bluuebunny/crossref_metadata_2025 huggingface dataset:
Exosomes‐mediated mTOR activation is involved in ovarian cancer cell aggression and migration
Authors: Madigan Audrey Pamela , Gallant Benjamin , Clark Matthew P , Wan Yinsheng , Zhang Wei , Lu Hongguang
Published: November 2023

Ovarian cancer remains difficult to treat, with the remission rate still high and its drug resistance obvious. Our previous studies have demonstrated that ovarian cancer cells resist chemotherapeutic drugs via activation of EGFR‐PI3K‐AKT mTOR cell survival signaling pathway. Recent studies have suggested that exosome biogenesis and release is associated with cancer cell aggression, migration and cell‐cell communication. We hypothesize that exosomes‐mediated mTOR activity is involved in ovarian cancer cell metastatic potential and migration. Using phagokinetic motility assay, we observed that inhibitors of PI3K/AKT, MEK/ERK, and mTOR reduced cell migration in ovarian cancer cells (CaOV3 cells). Through confocal microscopy and Western Blot we found that when CaOV3 cells are treated with FBS and/or EGF, higher amounts of CD‐63 and p‐S6 are found. We also found that inhibitors of exosome biogenesis and release, GW4869 and Brefeldin A, attenuated EGF‐ and serum‐induced cell proliferation and migration. Western blot analysis showed that EGF induced expression of CD63, one of the markers of exosomes, in a dose dependent manner. It was also seen that Brefeldin A and Monensin reduce the presence of CD‐63 and p‐S6. Interestingly, while Brefeldin A blocked serum‐induced mTOR activation, GW4869 appeared to enhance mTOR activity in CaOV3 cells. Collectively, our data suggests that exosome biogenesis and release feedback regulates mTOR activity in ovarian cancer cells, with a result of aggression, migration, and drug resistance. Modulation of exosome activities may provide clinical therapeutic options for ovarian cancer.

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RI‐IBRE
Signaling and secretion of chemotactic factors regulated by calcium‐sensing‐receptor mutants found in breast cancer patients
Authors: Orduña Lennis Beatriz , Hidalgo‐Moyle Joseline Janai , Vázquez‐Prado José , Reyes‐Cruz Guadalupe
Published: November 2023

The calcium‐sensing‐receptor (CaSR) belonging to the class C of the GPCR family regulates cell proliferation, calcium homeostasis and secretion, among other processes, depending on the tissue in which this receptor is expressed. Its physiological importance is also evidenced by several disorders in the calcium homeostasis associated with a number of naturally mutations. The bioinformatic study of CaSR in the catalogue of somatic mutations in cancer (COSMIC) has documented 419 mutations of this receptor in patients with cancer. Sixteen of which are specifically found in breast cancer, four of them (CaSRN639K, CaSRT732A, CaSRR886Q and CaSRV894I) were generated and study in our laboratory to analyze their role in the secretion of chemotactic factors and the signaling events. Considering the potential coupling of CaSR to multiple heterotrimeric G proteins, we examined the effect of PTX (pertussis toxin), an inhibitor of Gi protein; H89, a PKA inhibitor; and U73122, a phospholipase C inhibitor on ERK activation. Our results demonstrated that CaSRWt signals through Gi and Gq proteins, however the mutants receptors were potentially linked to Gs as well as Gq, and the CaSRN639K could also activates Gi. Importantly, our results indicate that the mutant receptors promoted different patterns in the chemotactic factors secretion analyzed by the potential effect of conditioned media obtained from each mutant receptors to promote migration of porcine aortic endothelial cells (PAE) in Boyden chamber assays. The mutants CaSRT732A and CaSRV894I promoted the secretion of chemotactic factors in a calcium‐dependent manner comparable to the CaSRWt although these secretion is minor with respect to the wild type receptor. The mutant CaSRN639K increased the secretion in a calcium‐independent manner, and its stimulation does not have an effect on secretion. Finally, the CaSRR886Q mutant receptor increase the secretion of chemotactic factors independently of stimulus, and surprisingly receptor stimulation inhibited the secretion of chemotactic factors.

CONCLUSION

The calcium sensing receptor mutants found in breast cancer patients potentially change the G‐protein coupling and elicited different secretory patterns, probably due to change in the G‐protein coupling.

Support or Funding Information

This work was supported by Consejo Nacional de Ciencia y Tecnologia (CONACyT) grants 240119 (to G.R.C.) and 152434 (to J.V.P.) and Fundación Miguel Aleman, A. C. (to G.R.C.). Lennis Beatriz Orduña Castillo is graduate student supported by a CONACyT fellowship (scholarship: 455391)
A single EGF molecule activates a preformed EGFR dimer: a single‐molecule multi‐color TIRF microscopy study
Authors: Saita Ei‐ichiro , Mong Dingze , Maruyama Ichiro N
Published: November 2023

Epidermal growth factor receptor (EGFR) plays roles in cell proliferation, inhibition of apoptosis and angiogenesis among others. It has long been thought that binding of the ligand induces the dimer formation of monomeric EGFR (the “ligand‐induced dimerization model”). Tyrosine residues in the C‐terminal tail of active EGFR are phosphorylated, which triggers activation of downstream signaling cascades by recruiting effector proteins such as Shc1. However, many recent studies indicate that EGFR exists in a form of dimer (preformed dimer). To understand the molecular mechanism underlying activation of EGFR, we visualized the activation of EGFR by ligand binding at the single‐molecule level. Using a multi‐color TIRF microscope, we directly observed interaction among EGFR, EGF labeled with two different fluorescent dyes, and GFP‐tagged Shc1. Within 30 seconds of application of the dye‐conjugated EGF to cultured cells, three different fluorescent spots of EGF and Shc1 appeared on the cell surface. Unexpectedly, two different fluorescent colors derived from EGF do not co‐localize, and instead, single‐colored EGF binding induces Shc1 recruitment to EGFR. These results suggest that binding of single EGF molecule can activate preformed EGFR dimer, and are consistent with negative co‐operative binding between EGF to EGFR.

Support or Funding Information

This work was partly supported by funding to the Information Processing Biology Unit from the Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan.
A Novel Interaction between β‐arrestins and Nuclear Steroid Receptors
Authors: Petrillo Maria Grazia , Cidlowski John A
Published: November 2023

The β‐arrestin proteins play a well established role in the dampening of G‐protein coupled receptor (GPCR) signaling. In addition to being negative regulators of GPCRs, β‐arrestins are being increasingly appreciated as scaffold proteins, thus conferring novel signaling properties independent from GPCRs activity. By acting as scaffolds, the β‐arrestins interface with several proteins, thus generating a new wave of signaling pathways, including interfering with the ubiquitin‐proteasome machinery, and altering gene transcription. Recent studies have demonstrated that glucocorticoids (GCs) can regulate the transcription of β‐arrestin proteins, thereby contributing in redirecting the GPCR signaling profile. Glucocorticoid responses are mediated by the glucocorticoid receptor (GR), a member of the nuclear receptor family. By binding specific DNA responsive elements, the glucocorticoid receptor activates or represses the transcription of target genes, thus regulating numerous physiological processes, including development, metabolism, and immune response. We have recently discovered that β‐arrestin‐1 can interact with the glucocorticoid receptor. This finding led us to investigate the possibility that β‐arrestin‐1 may alter the activity of the glucocorticoid receptor. Whole transcriptome sequencing revealed that β‐arrestin‐1 has a wide impact on the glucocorticoid receptor signaling. The transcriptome analysis identified post‐translational modification as one of the top‐ranked cellular functions significantly altered by the lack of β‐arrestin‐1. Moreover, within the subset of genes enriched for post‐translational modification, lack of β‐arrestin‐1 reshaped the GR gene signature, regulating genes responsible for the activation of the ubiquitin‐proteasome machinery. In vitro data revealed that β‐arrestin‐1 affects glucocorticoid‐induced GR downregulation by altering its expression and controlling its proteasome‐mediated degradation. These results indicate a novel protein partnership between glucocorticoid receptor and β‐arrestin‐1.

Support or Funding Information

National Institute of Health (NIH)
Steroid Hormone Receptor Gene Expression as a Marker for Phenothiazine Induced Endocrine Disruption and Stress in Mummichog, Fundulus heteroclitus
Authors: Laperche Jacob M , Chiari John B , Patel Roshni , McGinnis Courtney L
Published: November 2023

Molecules and contaminants that function as endocrine disruptors from industrial products are known to contaminate ground and surface water through agricultural runoff and industrial dumping. These contaminants are not typically removed during water purification and may have significant effects on biological systems through the disruption of normal endocrine function. This study aims to determine the effects of phenothiazine (PTZ), a potential endocrine disruptor, on markers for endocrine disruption and stress in Fundulus heteroclitus. The following steroid hormone receptors were assessed: glucocorticoid receptor (GR), androgen receptor (AR), progesterone receptor (PR), estrogen related receptor alpha (ERα), estrogen receptor beta A (ERβA), and estrogen receptor beta B (ERβB). Phenothiazine was detected at levels between 1–2ppm via gas chromatography‐mass spectrophotometry (GC‐MS) in river water sampling conducted in southern Connecticut, but the biological impact of the compound is unknown. Phenothiazine has been used previously as an antipsychotic medication, as well as, industrially in plastics to add softness and flexibility. To assess this impact of phenothiazine, fish were exposed to environmentally relevant concentrations of phenothiazine for one week and sacrificed. Brain, liver, and gonad tissues were removed and pooled from each treatment group. Quantitative PCR (qPCR) was conducted on tissues and analysis showed significant changes in gene expression. There was an increase in mRNA expression of ERβB, PR, AR, and GR in gonadal tissue. There was an increase in mRNA expression of AR, PR, and ERβA in liver tissue as well. In brain tissue, AR, PR, ERα, ERβA and ERβB mRNA expression was upregulated. These findings indicate that phenothiazine may alter endocrine function through the steroid hormone receptors. Additionally, there was a downregulation in GR mRNA expression in brain tissue, which has been associated with depression. Collectively, these tissue specific results demonstrate how phenothiazine disrupts normal fish physiology. In addition, to determine the impact of phenothiazine on stress, cortisol levels in the blood were assayed via enzyme linked immunosorbent assay (ELISA). No significant changes in cortisol levels following phenothiazine treatment were observed.

Support or Funding Information

The Community Foundation for Greater New Haven and Quinnipiac University: College of Arts and Sciences
Bile Acid Binding Protein STARD5 Suppresses Doxorubicin‐mediated Apoptosis in H1792 Lung Adenocarcinoma Cells
Authors: Clark Barbara J , Dougherty Susan M , Hill Bradford G , Klinge Carolyn M
Published: November 2023

The bile acid chenodeoxycholic acid (CDCA) is a FXR ligand that has anti‐proliferative and pro‐apoptotic actions in gastrointestinal cancer cells. The cytotoxic actions of CDCA appear to be both FXR‐dependent and independent and cell‐type specific. Although FXR is expressed in lung and bile acid biosynthesis is associated with lung inflammation, the role of FXR in lung cancer is unknown. Herein, we have used non‐small cell carcinoma (NSCLC) cancer cell lines to test the effect of the bile acid binding protein, STARD5, on doxorubicin‐mediated apoptosis. Western blot analysis detected high levels of STARD5 protein in 5 lung adenocarcinoma cell lines compared to non‐detectable levels in normal human bronchial epithelial cells (HBEC). These lung adenocarcinoma cells have greater resistance to doxorubicin‐induced apoptosis, measured by caspase activation, compared to HBECs. Notably siRNA silencing of STARD5 in H1792 lung adenocarcinoma cells decreased cell viability and increased sensitivity to doxorubicin‐induced apoptosis. To address possible anti‐apoptotic mechanisms, we measured the mitochondrial function of H1792 cells after siRNA knockdown or transient overexpression of STARD5. Using the Seahorse XF24 analyzer we found that STARD5 knockdown had no effect on mitochondria function while STARD5 overexpression resulted in a 40% decrease in both the ATP‐linked oxygen consumption rate (OCR) and glycolytic reserve. The proton leak was unchanged, suggesting no change in the mitochondria permeability transition pore, an early marker of apoptosis. A decrease in ATP‐linked OCR mediated by STARD5, in the absence of proton leak, may reflect increased mitochondrial ROS production. Decreased mitochondria function and increased mitochondrial ROS would be consistent with suppressed FXR action. To test whether STARD5 affects FXR activity, we measured FXR‐luciferase reporter gene activity in siRNA‐STARD5 knockdown cells. STARD5 knockdown resulted in increased FXR activity, suggesting that STARD5 sequesters FXR ligands (CDCA) which contribute to FXR's pro‐apoptotic activity. Future studies will test the model that FXR signaling in NSCLC sensitizes cells to doxorubicin‐mediated apoptosis and that increased STARD5 in NSCLC mediates suppression of FXR signaling and contributes to acquired chemoresistance.

Support or Funding Information

This study was supported by a U of L SOM Collaborative Matching Grant to BJC and KY Lung Cancer Research Program to CMK.
Examining the Effects of Aldosterone on Putative Target Genes in Mouse Collecting Duct Cells
Authors: Nolan Brandon , Solocinski Kristen , Gumz Michelle L , Zies Deborah
Published: November 2023

Hypertension is the number one risk factor for premature death worldwide, and yet many cases present with no obvious underlying cause. Understanding the normal regulation of blood pressure may help determine these underlying causes and reduce hypertension rates and deaths. Aldosterone is a steroid hormone that binds to the mineralocorticoid receptor (MR) and acts as a primary blood pressure regulator. A recent microarray and preliminary gene expression data from the Gumz Lab suggested two novel aldosterone target genes: growth‐arrest specific 5 (GAS5) and ubiquitin specific protease 8 (USP8). This research explored the relationship between aldosterone and the two potential target genes. It has been shown that GAS5 acts as a decoy for the glucocorticoid receptor (GR) and reduces its ability to activate target gene transcription in the presence of cortisol. Additional data suggests that GAS5 may do the same for aldosterone and MR. USP8 deubiquitinates ENaC and therefore the upregulation of USP8 by aldosterone would facilitate increased activity of ENaC and increased blood pressure. Based on these preliminary data it was hypothesized that aldosterone will first act to increase the expression of both GAS5 and USP8, but as GAS5 accumulates it will act as a decoy for aldosterone bound MR and thereby reduce its own expression as well as that of other aldosterone regulated genes, including USP8. To test this hypothesis a time course of 0, 4, 8, and 24 hour aldosterone treatment was administered to mouse inner medullary collecting duct (IMCD3) and mouse principal kidney cortical collecting duct (mpkCCD) cells. Total RNA from each time point was isolated and converted to cDNA. Quantitative PCR (qPCR) has been completed for GAS5, SGK1, and actin. Assays are currently underway to measure the expression of USP8 and ENaC. Results showed no increase in GAS5 expression in both mpkCCD and IMCD3 cells at any time point (ANOVA, p=0.298, 0.612). In contrast, the expression of SGK1, a known target of aldosterone, was significantly increased in both mpkCCD and IMCD3 cells (ANOVA, p=0.006, 0.01). To further our understanding of the regulation of GAS5 by aldosterone, we cloned the promoter region of the GAS5 gene into a pGL3 expression vector (Promega, Inc.). Current work is aimed at testing the effects of aldosterone treatment on the GAS5 promoter in a luciferase assay system.
Identification of a Novel Gene in the Shade Avoidance Response in Plants
Authors: Wershoven Nicole , Giddings Emily , Clark Amber Elizabeth Miller , Palmer Christine
Published: November 2023

Plants rely on light as their source of food and elicit a series of coordinated responses known as the shade avoidance syndrome (SAS) when faced with light challenge. These molecular and developmental responses include elongation of petioles, accelerated flowering, and decreased germination. Here we examine seven candidate genes in the model plant Arabidopsis thaliana for their role in the SAS. We conducted qPCR in mutant lines lacking each of these genes to observe any alteration in expression of pathways including light perception, transcriptional regulation, plant growth, and immune response. Preliminary results indicate that At1g677960, a gene of previously unknown function, may play a role in the SAS. Loss of this gene leads to impaired induction of shade avoidance genes and plants fail to show increased petiole length under shade conditions. Mutants are still able to induce early flowering, suggesting that this novel gene is required in the shade avoidance response but may act downstream or in parallel to the primary response. Further investigation of the role of At1g677960 will allow us to better understand the molecular mechanics of shade avoidance, and potentially offer insight into improving the success of vital crop species.

Support or Funding Information

Research reported here was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103449. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
Finding the Differential Interactome of active vs inactive Small Molecular Weight GTPases
Authors: Peterson Tabitha , Piper Robert C
Published: November 2023

The family of Ras‐related small molecular weight GTP‐binding proteins encompasses over 160 members. Together, these members control myriad aspects of cell biological processes by binding effector proteins in a manner that is dependent on whether they are bound to GTP or GDP. Ras‐family GTPases are built on a conserved structural template that dictates the mode of nucleotide binding. This template provides the opportunity to create mutations within conserved residues that force the GTP‐binding protein into the GDP‐bound conformation, which is typically inactive, or a GTP‐bound conformation, which is typically “active” in the sense that it now binds more favorably to its effectors. Each member of the Ras superfamily is thought to have its own particular set of interacting proteins that allow it to define and execute its function. This specificity is accounted for with fairly subtle sequence differences, typically clustered on small regions on an overall highly structurally conserved skeleton. Here we show further development of the DEEPN approach, which uses next‐generation sequencing to compare yeast‐2 hybrid interactions in batch across protein variants to computationally define set of interacting proteins specific for one protein variant over another. We show the application of these improved tools and methods on a subset of Ras‐family GTPases, where we uncover interactions that are specific for the GTP‐bound vs GDP‐bound conformations. Moreover, we describe new libraries and other key reagents that streamline this approach so that it can be widely adapted by other laboratories. Finally, we show how these efforts can be applied in a undergraduate teaching lab to expose students to both molecular and genetic techniques as well as computational approaches.

Support or Funding Information

NSF Research Project Grant: 1517110
Development of an improved in vitro culture system for Cryptosporidium parvum
Authors: Wilke Georgia , Funkhouser‐Jones Lisa , Ravindran Soumya , Kuhlenschmidt Mark , Stappenbeck Thad , Sibley L. David
Published: November 2023

The protozoan parasite Cryptosporidium parvum is a leading cause of pediatric diarrhea in developing countries, yet effective treatments for the disease are lacking due to the difficulty of studying Cryptosporidium in the laboratory. Specifically, current culturing methods rely on adenocarcinoma cell lines that do not support continual growth of C. parvum in vitro, hindering the development of reagents such as stage‐specific antibodies and genetic tools for this parasite. To better mimic the native intracellular niche of C. parvum, we have utilized primary intestinal epithelial cell (IEC) monolayers as an improved system for the study of C. parvum. We have found that human and mouse IECs support enhanced growth of C. parvum compared to adenocarcinoma cell lines. Importantly, parasite growth in mouse IECs, the first mouse cell line to support robust asexual C. parvum growth, enabled us to generate a unique panel of monoclonal antibodies against the intracellular life cycle stages of the parasite. We have also developed quantitative PCR assays to measure expression of genes associated with sexual‐stage development of the parasite independent of absolute parasite replication. With the development of these new tools and the enhanced culture system, we are now able to screen culture conditions mimicking the hypoxic, reducing environment of the small intestine to identify conditions that further improve C. parvum growth and/or complete development of the life cycle in vitro. Additionally, we are also screening a panel of gut microbial metabolites for their effects on C. parvum growth to gain insight into the complex community interactions that may contribute to C. parvum infection in vivo. In all, these new tools and improved culture system will facilitate the study of Cryptosporidium in vitro, allowing us to gain a better understanding of the parasite's biology as a means to more effective therapeutics for the disease in the future.

Support or Funding Information

This work is supported by the Bill and Melinda Gates Foundation.
Recognition and selectivity of peptide pheromones by ComR in the regulation of natural competence among Streptococcus species
Authors: Prehna Gerd , Shanker Erin , Morrison Donald A , Talagas Antoine , Nessler Sylvie , Federle Michael J
Published: November 2023

Bacteria transmit chemical signals to each other in a process known as quorum sensing. This adaptation is central to pathogenesis as it allows bacteria to coordinate a group response, such as when to secrete virulence factors or uptake new DNA. Quorum sensing in many species of Gram‐positive bacteria utilizes secreted short peptide signals that are sensed by protein receptors that alter gene expression. The ComR‐ComS receptor‐signal pair is a regulator of competence for genetic transformation that is broadly conserved in a majority of Streptococcus species. Despite this conservation, we observe sequence diversity among ComRS orthologs that raises questions relating to sensory specificity, potential cross‐talk between species, and the molecular mechanism of peptide recognition. To address this, we developed a ‘test‐bed’ assay to measure the activity of ComR proteins in response to cognate and heterologous XIP (mature ComS) peptides in vivo. This led to the identification of three categories of ComR receptors displaying strict, modest, and promiscuous responses. To elucidate the molecular mechanism of receptor specificity we determined an x‐ray crystal structure of ComR in the apo‐conformation and probed the XIP binding pocket by site‐directed mutagenesis both in test‐bed cultures and biochemically in vitro. From the structural and biochemical data, we observe a conserved face of the ligand pocket important for peptide binding and a variable face that functions in peptide specificity. Finally, the basic criteria necessary for peptide responses were used to redesign active peptides from inactive templates. Together, our results provide a model for XIP recognition and specificity, and serves as a template for the generation of novel XIP peptides.

Support or Funding Information

This work was supported by NIH grant R01‐AI091779 to MJF, Burroughs Wellcome Fund, NIH Fellowship 5F30AI110080 to ES, and funding provided by the University of Illinois at Chicago, Research Resources Center.
Investigation of Controlled Expression of MS2 Lysis Protein
Authors: Rasefske Kelsey Anne , Piefer Andrew J.
Published: November 2023

Bacteriophage are a class of virus that exclusively infect bacteria cells, with MS2 a group 1 RNA coliphage (affecting the bacteria Escherichia coli). The viral life cycle is comprised of six stages, the first two involving attachment and injection of the RNA genome directly into the host. Lysis is the final stage; including compromise of host cell membrane integrity and release of progeny phage, it is mediated by the lysis, or “L” protein. Consisting of seventy‐five amino acids,(1) L is one of four proteins coded for by the simple MS2 genome, approximately 3500 nucleotides in length.(2) It produces a larger hole in the host cell membrane compared to other bacteriophage lysis proteins and L retains lysis ability, although less efficient, in truncated form.(1) The virus‐free expression of L and the lysis process it elicits is the focus of this study. Toward this goal, a synthetic L‐gene was cloned and ligated to an inducible arabinose operon and RBS using “Bio‐brick” technology. After induction, lysis will be induced and relativity cell density monitored over time via spectrophotometry. A C‐terminally‐fused L‐GFP construct, to track protein localization within the cell and to provide a “handle” for future pull‐down experiments, will also be constructed. Investigation into the role of bacterial host proteins involved in the lysis process has not been previously reported.

Support or Funding Information

This study was graciously funded by Drs. Allen and Judy Freedman. (Hartwick College Freedman Prize ‐ Physical and Life Sciences)
Gene and Protein Profiling of Effects of Tart Cherry Anthocyanins in Preadipocytes
Authors: Jayarathne Shasika Harshani , Scoggin Shane , Claycombe Kate , Kottapalli Rao , Zabet Masoud , Bhawal Ruchika , Moustaid‐Moussa Naima
Published: November 2023

Several dietary bioactive compounds possess anti‐inflammatory and anti‐obesity properties and could potentially reduce obesity‐associated cardiovascular diseases, diabetes and other metabolic inflammatory diseases. We are specifically interested in tart cherry anthocyanins (ACY) and understanding mechanisms mediating their beneficial effects in metabolic diseases. Hence, we hypothesized that tart cherry ACY reduce obesity‐associated inflammation through direct anti‐inflammatory and lipid metabolizing effects in adipocytes. We first analyzed major ACY in tart cherry by Liquid Chromatography Mass Spectrometry (LC‐MS) and identified cyanidin‐3‐O‐glucoside (C3G), cyanidin‐3‐O‐rutinoside (C3R), cyanidin as well as the polyphenol p‐Coumaric acid with C3G being the most abundant ACY in tart cherry. To further gain insight into global effects of tart cherry ACY on adipocyte metabolism, we conducted RNA sequencing (using Illumina Hi‐Seq platform) and proteomics (using LC‐MS). We used 3T3‐L1 preadipocytes and treated them for 4hrs with culture media supplemented with or without tart cherry extracts (~18ug ACY/ml). Cells are then treated for 18hrs with media containing 200ng/ml Lipopolysaccharide (LPS) to stimulate inflammation in the absence or continuous presence of the tart cherry extracts. Media were used to determine changes in secreted adipokines by Enzyme‐Linked Immunosorbent Assay (ELISA). Tart cherry significantly reduced LPS‐induced secretion of the inflammatory cytokine (Interleukin‐6, IL‐6). Whole genome mRNA sequencing identified several genes differentially regulated by tart cherry ACY. Among 36,919 expressed Mus musculus genes, 2359 genes were identified as differentially expressed at the fold change of >2 at p≤0.05 and 95% confidence level. Of these, 14 genes were up regulated and 2345 genes were down regulated by tart cherry ACY. Using Ingenuity Pathway Analysis (IPA®), we observed predicted inhibition of inflammatory, immune and oxidative stress‐related pathways and genes by tart cherry. Signaling pathways regulated by tart cherry included Protein Kinase, TNFR2 and B cell activating factor signaling. Differentially expressed genes by tart cherry included Casp14, 14‐3‐3, c‐Fos, and c‐Jun. Using proteomics, we identified 62 differentially regulated proteins. Among these, inflammatory, oxidative stress and apoptosis‐related proteins were significantly down‐regulated by tart cherry at p≤0.05 (e.g. Nrf2, Gstp1, Asns, and Hsp90α). In conclusion, our results demonstrate that tart cherry ACY reduce expression of preadipocyte genes, proteins and pathways that are primarily associated with inflammation and oxidative stress. Global gene and protein profiling approaches in preadipocytes and other adipose cells may help identify novel pathways that may potentially serve as new targets for obesity and inflammation.
Comparison of the influence of pre‐exercise BCAA supplementation on serum HMB level between endurance and resistance exercises
Authors: Miyazaki Teruo , Ra Song‐Gyu , Ishikura Keisuke , Ohmori Hajime , Ikegami Tadashi , Matsuzaki Yasushi , Honda Akira
Published: November 2023

Aims

Branched‐chain amino acids (BCAA) play the certain beneficial roles on the resistance exercise‐induced muscle damage and soreness, and are also metabolized for energy production in the skeletal muscle during endurance exercise. In addition to the acetyl‐CoA production in the mitochondria of skeletal muscle, leucine is also metabolized in the cytoplasm to β‐hydroxy‐β‐methylbutyrate (HMB), which enhances protein synthesis through mTOR activation. However, it is unclear whether HMB is synthesized from the supplemented leucine by any kinds of exercises. This study investigated the influence of pre‐exercise BCAA supplementation on serum HMB level after exercises by comparison between endurance and resistance exercises.

Methods

Study 1) BCAA (3.2g) or placebo supplementation were given after every meal from a day to immediately before a full‐marathon race (N=14/group). Blood was collected at two days, immediately after and one day after the race.

Study 2) BCAA (3.2g) or placebo supplementation were given after every meal for three days prior to a repeated eccentric resistance exercise in the upper arm (N=5/group). Blood was collected at before supplementation, immediately after the exercise, and everyday for three days after the race. Serum concentrations of HMB and valine intermediate β‐hydroxyisobutyrate (HIB) that is a marker of BCAA catabolism for energy production were measured by LC‐MS/MS system.

Results

Study 1) Both HMB and HIB concentrations in serum immediately after the marathon race were significantly increased, and then, were returned to the base levels at next day. At the immediately after the race, the increased levels of the both BCAA metabolites were significantly higher in the BCAA group than in the placebo group. Study 2) Serum HMB concentration was significantly increased immediately after the eccentric resistance exercise only in the BCAA group but not in the placebo group, and was returned to the base level at next day. There was no change in serum HIB concentration in the both groups during the experiment.

Conclusion

The resistance exercise after the BCAA supplementation may activate the HMB metabolism from leucine, while HMB is also synthesized in the endurance exercise accompanied with BCAA catabolism for energy production. The beneficial effects of BCAA supplementation on the exercise‐induced muscle damage and soreness might be partly contributed by HMB derived from the supplemented leucine.

Support or Funding Information

This study was supported in part by an educational grant from the Seikatsu Bunkasya Co. Inc. (Tokyo, Japan), and by Kakenhi grants (25750334, 16K01734, 15J00614, 16K16571) from the Japan Society for the Promotion of Science.
Lysosome enlargement in PIKfyve inhibited cells proceeds through homotypic lysosome fusion rather than growth of individual lysosomes
Authors: Saffi Golam
Published: November 2023

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

Support or Funding Information

This work was made possible by NSERC and Canada Research Chair Program.
Stereochemical characterization of acyl‐phosphatidylglycerol and bis‐(monoacylglycerol)phosphate using nuclear magnetic resonance
Authors: Peros Colin S , Garrett Teresa A
Published: November 2023

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

Support or Funding Information

National Science Foundation Research at Undergraduate Institutions Grant #1516805 and #1152463, and National Science Foundation Major Instrumentations Grant #1039659 and #1526982
Structure and Organization of Mitochondrial Cristae Membranes
Authors: Ryan Robert O. , Ikon Nikita
Published: November 2023

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

Support or Funding Information

NIH R37 HL64159NIH T32 DK061918
Characterizing Lipid Production in Chlorella Vulgaris Exposed to Sethoxydim, an Acetyl‐CoA Carboxylase Inhibitor
Authors: Smythers Amanda L. , Holland Aaron T. , Roberts Aaron , Kolling Derrick R.J.
Published: November 2023

Microalgae, with their ability to grow in adverse environments using a variety of water sources, can be a lucrative energy source that will not compete with agriculture (Sharma et al. 2012). In order to realistically produce fuel on an industrial scale, microalgal lipid production must be increased. Microalgae with 70% oil by biomass would satisfy 50% of the United States' transport fuel needs while using less than 2% of the existing cropping area used for fuel production (Chisti 2007).

The objective of this work is to characterize the process through which Chlorella vulgaris, a unicellular green alga, produced elevated lipid levels under acetyl‐CoA carboxylase (ACCase) inhibition by sethoxydim, a common herbicide. Previous work aimed to use applied natural selection, or directed evolution, to create a modified C. vulgaris culture with increased ACCase activity that would increase the overall lipid yield for use in biofuels. In the preliminary experiments, a puzzling phenomenon emerged: cells exposed to sethoxydim produced over 3X more lipids than unexposed cells. Furthermore, there were metabolic changes in sethoxydim‐exposed cells: rapid chlorosis indicated a decrease in photosystem II activity that was absent in the control. Further experimentation confirmed that this paradox is not an artifact, but rather a novel response due to the presence of sethoxydim.

The current experimental approach is twofold: to identify the chemical compounds in sethoxydim‐exposed lipid samples and to characterize the mechanism through which this lipid accumulation occurs. Gas chromatography‐mass spectrometry will be used for identification. This is particularly useful in determining the presence of non‐membrane fatty acids, including triacylglycerides, the lipid most desired for biodiesel. Since the herbicide inhibits ACCase, based on currently known biological information, there should be few fatty acids in the experimental sample as compared to the control. If that is the case, the next step will be to look for the presence of other lipids, such as isoprenoids.

Isoprenoids represent a variety of lipids formed from isopentenyl diphosphate (IPP), a 5‐C building block. Currently, two pathways are known for the biosynthesis of IPP, the mevalonate pathway and the methylerythritol phosphate pathway (Lohr et al. 2012). The mevalonate pathway is of particular interest to this experiment as it begins with the condensation of 3 acetyl‐CoA molecules. If acetyl‐CoA is building up in the cell and cannot be catalyzed by ACCase, it would make sense to follow this pathway toward the synthesis of isoprenoids.

The increased lipid mass under sethoxydim stress was highly unexpected and the mechanism through which it occurs is not yet found in literature. Identifying the chemical makeup of these lipid samples and characterizing this mechanism could lead to fundamental science that could potentially be used in industrial applications.
Stronger Together: The role of cysteine residues in Ebola viral matrix protein VP40
Authors: Baker Sarah Catherine Briann , Johnson Kristen , Stahelin Robert
Published: November 2023

Introduction

One hallmark of an intracellular Ebola infection is the formation of virus‐like particles, or VLPs, on the extracellular surface of infected cells. The formation of VLPs is known to be facilitated by oligomerization of the viral matrix protein VP40. The tendency of VP40 to oligomerize into dimers, hexamers, and octamers is well‐established, yet the mechanism of this oligomerization is not fully understood. An examination of the secondary structure of VP40 reveals the presence of only two cysteine residues, the positions of which are highly conserved across all five sub‐species of Ebola virus. In the protein's fully‐folded conformation, these cysteine residues would be solvent exposed in the dimer conformation. In the 4LDB VP40 crystal structure, these residues are shown to be at the interface of the VP40 tetramer.

Objective

We aim to better understand the role of the cysteine residues on VP40.

Methods

Site directed mutagenesis, protein purification, lipid binding assays, confocal imaging.

Results

When VP40 cysteines are reduced with DTT, lipid binding in‐vitro is changed. We aim to understand how this affects VP40 function in cells my comparing WT VP40 and VP40 with cysteine to alanine mutations. Our preliminary data suggests that VP40 cysteine residues are important for protein function. Future work in cellular and lipid‐binding assays with WT and mutant VP40 may shed further light on how the cysteine residues interact at the membrane, and analysis of wild‐type proteins exposed to reducing agents may elucidate the importance of disulfide bonds specifically.

Support or Funding Information

Financial Support: NIH AI081077
Ceramide Induced Regulated Alternative Translocation of TM4SF20 and Beyond
Authors: Denard Bray , Chen Qiuyue , Lee Ching‐En , Han Sungwon , Ye James , Ye Jin
Published: November 2023

TM4SF20 (transmembrane 4 L6 family 20) is a polytopic membrane protein that normally inhibits proteolytic processing of CREB3L1 (cAMP response element‐binding protein 3‐like 1), a membrane‐bound transcription factor that inhibits cell proliferation and activates collagen synthesis. Recently, we have discovered that ceramide stimulates CREB3L1 cleavage by inverting the topology of TM4SF20 in membranes. In the absence of ceramide, the N‐terminus of the first transmembrane helix of TM4SF20 is inserted into the endoplasmic reticulum lumen. In the presence of ceramide, the N‐terminus of the first transmembrane domain of TM4SF20 is exposed to cytosol. Consequently, the membrane topology of TM4SF20 is inverted, and this form of TM4SF20 stimulates CREB3L1 cleavage. We designate the mechanism causing regulated inversion of the membrane topology as “Regulated Alternative Translocation”.

We identified a GXXXN motif in the first transmembrane helix of TM4SF20 that is critical for Regulated Alternative Translocation of TM4SF20. Bioinformatics analysis revealed hundreds of membrane proteins containing this motif. Our result suggests that Regulated Alternative Translocation could be a general mechanism regulating a large family of transmembrane proteins.

Support or Funding Information

This work was supported by grants from the National Institutes of Health (GM‐116106, AI‐70116, and HL‐20948)