bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationNational 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 InformationThe 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 InformationThis study was supported by a U of L SOM Collaborative Matching Grant to BJC and KY Lung Cancer Research Program to CMK.
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 InformationResearch 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 InformationNSF 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 InformationThis 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 InformationThis 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 InformationThis study was graciously funded by Drs. Allen and Judy Freedman. (Hartwick College Freedman Prize ‐ Physical and Life Sciences)
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

AimsBranched‐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.MethodsStudy 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.ResultsStudy 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.ConclusionThe 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 InformationThis 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.