bluuebunny/crossref_metadata_2025 huggingface dataset:
Caspase Activity in Early Stage Regeneration of A. viride Regulated by the Wnt/β‐Catenin Signaling Pathway
Authors: Fok Sheridan
Published: November 2023

It has been widely known that invertebrates posses extraordinary regenerative ability to repair their lost tissues or organs. Aelosoma viride, a fresh water annelid, can complete anterior regeneration within 5 days post amputation. In addition, its relatively small body size (2mm in length) and transparent color can be easily manipulated for regeneration study. Regeneration processes of A. viride are similar to other model organisms, including wound healing, blastema formation, and tissue remodeling. Apoptotic caspase has been known to be required during early stage regeneration. A previous study done by RNA interference showed that A.viride could not complete its anterior regeneration when Avi‐caspase‐3/7 was knocked down. Recent studies have demonstrated that wnt/β‐Catenin signaling pathway can promote apoptosis through a caspase‐dependent pathway. Also, our previous studies have showed that gene expression of both Avi‐wnt‐4 and β‐Catenin elevated in the anterior region during early stage of regeneration. Thus, our future studies will try to realize whether or not caspase activity in early stage regeneration is regulated by the wnt/β‐Catenin signaling pathway in A. viride.
Expression and functional analysis of Fas ligand‐like protein from silkworm, Bombyx mori
Authors: Yanagida Kosuke , Yabu Takeshi , Shiba Hajime
Published: November 2023

In the silkworm, Bombyx mori, the degradation of larval tissues is an essential process for generating raw materials to build an adult body. It has been shown that most of the larval tissues disappear in the pupal stage upon activation of Bmcaspase dependent apoptosis. As an approach to identify the ligand responsible for initiating the death pathway, we searched Silk Base database and cloned a candidate cDNA fragment encoding a protein sequence similar to the mammalian Fas ligand in the Pichia Pastoris yeast expression system. The expressed recombinant protein was purified using Ni‐agarose affinity column and Sephacryl S‐300 gel filtration column. While the gel filtration column chromatography gave a molecular weight of 108,000, the subunit molecular weight was 38,000 as estimated by using SDS‐PAGE under reducing condition, indicating that the protein was purified as a homo‐trimer. To verify its capability of inducing apoptosis, the recombinant protein was injected into the larvae of V6 (6th day after entering fifth instar) at various amounts. All larvae died within 24 hours at the dosage of 16 ng or more protein. Inside the dead bodies, the lysis of the larval tissues was observed and a remarkable elevation of Bmcaspase activity was detected; Ac‐DEVD‐MCA, an artificial fluorescent substrate, was used. Western blot analysis revealed the occurrence of BmJNK phosphorylation in the same tissues. Our results clearly showed that the purified recombinant protein is able to induce Bmcaspase‐dependent apoptosis that causes disappearance of larval tissues, possibly through BmJNK signaling pathway.
Evaluating Tim12p as a potential target of the anti‐cancer compound Parthenolide
Authors: Babb Emily Katherine , Callahan Kevin P
Published: November 2023

The World Health Organization (WHO) expects that annual cancer rates will rise from 14 million in 2013 to 22 million by 2032. Statistics such as this one serve as motivation to discover and provide new cancer treatments that are targeted, effective, and less cytotoxic. Parthenolide (PTL) is a secondary metabolite of Tanacetum parthenium (Feverfew) with several anti‐cancer properties. PTL has demonstrated the ability to induce apoptosis in human Acute Myeloid Leukemia (AML) cells, a cancer of the early, immature myeloid line of blood cells, while sparring the normal hematopoietic cells. Over the past decade, Saccharomyces cerevisiae (yeast) has provided powerful methods to identify the mechanism of action of known drugs. A common screening technique used to identify targets of a compound is the Haplo‐insufficiency profiling (HIP) assay, in which a complete set of heterozygous deletion strains are grown in the presence of a compound of interest. The use of this technique allowed for the identification of many strains of yeast that are sensitive to PTL. Strains most sensitive to the compound often carry deletions in genes that encode the drug target. One of the possible targets of PTL identified in the assay is Tim12p, a component of the TIM22 complex of the inner mitochondrial membrane that is responsible for protein import into the yeast mitochondria. To validate the findings of the HIP assay we have obtained a strain lacking one copy of TIM12, confirmed its genotype via PCR, and tested its sensitivity to PTL. Future experiments will focus on testing the sensitivity of our tim12‐Δ strain to other chemotherapeutics in order to help determine if PTL specifically interacts with Tim12p.
Fine Tuning the Catalytic Activity of Caspase‐3 by Phosphorylation and Site Directed Mutagenesis of an Allosteric Site
Authors: Thomas Melvin Edward , Clark Clay
Published: November 2023

Caspases are cysteine‐dependent aspartate‐specific proteases that are well known for their role in carrying out cell death via apoptosis; however, they are also key proteases in many other non‐apoptotic processes. Caspase‐3 activity is required for terminal erythroid differentiation, but its activity is fine tuned to prevent apoptosis, leading to differentiation. There are three main ways cells regulate caspase‐3 activity: reduction, but not complete loss, of its catalytic activity by phosphorylation at the highly conserved Ser150 by p38‐MAPK; preventing its intersubunit linker (IL) cleavage and activation by binding of Hsp27 in the prodomain of caspase‐3; or directing its cellular localization. The mechanisms behind how these processes modulate caspase‐3 activity are poorly understood. The site of phosphorylation of caspase‐3 by p38‐MAPK is conserved in caspase‐8 and ‐7, which indicates an important regulatory relationship between the caspase cascade and the human kinome. The goals of this project are to reveal how the cell utilizes these three processes during erythroid maturation; and how phosphorylation regulates caspase‐3 is addressed in the following experiments. We have previously shown that destabilization of helix 3 in caspase‐3 propagates to the active site causing its destabilization, shifting the population to a less active state. The site of phosphorylation at S150 is at the base of helix 3, therefore we hypothesize this modification causes a similar mechanism, leading to a less active state. We performed point mutations in the background of cleavable, D2A, and uncleavable caspase‐3 (D3A) at S150 as well as other predicted phosphorylation sites to mimic phosphorylation events. We also performed kinase assays with p38‐MAPK to purify phosphorylated caspase‐3 (p‐cp‐3). Using in vitro cleavage assays we found that both p‐cp‐3 and S150D mutant have about a 10‐fold reduction in enzymatic activity compared to wt. We solved the structures of the cleavable and D2A phospho‐mimic mutants using X‐ray crystallography and saw no significant global structural changes but an increase in electrostatic interactions at the mutation site in S150D and a decrease in S150A. Time dependent loss of activity experiments, however, reveal that p‐cp‐3 and S150D causes the protein to populate an inactive conformation before shifting to an active conformation that promotes cell death. These data quantify the reduction in activity upon caspase‐3 phosphorylation provides a mechanism for how cells survive when caspase‐3 is phosphorylated. It also reveals S150D as a successful mimic of phosphorylation. The reduction, but not complete loss, of enzymatic activity supports our hypothesis that phosphorylation leads to destabilization of the active site, and shifts the population to a less active state.
A Tunable Allosteric Library of Caspase‐3 Identifies Coupling Between Conserved Water Molecules and Conformational Selection
Authors: Maciag Joseph J. , Mackenzie Sarah H. , Tucker Matthew , Schipper Joshua L. , Swartz Paul , Clark A. Clay
Published: November 2023

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformer, while targeting an allosteric site in the dimer interface selects an inactive conformer that contains disordered active site loops. Mutations and post‐translational modifications stabilize high‐energy inactive conformers, with mostly formed, but distorted, active sites. In order to examine the interconversion of active and inactive states in the ensemble, we used DRoP to analyze 4,995 waters in 15 high‐resolution (<2.0 Å) structures of wild‐type caspase‐3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of post‐translational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of nineteen caspase‐3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies over four orders of magnitude. Altogether, our database consists of 37 high‐resolution structures of caspase‐3 variants, and we demonstrate that each 10‐fold decrease in activity correlates with a loss of 23 conserved water molecules. The data show that the activity of caspase‐3 can be fine‐tuned through globally de‐solvating the active conformer within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine‐tune activity through conformational selection.
Pentachlorophenol and Dichlorodiphenyltrichloroethane Alter Secretion of Interleukin 1 &[Beta]eta (IL‐1β) from Human Immune Cells
Authors: Martin Tamara J , Whalen Margaret
Published: November 2023

Pentachlorophenol (PCP) and Dichlorodiphenyltrichloroethane (DDT) are environmental contaminants found in human blood. PCP has been used in fungicides, insecticides, herbicides, and antifouling paint. 4, 4′‐Dichlorodiphenyltrichloroethane (4, 4′‐DDT), has been banned in the U.S. however, it is still used in other countries as a pesticide to kill insects. A number of cancers are associated with exposures to these contaminants. Previous studies in our lab show that PCP and DDT inhibit the lytic function of highly enriched human natural killer (NK) lymphocytes and decrease the expression of several surface proteins on NK cells. Interleukin 1‐βeta (IL‐1β) is a cytokine produced by lymphocytes and monocytes. It is involved in stimulating cell proliferation, tissue repair, and present during the inflammatory response to injury. An increase or decrease in the level of Il‐1β may lead to immune dysfunction and increased potential for tumor development and invasiveness. The objective of this study was to examine if exposure of human immune cells to different concentrations of PCP and DDT alters the secretion of IL‐1β. Immune cells included Peripheral Blood mononuclear cells (PBMCS), monocyte‐depleted (MD) PBMCs, and enriched NK cells. Cells were exposed to concentrations of PCP ranging from 5 μM to 0.05 μM and DDT concentrations of 2.5 μM to 0.025 μM for 24 h, 48 h and 6 days. Secreted IL1‐β levels were measured using ELISA (enzyme‐linked immunosorbent assay). Results showed that PCP and DDT induced alterations in IL‐1β secretion from each of the human immune cell preparations. NK cells, MD‐PBMCs and PBMCs all showed increased secretion of IL‐1β after at least one length of exposure to at least one concentration of PCP. The concentrations and lengths of exposure at which these increases occurred varied among the donors. With DDT, all donors showed an increase in IL‐1β secretion after exposure to at least one concentration of DDT in all types of cell preparations. These results suggest that PCP or DDT‐induced alterations in IL‐1β secretion may have the potential to cause immune system dysregulation.

Support or Funding Information

Supported by NIH grant 5U54CA163066
Tributyltin Alters Secretion of Interleukin 6 (IL‐6) From Human Immune Cells
Authors: Wilburn Wendy JoAnne , Brown Shyretha , Whalen Margaret
Published: November 2023

Tributyltin (TBT) is a widespread environmental contaminant due its use as a biocide (including as an antifungal agent). TBT has been found in human blood samples and has been shown to increase tumors in mammals. IL‐6 is both an anti‐inflammatory and pro‐inflammatory protein that is produced by T lymphocytes (as well as other cells). It regulates cell growth, tissue repair, and immune functions. Previous studies have shown that TBT alters the secretion of cytokines IL‐1β, TNFα, and INFγ from human immune cells. Due to the important role IL‐6 plays in immune responsiveness, it is important to understand whether exposures to TBT are able to disrupt its secretion. Natural killer (NK) cells, monocyte‐depleted (MD) human peripheral blood mononuclear cells (PBMCs), PBMCs, PBMC+granulocytes, and granulotcytes were exposed to TBT at concentrations of 0–200 nM and monitored for secretion of IL‐6 at 24 h, 48 h, and 6 days using enzyme‐linked immunosorbent assay (ELISA). After 24 h exposures to TBT at concentrations of 100 and 200 nM, NK cells, MD‐PBMCs, and PBMCs showed significant decreases in secretion of IL‐6 while PBMCs‐granulocytes exposed to 100 nM TBT showed increased secretion of IL‐6 compared to control. Additionally, PBMCs exposed to 2.5 nM TBT showed increased in IL‐6 secretion compared to controls. These results indicate that TBT is able to disrupt IL‐6 secretion from immune cell preparations and the nature of those disruptions are dependent on the type of immune cell that is being tested as well as the concentration of TBT.

Support or Funding Information

Supported by NIH grant 5U54CA163066.
Combination of Aurora A and Haspin inhibitors enhances p53 expression and tumor growth inhibition in human colorectal cancer
Authors: Chang Yun‐Hsuan , Lin Chien‐I , Chen Zan‐Chu , Liu Kuang‐Kai , Chao Jui‐I
Published: November 2023

Colorectal cancer (CRC) is one of the leading causes and mortality in the world. Aurora A and Haspin are important mitotic kinases for promoting cancer progression. Here, we show the combination of an Aurora A inhibitor MLN8237 and a Haspin inhibitor CHR6494 that can enhance anticancer activities to CRC. Both MLN8237 and CHR6494 induced abnormal chromosome segregation and mitotic catastrophe. Interestingly, the combination of MLN8237 and CHR6494 enhanced the cell growth inhibition and apoptosis induction. Meantime, MLN8237 and CHR6494 reduced the protein levels of survivin. However, over‐expression of survivin by an ectopic survivin vector resisted the MLN8237 and CHR6494 induced cell death. Moreover, MLN8237 and CHR6494 induced higher apoptosis in the p53‐wild type cells than the p53‐null cells. MLN8237 and CHR6494 increased the total p53, phospho‐p53 (Ser46) and Bax for apoptosis induction. Combination of CHR6494 and MLN8237 enhanced the inhibition of xenografted human CRC tumors in nude mice. Together, these results demonstrate that targeting on Aurora A and Haspin by the inhibitors will be potentially developed as a novel strategy for CRC therapy.
Structural analysis of PTM hotspots reveals large body of putative regulatory elements with predicted biological function in eukaryotic protein families
Authors: Torres Matthew , Dewhurst Henry , Choudhury Shilpa
Published: November 2023

Post‐translational modifications (PTMs) regulate protein behavior through modulation of protein‐protein binding and recognition site interactions, allosteric regulation, and conformational switching. In order to explore the structure/function relationships of large‐scale PTM data, we developed SAPH‐ire (Structural Analysis of PTM Hotspots), which has been used effectively to reveal novel PTM regulatory elements in discrete protein families (Dewhurst et al., 2015). Here, we proceed by applying SAPH‐ire to the comprehensive set of eukaryotic protein families containing experimental PTM and 3D structure data – capturing 1,864 protein families with 60,756 unique PTM sites organized into 40,493 PTM hotspots, of which 2,659 possess known biological function (data obtained from PhosphoSitePlus), analyzed across 51,000 crystalized molecular structures. PTM hotspots with a known biological function undergo up to 6‐fold enrichment using SAPH‐ire ranking methods. Moreover, we find that a high percentage of top‐ranked hotspots lacking functional evidence in our input data could be linked to biological function by in‐depth literature searches. In contrast, ~10% of PTM hotspots predicted to be biologically impactful have yet to be verified experimentally ‐ representing putative regulatory elements. We provide experimental evidence for one such element in which we discovered a previously un‐described feedback phosphorylation mechanism in GPCR signal transduction systems. Taken together, these results highlight the utility of SAPH‐ire as a predictive tool for the identification of biologically important PTMs and provide a model through which the structural topology of PTMs may be explored.

Support or Funding Information

This work was funded by the Georgia Tech School of Biology (M.T.) as well as the National Institutes of Health grant 4R00 GM094533‐03 (to M.T.).
Chlorinated Lipids Activate Human Neutrophils
Authors: Palladino Elisa N. D. , Ford David A
Published: November 2023

Neutrophil activation is orchestrated by a complex array of mechanisms including phosphorylation and activation of NADPH oxidase and subsequent hydrogen peroxide consumption by myeloperoxidase leading to hypochlorous acid (HOCl) production. Our research group has previously shown that the vinyl ether bond of plasmalogen phospholipids is targeted by HOCl in activated leukocytes, which results in the production of 2‐chlorofatty aldehyde (2‐ClFALD). 2‐ClFALD is also oxidized to 2‐chlorofatty acid (2‐ClFA) in neutrophils. The role of these two chlorinated lipids on neutrophil activation was investigated in the present study. 2‐ClFALD production was used as an indicator of the transition from quiescent to activated neutrophils when treated with chlorinated lipids. Treatment with the 2‐ClFA, 2‐chlorohexadecanoic acid (2‐ClHA), resulted in neutrophil 2‐ClFALD production, which included both 2‐chlorohexadecanal (2‐ClHDA) and 2‐chlorooctadecanal (2‐ClODA) molecular species. In contrast, 2‐ClHDA did not result in 2‐ClFALD production (monitored by 2‐ClODA production). 2‐ClFALD production elicited by 2‐ClHA was less than that elicited by phorbol myristate acetate (PMA), but concomitant PMA and 2‐ClHA treatments led to a greater amount of 2‐ClFALD production compared to the individual treatments. Employing selective pathway inhibitors, it was shown that unlike the neutrophil activation pathway stimulated by PMA, the pathway activated by 2‐ClHA is PKC‐, ERK1/2‐ and calcium‐independent. Further studies comparing human neutrophils and neutrophil‐like HL‐60 cells, demonstrated that these cells disparately respond to PMA and 2‐ClHA suggesting that further detailed comparisons between these cell types may provide additional insights into the mechanisms mediated by 2‐ClFA‐mediated neutrophil activation. In summary, 2‐ClFA may be an important mediator produced during inflammation, which is responsible for the regulation of one of the major defense mechanisms used by innate immunity.

Support or Funding Information

National Institutes of Health Grant GM115553
N‐Docosahexaenoylethanolamine ameliorates LPS‐induced brain inflammation through suppression of neutrophil/macrophage as well as microglia activation
Authors: Park Taeyeop , Lee Jiwon , Chen Huazhen , Kim Hee‐Yong
Published: November 2023

Inflammation is a widely accepted common factor among various neuropathological processes and has been implicated as a critical mechanism responsible for the progression of neurodegeneration. Brain inflammation is characterized by glial cell activation accompanied by production of inflammation‐related cytokines, chemokines and nitric oxide (NO).

Synaptamide (N‐docosahexaenoylethanolamine) is synthesized from docosahexaenoic acid (DHA) in the brain and potently induces neurite growth and synaptogenesis in developing neurons and neurogenic differentiation of neural stem cells (NSCs). Growing evidence also suggests that DHA‐derived metabolites have anti‐inflammatory and pro‐resolving effects, however, the role of synaptamide in the brain inflammation is unknown. Thus, we investigated synaptamide effects on lipopolysaccharide (LPS)‐induced cytokine, chemokines, and iNOS expression in the brain, along with possible mechanisms.

We found that synaptamide significantly reduces LPS‐induced production of TNF‐α and Nitric oxide in microglia cells with concomitant induction of cAMP/PKA phosphorylation. Conversely, adenylyl cyclase or PKA inhibitors abolished the synaptamide effect on TNF‐α expression. More importantly, we found that in vivo administration of synaptmide almost completely suppresses brain inflammatory response, including morphological activation of microglia and astrocytes, mRNA expression of inflammatory cytokines, chemokine and iNOS in the brain. Interestingly, we also found that synaptamide exerts anti‐inflammatory effect on neutrophil and macrophage cells. Taken together, our findings suggest that synaptamide can reduce LPS‐induced brain inflammation through suppression of microglia, neutrophil and macrophage activation. The synaptamide‐mediated mechanism contributes to the protective effects of DHA against neuroinflammation, and may provide a new therapeutic target to ameliorate the inflammation‐induced brain dysfunction.
Brain stearoyl‐CoA desaturase‐1 deficiency protects against high‐fat diet‐induced metabolic derangements
Authors: Zdechlik Alina Catherine , Harrington Kristin R. , Burhans Maggie S. , Guo Chang‐An , Bond Laura M. , Ntambi James M.
Published: November 2023

The high prevalence of obesity has classified it as a significant health problem due to the potential for it to increase the risk for development of other metabolic disorders including insulin resistance and hepatic steatosis. Maintenance of metabolic health requires extensive crosstalk among numerous tissues, including brain, adipose and liver. Previous work on the role of brain fatty acids and systemic metabolic regulation has focused on effects of exogenous fatty acids while the role of endogenously synthesized brain fatty acids is not well described. We previously demonstrated that whole‐body deficiency of stearoyl‐CoA desaturase (Scd)‐1 in mice protects from high‐fat diet (HFD)‐induced obesity, hepatic steatosis and insulin resistance. However, the contribution of brain‐specific Scd1 deficiency in protection from these diet‐induced metabolic disorders has not yet been described. Our lab developed an Scd1 brain knockout (BKO) mouse model using the cre‐Lox system in which cre expression was driven by the Nestin promoter. BKO mice gain significantly less weight and exhibit reduced hepatic triglyceride accumulation after 18 weeks of HFD feeding. Further, high‐fat diet‐fed BKO mice have improved glucose tolerance compared to Lox controls, suggesting increased glucose disposal in skeletal muscle and adipose tissue.
Hepatic Stearoyl‐CoA Desaturase deficiency suppresses gluconeogenesis by regulating lipid profile of mitochondria
Authors: Guo Changan , Ntambi James M
Published: November 2023

Obesity plays a central role in the metabolic syndrome, which includes hyperinsulinemia, hypertension, hyperlipidemia and an increased risk of atherosclerotic cardiovascular disease. A common health complication observed in obese patients is insulin resistance, a condition in which increased insulin levels are required to maintain normal blood glucose levels. Without proper intervention, the condition often deteriorates into type 2 diabetes mellitus, characterized by increased hepatic glucose production, and decreased glucose clearance by insulin‐responsive tissues. It has been suggested that high glucose is associated with increased oxidative stress, enhanced leukocyte‐endothelial interaction, and glycosylation of lipoproteins, apolipoproteins, and clotting factors. Identification of new therapeutic approaches to improve glycemic management via the inhibition of hepatic glucose production will be beneficial for diabetes, cardiovascular diseases and related metabolic disorders.

Stearoyl‐CoA desaturase (SCD) expressed at high levels in several human tissues is required for the biosynthesis of oleate (18:1) and palmitoleate (16:1), which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides and cholesterol esters. Given the multiple roles of monounsaturated fatty acids, variation in stearoyl‐CoA desaturase activity in mammals would be expected to have a major effect on lipid metabolism. We and others have reported that global deletion of SCD1 in mice dramatically increases whole body energy expenditure and the loss of SCD1 activity in the liver suppresses hepatic glucose production when mice were fed a lipogenic diet. We hypothesize that the deficiency of SCD alters energy metabolism at the cellular level via the regulation of mitochondria function. Our preliminary data suggest that hepatic SCD1 deficiency alters the lipid profile of mitochondria membrane, therefore alters the membrane potential and metabolic rate of the cell. In addition, we show that the pharmaceutical inhibition of SCD activity reduces hepatic glucose production in the cell culture conditions. These studies provide essential information on the role of SCD on energy metabolism and, at the same time validate hepatic SCD enzymes as therapeutic targets against hyperglycemia in disease conditions, such as diabetes and metabolic syndrome.

Support or Funding Information

American Diabetes Association 7‐13‐BS‐118
Diacylglycerol Kinase ζ Produces 16:0‐containing Phosphatidic Acid Molecular Species during Neuroblastoma Cell Differentiation
Authors: MIZUNO Satoru , Sakane Fumio
Published: November 2023

Phosphatidic acid (PA) is one of the phospholipids composing the plasma membrane and acts as a second messenger to regulate a wide variety of cellular functions, including cell growth, migration and differentiation. PA consists of various molecular species with different acyl chains at the sn‐1 and sn‐2 positions. However, it was unclear which PA molecular species were produced during cell stimulation. In this study, we identified the PA molecular species produced during neuroblastoma cell differentiation and the PA species production pathway using our previously developed liquid chromatography (LC)/mass spectrometry (MS) method (1).

First, mouse neuroblastoma Neuro‐2a cells were differentiated by treatment with 20 μM retinoic acid (RA) for 24 hours. LC/MS analysis showed that the amount of total PA was significantly increased in RA treated Neuro‐2a cells (1.69‐fold). Notably, the amount of 30:0‐ and 32:0‐PA species were dramatically increased in RA treated Neuro‐2a cells (2.13‐, 4.67‐ and 1.85‐fold, respectively). Similar results were obtained when Neuro‐2a cells were treated with serum starvation for 24 hours. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). Next, we examined the production pathway of 30:0‐ and 32:0‐PA. FIPI, phospholipase D‐specific inhibitor, did not inhibit the production of these PA species. RT‐PCR analysis showed that diacylglycerol kinase (DGK) δ and DGKζ were highly expressed in Neuro‐2a cells. The silencing of DGKδ expression did not decrease the production of the PA species. However, DGKζ‐specific siRNA significantly decreased the production of 30:0‐ and 32:0‐PA. In addition, DGKζ‐specific siRNA substantially attenuated neurite outgrowth/differentiation. These results suggest that DGKζ produces 16:0‐containing 30:0‐ and 32:0‐PA species and regulates neurite outgrowth during neuroblastoma cell differentiation.

Support or Funding Information
Sub‐Chronic Exposure To Cadmium At Low Levels Alters Microsomal Lipid Storage in Rat
Authors: Ogunrinola Olabisi O , Wusu Dorcas A , Afolabi Kayode , Balogun Esther , Ademuyiwa Oladipo , Odukoya Olusegun
Published: November 2023

Cadmium (Cd) is a ubiquitous environmental pollutant of increasing worldwide concern. Epidemiological studies suggest an association between cadmium in drinking water and many harmful effects in the health of living organisms. However, the precise cadmium mechanism of action remains enigmatic. This study was undertaken to investigate the effect of cadmium on microsome lipid storage of Wister male albino rats by exposing the animals (n = 24; mean weight = 250g) to 100, 200 and 300ppm cadmium doses for 12 weeks in their drinking water. Control animals (n = 8) received distilled water for the same period. At the end of 12 weeks, blood, liver and brain were removed from the animals and lipid dynamics were investigated spectrophotometrically. Exposure to cadmium resulted in accumulation of cadmium in the liver (from 6.14±0.22 μg Cd/g tissue in control to 54.87±4.86 μg Cd/g tissue in 300ppm dose). Induction of cadmium doses at all doses produced different patterns of microsomal lipid storage. Dose‐dependent up‐and‐down regulation characterized the effect of cadmium exposure at all doses. Exposure to cadmium at the highest dose significantly (p > 0.05) inhibited both hepatic and brain microsomal lipid profiles. On the other hand, brain cholesterol (65%) and triacylglyceride (50%) were dose‐dependently decreased by cadmium exposure, respectively, whereas phospholipid (38%) was increased by cadmium exposure. Hepatic cholesterol and phospholipid were decreased by 55% and 32%, while triacylglyceride was enhanced by 52%. We observed negative associations between tissue cadmium levels, brain and hepatic microsomal lipid profile. The results suggest that exposure to different doses of cadmium has differential effects on storage lipid profiles of rat brain and hepatic microsomes. This might unravel another aspect of the underlying mechanism of cadmium toxicity.
Choline Deficiency Alters Lipid Droplet Composition and Size
Authors: Brown Elizabeth , Townsend Elizabeth , Hains Anastasia , Listenberger Laura
Published: November 2023

Excess lipid is stored in organelles known as lipid droplets. The surface of a lipid droplet is a phospholipid monolayer composed primarily of two phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Specific proteins decorate the surface of each lipid droplet and regulate its activity. We aimed to determine whether the phospholipid composition of the lipid droplet surface influences the number and type of lipid droplet proteins. We cultured NIH 3T3 cells in choline deficient media and measured a decrease in the PC:PE ratio in lipid droplet fractions. While total levels of lipid droplet proteins were not changed, choline deficient media altered the lipid droplet binding of a subset of proteins. For example, cells grown in choline deficient media had 1.4 and 1.6‐fold more perilipin 2 and 3 fractionating with lipid droplets. Moreover, cells grown in choline deficient media accumulated larger and more numerous lipid droplets following oleate supplementation than cells grown with choline. Our studies link lipid droplet phospholipid composition and protein binding to cellular lipid homeostasis.

Support or Funding Information

This research was funded by St. Olaf College CURI, the Mindlin Foundation, and an ASBMB undergraduate research award (to EB).
Screening for Genes Required for Coenzyme Q Uptake and Trafficking in S. cerevisiae
Authors: Kampmeyer Christopher , James Andrew M. , Murphy Michael P. , Clarke Catherine F.
Published: November 2023

Coenzyme Q (ubiquinone or Q), is a critical component of the electron transport chain in mitochondria where it functions as an electron carrier and as a lipid antioxidant. Q has as a result garnered attention as a nutritional supplement in treating coenzyme Q deficiencies and as a potential therapy in the treatment of cardiovascular disease and neurodegenerative disorders. Previous work identified several yeast genes required for the rescue of Q‐less coq yeast mutants by exogenous supplementation with coenzyme Q6, the Q isoform synthesized by Saccharomyces cerevisiae. While it is known that transport of coenzyme Q6 requires the endocytic pathway, the mechanism of exogenous Q uptake into the cell is still poorly understood. In mutant strains of the well‐characterized model, Saccharomyces cerevisiae, Q is nonessential for growth, owing to yeast's ability to grow anaerobically on fermentable carbon sources; on nonfermentable carbon sources, yeast mutants unable to synthesize Q exhibit a growth defect unless Q is exogenously supplied and trafficked to the mitochondria. This characteristic recovery by exogenous Q forms the basis for the screen to identify genes required for Q uptake.
PGC‐1α‐mediated Changes in Phospholipid Profiles of Exercise‐trained Skeletal Muscle
Authors: Miura Shinji , Senoo Nanami , Miyoshi Noriyuki , Goto‐Inoue Naoko , Minami Kimiko , Yoshimura Ryoji , Morita Akihito , Sawada Naoki , Matsuda Junichiro , Ogawa Yoshihiro , Setou Mitsutoshi , Kamei Yasutomi
Published: November 2023

Exercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber‐type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC‐1α, PGC‐1α may contribute to the exercise‐mediated change in phospholipid fatty acid composition. To determine the role of PGC‐1α, we performed lipidomics analyses of skeletal muscle from genetically modified mice that overexpress PGC‐1α in skeletal muscle or that carry knockout alleles of PGC‐1α. The overexpression of PGC‐1α in the skeletal muscle caused a significant change in the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) profiles of the muscle. The PC and PE profile of EDL from PGC‐1α‐Tg mice, which showed oxidative characteristics, was similar to the profile of the originally oxidative muscle, such as the soleus. We also found that PGC‐1α increased several phospholipid species in glycolytic muscle, namely PC (18:0/22:6) and PE (18:0/22:6). Exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC‐1α was required for these alterations. Since phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training‐mediated functional changes in the skeletal muscle.

Support or Funding Information

This study was supported by the Council for Science, Technology and Innovation (CSTI), Cross‐ministerial Strategic Innovation Promotion Program (SIP, No.14533567), and “Technologies for creating next‐generation agriculture, forestry and fisheries” (funding agency: Bio‐oriented Technology Research Advancement Institution, NARO), The Tojuro Iijima Foundation for Food Science and Technology (Chiba, Japan), Grants‐in‐Aid for Scientific Research (KAKENHI, No. 26282184, 26560400, 21300240) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT, Tokyo), The Uehara Memorial Foundation (Tokyo, Japan), The Kao Research Council for the Study of Healthcare Science (Tokyo, Japan, No. A‐31006), and University of Shizuoka Grant for Scientific and Educational Research.
Molecular determinants of the localization of the phosphatidylinositol/phosphatidic acid transfer protein, Nir2 to ER‐PM contact sites
Authors: Kim Yeun Ju , Guzman‐Hernandez Maria Luisa , Wisniewski Eva , Echeverria Nicolas , Balla Tamas
Published: November 2023

We have recently described that Nir2, a homolog of the Drosophila RdgB protein and a mammalian phosphatidylinositol (PI) transfer protein, is required for the transfer of phosphatidic acid (PA) from the plasma membrane (PM) to the ER and for the transport of PI in the other direction at ER‐PM contact sites. Nir2, which is localized to the ER in quiescent cells, translocates to ER‐PM contacts sites upon phospholipase C (PLC) activation. Here, the transport of PI and PA in the opposite direction is critical for the maintenance of PI(4,5)P2 levels and hence, sustains signaling during PLC action. The ER localization of Nir2 has been shown to be mediated by interaction with the ER‐resident VAP‐A and –B proteins via an FFAT domain, whereas the PM localization is induced by interaction with the lipid products of PI(4,5)P2 hydrolysis, namely, diacylglycerol (DG) and PA. Here we investigated which part(s) of Nir2 are responsible for PM localization and how mutations in VAP‐B protein, identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou‐Gehrig's disease) affect Nir2 localization in the ER. We will present data on our truncation and mutation studies addressing DG and PA recognition by Nir2 and show that mutations of VAP‐B not only cause mislocalization and aggregation of the VAP protein, but also impairs its binding to Nir2.
Substrate selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli
Authors: Lin Yibin , Bogdanov Mikhail , Tong Shuilong , Guan Ziqiang , Zheng Lei
Published: November 2023

Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2‐ acyl lyso‐PE recycling in gram‐negative bacterium. This work identified the potent role of LplT in maintaining membrane stability and integrity in E.coli envelope. We demonstrated the involvement of LplT in the recycling of three major bacterial phospholipids by using combination of in vitro lysophospholipid binding assay with purified protein and transport assays with E.coli spheroplasts. Our results showed that lyso‐PE and lyso‐PG, but not lyso‐PC, were taken up by LplT for reacylation by acyltransferase/acylACP synthetase (Aas) on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A2 to form diacylated cardiolipin and eventually the completely deacylated head group. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate tri‐acylated cardiolipin by Aas, demonstrating the first evidence of cardiolipin remodeling in bacterium and supporting a notion that fatty acid chain is not required for LplT transport. We also found that LplT cannot transport lyso‐PA and its substrate binding was not inhibited by either orthophosphate or glycerol‐3‐ phosphate, revealing that glycerol or ethanolamine head group is the chemical determinant for substrate recognition. Any diacyl form of PE, PG or tetra‐acylated form of cardiolipin could not serve as competitive inhibitors in vitro. Based on an evolutionary structural model, a proposed “sideways sliding” mechanism explains how a conserved membrane‐embedded α‐helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophosholipid across the cell membrane.

Support or Funding Information

This work was supported by NIH grant R01 to Lei Zheng.
Investigation of Molecular Interactions Between Lysophospholipase A2 and its Diverse Bioactive Lipid Substrates
Authors: Wepy James Anthony , Xu Shu , Kudalkar Shalley Nadish , Marnett Lawrence J
Published: November 2023

Lysophospholipase A2 (LYPLA2) is a serine hydrolase responsible for the hydrolysis of lysophospholipids, including lysophosphatidic acids, lysophosphatidylcholines and lysophosphatidylserines. These lysophospholipids are known to elicit a variety of physiological effects, such as the activation and recruitment of phagocytes, induction of cytokine expression, aggregation of platelets, calcium mobilization, and proliferation of vascular smooth muscle cells. We have also recently identified LYPLA2 as the major prostaglandin glyceryl ester (PGG) hydrolase in human cancer cells. These metabolites of the endocannabinoid species, 2‐arachidonoylglycerol (2‐AG), are precursors to the bioactive prostaglandin species, which also elicit a variety of effects, including vasoregulation, hyperalgesia, and inflammatory responses.

With purified recombinant LYPLA2, we have used mass spectrometric approaches to evaluate and compare the kinetic rates of hydrolysis of its various substrates, including PGGs, lysophospholipids, and the prostanoid precursors, 2‐AG and arachidonoylethanolamide. With these data, we have shown that LYPLA2 preferentially hydrolyzes 1‐glyceryl esters of esterified lipids, and selectively metabolizes PGGs over their endocannabinoid precursor, 2‐AG. We have recently solved the first crystal structure of LYPLA2 and used this data to develop site‐directed mutants, providing new insights into the binding site of this enzyme. Structural investigations of LYPLA2 reveal a large channel at the surface of the protein, as well as a mobile loop near its catalytic triad. To further investigate the binding site interactions of LYPLA2 with its structurally diverse substrates, site‐directed mutagenesis was employed to express, purify and assess the hydrolytic activity of mutants at these hypothesized binding sites. Mutations of amino acids located in the surface channel were mutated to large tryptophan residues to sterically hinder binding of lipid substrates in the channel, and have shown to significantly inhibit the enzyme's hydrolytic activity. Here we describe the first kinetic analyses of this enzyme's ability to hydrolyze a wide range of PGG, endocannabinoid, and lysophospholipid substrates. Collectively, these data provide the first structural interpretation of the interactions of LYPLA2 with its bioactive lipid substrates.

Support or Funding Information

Authors would like to acknowledge sources of funding for this research including:

Center in Molecular Toxicology Predoctoral Training Grant (NIEHS T32 ES007028)

National Institute of Health Research Grant (GM15431)
FauxSeqEvolver (FaSE) Software Automates the Electronic Creation of Novel Pseudo Biological Sequences, Generating Truly Novel “Unknown” Sequences For Use In Bioinformatic Education
Authors: Rutherford Robert
Published: November 2023

Some faculty ask their students to characterize “unknown” biological sequence(s) to assess a student's skills in bioinformatics analysis and/or foster student engagement. Unfortunately, the ever expanding set of easily accessible, well characterized biological databases and student social media makes maintaining a set of “unknown” sequences challenging and tedious. In response, we have created FauxSeqEvolver(FaSE) to automate the electronic creation of biologically credible sequences whose genuine novelty will afford each student the full benefit of characterizing a unique and un‐annotated sequence. Faculty can customize the sequences FaSE creates by inputting one or more seed sequence(s) (which students shall hopefully uncover later as candidate homologs…), and by defining the timeline for evolutionary divergence and degree and nature of selective pressure. The software, which is implemented in BioPython and C++, iteratively mutates seed sequence(s) to simulate biomolecular evolutionary events. Upon completion, the software outputs include: 1) novel faux biological sequence(s) appropriate to challenge students to do their own novel bioinformatics characterization, and 2) a concise record of each sequence's unique attributes for the instructor's use in assessing the student's forthcoming characterization work. The author welcomes discussions with faculty who might desire to use FaSE or suggest ways to further enhance its utility.
Is More Activity Always Better? A Department‐Wide Study of Relationships Between Classroom Practices and Student Performance
Authors: Barker Megan Kayte , Weir Laura , McDonnell Lisa , Schimpf Natalie , Rodela Tammy , Schulte Patricia
Published: November 2023

Extensive research into science education has established that active learning practices positively impact student learning1. From these findings, we can now move forward to explore how to best design active classroom practices, and assess the most effective use of instructional time. To address these issues on a large scale within biology, we investigated the relationships between specific classroom practices and student learning in a biology department‐wide study at a large research university in the Pacific Northwest. For 31 different biology course sections ranging from freshman to senior year, and on topics from molecular biology and biochemistry to ecology and physiology, we collected classroom activity and diagnostic test data. Classroom observations captured the student and instructor behavior using the validated tool COPUS2; the diagnostic assessments were multiple‐choice pre‐ and post‐tests aligned to each course's content, consisting largely of validated questions from the concept inventory literature. Our observational data analysis showed a wide variety of instructor practices across the department, consistent with other large‐scale characterizations, which allow for clustering this multidimensional classroom data into broader instructional styles3,4. Notably, students in “Collaborative” classrooms significantly outperform students in “Peer Instruction” and “Lecture” styles, though there is no difference between the performance in the two latter styles. More finely‐grained, the amount of class time spent on clicker questions or worksheets are each significant and positive predictors of student performance. Interestingly, we see a negative relationship between unstructured group discussions (using neither clicker nor worksheets) and performance, highlighting the need for targeted approaches to engage students in productive active learning. Thus, building on current research that describes large‐scale teaching practices, we can now quantitatively link program‐wide COPUS observational data with student outcomes. The results of this work describe a platform by which others might evaluate learning and classroom practices, inform future directions for instructor development, and provide further insight into the types of classroom activities that have the greatest impact on student learning.