit at the University of New Mexico, with the aim of predicting mortality based on initial measures of lactate, glucose, and kidney function in sepsis. Many of these parameters, e.g. blood glucose, follow similar distributions in survivors as compared to non‐survivors. Recent randomized controlled trials aimed at normalizing these parameters have generally failed to improve survival. These lines of evidence indicate that many sepsis “abnormalities” are in fact adaptive, not dysfunctional. This presentation will give examples of how evolutionary and evidence‐based medicine can work in concert to produce better outcomes in sepsis and critical care.

Support or Funding Information

Valente Foundation and the University of New Mexico Department of Emergency Medicine
Structural and Functional Characterization of Outer Membrane Usher Activation in Uropathogenic E. coli
Authors: Omattage Natalie , Deng Zengqin , Yuan Peng , Hultgren Scott J.
Published: November 2023

Gram‐negative bacteria assemble extracellular fibers termed chaperone‐usher pathway (CUP) pili to adhere to host tissues and promote colonization. This has been best studied in the establishment of urinary tract infections (UTIs), which affect 60% of women in their lifetime. Uropathogenic Escherichia coli (UPEC) is the causative agent of 85% of community‐acquired UTIs. UPEC utilize the type 1 pilus, via its tip‐located FimH adhesin, to bind and adhere to mannosylated receptors on the surface of bladder epithelium. Type 1 pilus assembly is an intricate molecular process that requires the passage and polymerization of hundreds of subunits across both the inner and outer membranes of the bacterium. To accomplish this, the dedicated periplasmic chaperone, FimC, binds to and facilitates folding and maintenance of pilus subunits in a high‐energy conformation suitable for polymerization; and the outer membrane usher, FimD, serves as a multi‐domain assembly platform to catalyze subunit polymerization. Pilus assembly is initiated by the high‐affinity binding of the chaperone‐adhesin complex (FimCH) to the N‐terminal periplasmic domain of FimD. By some unknown mechanism, this binding event results in the “activation” of the usher for its catalytic activity. Usher activation results in marked rearrangements within the transmembrane and periplasmic FimD domains, but it remains unclear how chaperone‐adhesin binding to FimD triggers these conformational changes. We have conducted extensive structure‐function studies to gain insight into the molecular mechanism by which the chaperone‐adhesin primes the FimD usher for activation. Using mutagenesis and in vivo functional assays, we have identified interaction interfaces important for usher activation. We have developed systems for large‐scale expression and purification of the FimD usher and for reconstitution of pilus assembly intermediates in vitro. We are currently characterizing the structural basis for chaperone‐adhesin complex engagement of the N‐terminal domain of the FimD usher using X‐ray crystallography, single particle cryo‐electron microscopy (cryo‐EM) and small angle X‐ray scattering (SAXS) methods. Taken together, these biochemical and biophysical studies will provide unprecedented insight into how allostery primes the usher and drives pilus biogenesis. Ultimately, these studies have the potential to inform the development of innovative small molecules that block events at the outer membrane usher and prevent assembly of one of the bacterium's major virulence factors.

Support or Funding Information

NSF GRFP DGE‐1143954 (NSO) and NIH R01 AI029549‐24 (SJH)
In vitro Activity of a Purified Natural Anion Channelrhodopsin
Authors: Li Hai , Sineshchekov Oleg , Wu Gang , Spudich John
Published: November 2023

Natural anion channelrhodopsins (ACRs) recently discovered in cryptophyte algae are the most active rhodopsin channels known. They are of interest both because of their unique natural function of light‐gated chloride conductance, and their unprecedented efficiency of membrane hyperpolarization for optogenetic neuron silencing. Light‐induced currents of ACRs have been studied in HEK cells and neurons, but light‐gated channel conductance of ACRs in vitro has not been demonstrated. Here we report light‐induced chloride channel activity of a purified ACR protein reconstituted in large unilamellar vesicles (LUVs). Electron paramagnetic resonance (EPR) measurements establish that the channels are inserted uniformly ‘inside‐out’ with their cytoplasmic surface facing the medium of the LUV suspension. We show by time‐resolved flash spectroscopy that the photochemical reaction cycle of a functional purified ACR from Guillardia theta (GtACR1) in LUVs exhibits similar spectral shifts indicating similar photocycle intermediates as GtACR1 in detergent micelles. Furthermore, the photocycle rate is dependent on electric potential generated by chloride gradients in the LUVs in the same manner as in voltage‐clamped animal cells. We confirm with this system that, opposite to cation‐conducting channelrhodopsins, opening of the channel occurs prior to deprotonation of the Schiff‐base. However, the photointermediate transitions in the LUVs exhibit faster kinetics. The ACR‐incorporated LUVs provides a purified defined system amenable to EPR, optical and vibrational spectroscopy, and fluorescence resonance energy transfer measurements of structural changes of ACRs with the molecules in a demonstrably functional state.

Support or Funding Information

This work was supported by NIH grant R01GM027750 and Endowed Chair AU‐0009 from the Robert A. Welch Foundation.
Probing the structural basis of P‐glycoprotein transport of μ‐opioid receptor agonists: methadone and loperamide
Authors: Gibbs Morgan Elizabeth , Ledwitch Kaitlyn , Roberts Arthur
Published: November 2023

P‐glycoprotein (Pgp) protects the brain from toxic insults by acting as a gatekeeper at the blood‐brain barrier. In addition to effluxing toxins, Pgp can also limit penetration of therapeutics into the brain for the treatment of central nervous system (CNS) disorders and diseases, including μ‐opioid receptor agonists that are used in pain management. Although they are structurally similar, the μ‐opioid receptor agonist loperamide (Imodium®) is effluxed out of the CNS by Pgp at four times the rate of methadone. Because of these differences, methadone and loperamide were used as model drugs to probe the mechanism of Pgp‐mediated μ‐opioid receptor agonist efflux. We hypothesize that higher Pgp‐mediated efflux rates of loperamide are the result of multiple loperamide binding to Pgp, allowing more drug to be transported per ATP hydrolyzed. We found that loperamide was a more potent activator of Pgp‐mediated ATP hydrolysis than methadone. Consistent with our hypothesis, the loperamide‐induced activation of ATP hydrolysis suggested at least two loperamide binding sites on Pgp, while methadone‐induced activation of ATP hydrolysis suggested a single methadone binding site. NMR and fluorescence were used to investigate the interactions of these drugs with the transporter and develop a structural model for transport. The saturation transfer double difference (STDD) NMR technique identified the functional groups involved in molecular recognition of Pgp and revealed that loperamide had more interactions with the transporter than methadone, which is consistent with the higher transport rate of loperamide. Acrylamide quenching of Pgp fluorescence to probe transporter conformation indicated that loperamide shifts Pgp into a conformation that promotes ATP hydrolysis and transport, while methadone shifts Pgp into an inhibitory conformation. This information combined with paramagnetic relaxation enhancement (PRE) NMR experiments and computer modelling was used to pinpoint the binding locations of loperamide and methadone on Pgp. The drug‐bound Pgp models suggest that multiple drug binding and conformational changes play a critical role in Pgp transport of this class of drugs.

Support or Funding Information

This work was funded and supported by the National Institute of Health R15 Area Grant (1R15GM107913‐01A1).
Investigating the role of GlfT2 in mycobacterial cell wall assembly
Authors: Kim Lili M. , Justen Alexander M. , Kiessling Laura L.
Published: November 2023

The disease tuberculosis has remained difficult to treat to many small molecule treatments. One feature thought to contribute to this is the complex, impermeable cell wall of Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis. A core component of this cell wall is a linear carbohydrate polymer known as the galactan. The galactan, comprised of galactofuranose residues, serves as an essential covalent connector between the waxy mycolic acids bilayer and the peptidoglycan. In M. tb, the elongation of the galactan is mediated by a galactofuranosyl transferase, GlfT2. Using a combination of data derived from in vitro enzymatic assays and in vivo cell wall composition assays, we have arrived at the hypothesis that GlfT2 controls both length and linkage patterns of the galactan chain intrinsically. However, the hypothesis that GlfT2 is the only determining factor in length and linkage distribution has yet to be tested in vivo. The role of GlfT2 in galactan elongation is evolutionarily conserved; however, galactan length and linkage distribution varies across species. By transferring GlfT2 homologues from closely related organisms into Mycobacterium smegmatis (M. smeg) and studying the resulting growth patterns and cell walls, we can determine whether GlfT2 alone controls polymer length and linkages. To do this, we have developed and confirmed a GlfT2‐deficient strain of M. smeg using recombineering methods. We have also cloned a small library of GlfT2 homologues to be transformed into this strain. Preliminary results with Corynebacterium glutamicum (C. glut) GlfT2 have demonstrated rescued, but slower, growth in the deficient strain. We are particularly interested further assessment toward how changes to the galactan affect mycobacterial physiology.
Combining Same‐Day Student Food Diaries With ASA‐24 Interviews and Coding to Accurately Assess the in‐School Food Consumption of 4th to 6th Graders
Authors: Niland Katherine , Cohen Rhoda , Briefel Ronette , Hu Mindy , Magness Allison , Crepinsek Mary Kay
Published: November 2023

Accuracy in the collection of children's food consumption remains an important issue in nutrition research and policy. For an evaluation of the Canned, Frozen, and Dried pilot project for the Fresh Fruit and Vegetable Program, we collected student dietary recalls and menu surveys of school meal and FFVP offerings in 34 pilot schools in 4 pilot states in school year 2014–2015. We collected in‐school dietary recalls for elementary school students in grades 4–6 (1,255 in the fall semester and 1,236 in the spring semester). We implemented a dietary recall method that engaged students, minimized time away from class, and improved accuracy in coding foods and their amounts. During data collection for a target week in each school, trained interviewers met with selected students in the morning to describe how to list the items consumed in school on the Foods Eaten in School Today (FEST) food diary. During the school day, trained interviewers also completed a menu survey about the school meal and FFVP offerings with the school's foodservice manager. At the end of the school day, the same interviewer met individually with each student to administer the recall interview, using the foods listed on the diary and school menu survey as a guide. The interviewer recorded the foods and their amounts consumed using the Automated Self‐Administered 24—Adult Version, 2014 (ASA‐24) software developed by the National Cancer Institute. We selected the ASA‐24‐Adult version because it contained more detailed food‐level probes than the child version (ASA‐24‐Kids). We also modified the ASA‐24 to capture specific school sources of foods as: school breakfast, FFVP snack, school lunch, home, or other. USDA's Survey Net system (version 4.0) was used to manually code hard copy menu survey items including all FFVP snacks as well as school lunch and breakfast items. To provide standardized food codes and more accurate portion size information for the in‐school dietary intake, school foods reported in the dietary recall were matched to the coded school menu survey file for the dietary recall student‐level food and nutrient level analysis. We believe this represents the first time that student record‐keeping (the FEST form) combined with ASA‐24 administration by interviewers was used in a large scale study of U.S. elementary school students.

Support or Funding Information

Evaluation funded by the Food and Nutrition Service, USDA.