bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structure of the two‐component response regulator RcsB‐DNA complex
Authors: Filippova Ekaterina V. , Zemaitaitis Bozena , Wolfe Alan J. , Anderson Wayne F. 
Published: November 2023

The Rcs phosphorelay is a complex two‐component system in enteric pathogens that achieves transmission of molecular signals through a phosphorylation cascade between two hybrid sensor kinases RcsC‐RcsD and the response regulator RcsB. The latter can form either homodimers or heterodimers with other transcriptional factors. These heterodimers can be phosphorylation‐dependent or –independent. This complexity allows RcsB to regulate the transcription of a diverse array of genes essential for pathogen survival and host invasion. Despite increasing appreciation of the importance of RcsB in pathogenicity, the molecular mechanisms by which RcsB forms homo/heterodimers to facilitate DNA binding in response to different environmental stresses are not well understood. Here, we present the first X‐ray crystal structure of the response regulator RcsB from Escherichia coli in complex with DNA determined at a resolution of 3.4 Å. The structure reveals that non‐phosphorylated RcsB forms an unusual asymmetric dimer in the DNA‐bound state. This asymmetry likely provides conformational flexibility that allows RcsB to interact with a large number of genes and to form heterodimers with other transcriptional regulators. Combining our structural data with electrophoretic mobility shift assays, surface plasmon resonance, and size exclusion chromatography with multi‐angle light scattering analysis provides important details about the molecular mechanism by which the universal two‐component response regulator RcsB recognizes DNA.Support or Funding InformationCSGID is funded by the National Institute of Allergy and Infectious Diseases of NIH under Contracts No. HHSN272200700058C and HHSN272201200026C
Novel Genetic Variants in the Oxytocin Receptor Predict Oxytocin Non‐Responsiveness in Pregnant Women
Authors: Lee Grace Y. , Raghuraman Nandini , Reinl Erin L. , England Sarah K. 
Published: November 2023

BackgroundOxytocin is used on 43% of all women in the US to induce or augment labor. The maximum oxytocin dose varies between women from ≤2mU/min to ≥20mU/min. Those requiring ≥20mU/min of oxytocin are defined as “non‐responsive.” The biological cause underlying these individual differences in oxytocin response is unknown, however, maternal genetics is suspected to be a factor. Our laboratory has identified three gene variants (H173R, R150L, and R151C) in the oxytocin receptor (OXTR) gene in women who are non‐responsive to oxytocin.HypothesisWe hypothesize that variants H173R, R150L, and R151C of the OXTR gene impair ligand binding and receptor signaling and underlie oxytocin non‐responsiveness seen in certain pregnant women.MethodsOXTR variants were created via mutagenesis and transfected into HEK293T cells. The oxytocin binding and signaling abilities of the wild‐type OXTR and OXTR variants were then assessed with an ELISA assay using IP1 accumulation as the readout for OXTR signaling.ResultsH173R, R150L, and R151C are located in the binding and signaling domains of the OXTR and were predicted by the Sorting Intolerant From Tolerant (SIFT) algorithm to be damaging to protein function. Wild‐type OXTR were functional as measured by ELISA and Ca2+ imaging. Increasing concentrations of oxytocin led to greater stimulation of the OXTR and greater IP1 accumulation.ConclusionOur studies will provide evidence as to whether oxytocin responsiveness can be predicted. This can help improve clinical methods and treatment of women during labor and delivery.Support or Funding InformationSupported by the American Physiological Society (to G.Y.L) NIH 1R21 HD076677 (to S.K.E.) and F31 HD079148 (to E.L.R).

Transfected with:
Oxytocin concentration
IP1 concentration (nM)

Wild‐type OXTR
0nM
17.57

30nM
27.42

100nM
29.35

1uM
35.07

Un‐transfected
0nM
8.42

1uM
7.97
Wnt and Ca2+ signalling changes in LRRK2 Parkinson's disease models
Authors: Harvey Kirsten , Wetzel Andrea , Hughes Michael , McKay Tristan , Waddington Simon , Rahim Ahad 
Published: November 2023

ObjectiveExamination of the influence of leucine‐rich repeat kinase 2 (LRRK2) on Wnt and Ca2+ signalling changes in Parkinson's disease.BackgroundMutations in LRRK2 are a major cause of Parkinson's disease. Changes in LRRK2 activity were shown to cause alterations in several cell signalling pathways. Wnt and Ca2+ signalling changes are some of the most reproducible reported observations to date. LRRK2 was shown to modulate Ca2+‐dependent immune responses, autophagy and mitochondrial function. In addition, LRRK2 modulated canonical Wnt signalling was shown to be affected in opposite ways by protective and pathogenic genetic Parkinson's disease variants, suggesting a clear correlation between Wnt signalling activity and neurodegenerative disease. This suggests the prospect of targeting Wnt signalling pathways to slow or halt the progression of Parkinson's disease.MethodsUse of LRRK2 knockout and G2019S knock‐in models to conduct functional Wnt signalling assays. We introduced lentiviral biosensors into mouse brains to record Wnt and Ca2+ cell signalling changes in the brains of live animals from one week to six months of age using an advanced imaging technology (IVIS).ResultsHere, we present the analysis of Wnt and Ca2+ signalling activity using the exciting new technology of tissue‐targeted delivery of lentiviruses containing a transcription factor activated luciferase reporter to newborn mice. This allows quantification of transcription factor activity over the lifetime of an animal. We compare wild type mice with LRRK2 knockout mice and with mice harbouring the most prevalent familial LRRK2 G2019S mutation. Our data collectively support the idea of LRRK2 as a modulator of Wnt and Ca2+ signalling.ConclusionsIncreasing our understanding of the role of LRRK2 in Wnt and Ca2+ signalling pathways during neuronal development and aging might contribute to the generation of novel PD treatments targeting early events in the pathogenesis of PD. Decrease in Wnt signalling activity has previously been linked to neurodegeneration and provides therefore a plausible mechanism and a good therapeutic target in the Parkinson's disease process.Support or Funding InformationMedical Research Council, Wellcome Trust
Small size of membrane‐tethered ligands regulates kinase‐phosphatase segregation in gamma/delta TCR triggering.
Authors: Choudhuri Kaushik 
Published: November 2023

Ligand recognition by the T cell antigen receptor (TCR) is a central event in initiating T cell‐mediated immunity. While ab T cells recognize small peptides in association with major histocompatibility complex molecules (pMHC), gd T cells do not undergo canonical thymic selection, and consequently are not restricted by pMHC antigens or the co‐receptors CD4/8. Some of these unconventional T cells recognize invariant stress‐inducible membrane‐tethered ligands, and their rapid activation and recruitment to sites of inflammation is thought to serve as a ‘first line of defense’ against incipient infection. In light of the substantial structural and stoichiometric similarities between ab and gd TCRs, we sought to determine if gd TCR ligand‐recognition shared basic physicochemical constraints to triggering that have been identified for conventional ab TCRs. We chose as a model system the stress‐inducible surface protein T22 that is recognized by the G8 gd TCR, since this receptor‐ligand pair has been characterized biophysically, and the T22/G8 structure has been solved at atomic resolution. Since T22 is similar in size to pMHC molecules, we tested whether ‘size‐based’ kinase‐phosphatase segregation, following engagement of G8 TCR by T22, controls initiation of TCR signaling. In our ongoing studies, we have established that the small axial size of T22 is critical for G8 triggering. Elongation of the T22 ectodomain leads to failure of TCR phosphorylation, and IL‐2 responses. Using optical and electron microscopy we show that elongated T22/G8 complexes increases access of inhibitory phosphatases to sites of TCR engagement at cell‐cell contacts. We are testing whether truncation of the ectodomain of the large membrane phosphatases CD45 and CD148, which are normally sterically segregated from engaged TCR, abrogates TCR phosphorylation by membrane‐associated Src‐kinases. Finally, we are adapting these strategies, to investigate the principles of triggering in gd T cells which recognize small‐molecule chemical haptens.Support or Funding InformationAI093884 (NIH/NIAID)
TAK1/TRAF6 signalling in regulation of skeletal muscle mass
Authors: Kumar Ashok , Hindi Sajedah M 
Published: November 2023

Loss of skeletal muscle mass is a devastating consequence of a large number of chronic disease states and several other conditions such as starvation, denervation, immobilization, and aging. However, the signaling and molecular mechanisms that regulate skeletal muscle growth and atrophy remain less understood. It is now evident that a complex array of signaling pathways regulates skeletal muscle mass primarily through modulating the rate of protein synthesis and degradation. Transforming growth factor β (TGF‐β) activated kinase 1 (TAK1), a member of the MEK kinase family, is an important signaling protein which activates multiple signaling pathways in response to cytokines, growth factors, and microbial products. In many signaling cascades, TRAF6 ubiquitylates and activates TAK1. We have previously reported that TRAF6 mediates skeletal muscle atrophy in response to denervation, starvation, and cancer growth. We have now investigated the role of TAK1 in the regulation of adult skeletal muscle mass. Our results demonstrate that inducible inactivation of TAK1 leads to severe muscle wasting and development of kyphosis in adult mice. Moreover, denervation‐induced muscle atrophy is exacerbated in skeletal muscle‐specific TAK1 knockout mice. TAK1 is required for the activation of specific intracellular signaling pathways which promote skeletal muscle growth and prevent atrophy. Inactivation of TAK1 inhibits protein synthesis, induces protein degradation, and causes oxidative stress in skeletal muscle of adult mice. We have also found that deletion of TAK1 leads to accumulation of dysfunctional mitochondria in skeletal muscle potentially due to the inhibition of mitophagy. Intriguingly, phosphorylation of TAK1 is dramatically induced in skeletal muscle of mice undergoing load‐induced hypertrophy. Importantly, targeted inactivation of TAK1 blunts skeletal muscle hypertrophy in response to functional overload. Overall, our experiments suggest that TAK1 is a key regulator of skeletal muscle mass.Support or Funding InformationNational Institute of Health grants AR059810, AR068313, and AG029623
General Amino Acid Control and 14‐3‐3 Proteins Bmh1/2 Are Required for Nitrogen Catabolite Repression‐Sensitive Regulation of Intracellular Gln3 and Gat1 Localization
Authors: Cooper Terrance G. , Tate Jennifer J. , Buford David , Rai Rajendra 
Published: November 2023

Nitrogen Catabolite Repression (NCR), the ability of Saccharomyces cerevisiae to utilize good nitrogen sources in preference to poor ones, derives from nitrogen‐responsive regulation of the GATA family transcription activators Gln3 and Gat1. In nitrogen replete conditions the GATA factors are cytoplasmic and NCR‐sensitive transcription minimal. When only poor nitrogen sources are available, Gln3 is nuclear dramatically increasing GATA factor‐mediated transcription. In addition, to these two growth conditions, Gln3 localization responds to short‐ and long‐term nitrogen starvation as well as rapamycin and methionine sulfoximine (glutamine synthetase inhibitor) treatments. Originally these responses were all attributed to mTorC1‐mediated control of Gln3. We subsequently showed that this was not the case, but that there were at least three distinct regulatory pathways controlling Gln3 localization. Two regulatory systems act cumulatively to maintain cytoplasmic Gln3 sequestration; one is TorC1. The second regulatory system has remained elusive. A third regulatory pathway takes over once Gln3 enters the nucleus. This pathway is glutamine specific. When glutamine levels are low, Gln3 must bind to its DNA target promoter sequences (GATAA) before it can exit from the nucleus. On the other hand, when glutamine (or glutamine analogue) levels are high, Gln3 can exit from the nucleus in the absence of DNA binding. Present experiments identify Gcn2‐dependent general amino acid control as the regulatory system that has thus far escaped detection. We show that the uncharged tRNA‐activated protein kinase Gcn2 is required for NCR‐sensitive nuclear Gln3‐Myc13 localization but not for short‐ and long‐term nitrogen starvation or responses to Msx and rapamycin treatments. From epistasis experiments, it appears that Gcn2 functions upstream of Ure2. Ure2 sequesters Gln3 in the cytoplasm in nitrogen replete conditions. 14‐3‐3 proteins Bmh1/2 are also required for nuclear Gln3‐Myc13 localization and GATA factor‐mediated transcription. In contrast with Gcn2, however, Bmh1/2 appear to function downstream of Ure2. Overall Gln3 phosphorylation levels decrease upon loss of either Gcn2 or Bmh1/2. Our results add a new dimension to the cumulative participation of these three major pathways in the nitrogen‐responsive control of GATA transcription factor localization and NCR‐sensitive gene expression in yeast.Support or Funding InformationNIH GM‐35642‐27
Signaling Pathways Involved in Tributyltin‐Induced Increases in Interleukin 6 Production by Lymphocytes
Authors: Hamza Nafisa , Brown Shyretha , Whalen Margaret 
Published: November 2023

Tributyltin (TBT) is a toxic compound that has industrial uses, such as slime control on masonry, wood preservation, and disinfection of circulating industrial cooling water and is most frequently used in antifouling paints on the hulls of ships. As a result of TBT's lipophilic character, it absorbs readily into organisms and contaminates the environment and is found in human blood in concentrations as high as 261 nM. TBT decreases the lytic function and secretion of interferon gamma (IFNγ), tumor necrosis factor alpha and interleukin 1 beta (IL‐1β) from human lymphocytes. IL‐6 is a cytokine which regulates the function of many cells including tumor cells. Recent studies in our lab have shown that TBT alters production of IL‐6 by lymphocytes. The current study aims to determine whether TBT utilizes MAPK signaling pathways (ERK 1/2, p38) to cause alterations in IL‐6 production. TBT‐induced production of IL‐6 in human lymphocytes was measured in the presence and absence of ERK1/2 pathway (PD98059) and p38 (SB202190). Results indicated that the stimulation of IL‐6 production by TBT decreased in the presence of each of the MAPK pathway inhibitors. These data suggest that TBT may be causing increased IL‐6 production by activating ERK1/2 and p38 MAPKs.Support or Funding InformationSupported by NIH grant 5U54CA163066
Role of MAPKs and NFκB in Tributyltin‐stimulated Interleukin 1 Beta Secretion and Production from Human Immune Cells
Authors: Boules Mariam , Brown Shyretha , Whalen Margaret 
Published: November 2023

Tributyltin (TBT) contaminates the environment due to its many uses and has been found in human blood samples at levels as high as 261 nM. TBT interferes with the lytic function of human natural killer lymphocytes and alters the secretion of interleukin 1 beta (IL‐1β), Interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) from human leukocytes. IL‐1β regulates cellular growth and immune responsiveness. Elevated levels of IL‐1β have been shown to increase the invasiveness of tumors. Our recent studies showed that TBT exposures of 5–25 nM (levels that are seen in human blood samples) caused increased secretion of IL‐1β from monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs). In this study we examine the roles of the MAPKs, (ERK1/2, p38, and JNK) and NFκB in TBT‐induced increases in IL‐1β secretion and intracellular levels to assess their role in TBT‐induced production of IL‐1β. MD‐PBMCs were treated with the MEK1/2 inhibitor PD98059, p38 inhibitor SB202190, JNK inhibitor BI78D3 or NFκB inhibitor BAY 11‐7085 for 1h prior to exposure to TBT concentrations of 25, 10, and 5 nM for 24 h. Secretion of IL‐1β was measured using ELISA and intracellular protein levels were monitored using western blot. Results indicated that the TBT‐induced secretion of IL‐1β requires the ERK1/2 and p38 pathways. But does not require the NFκB or JNK pathways. The increases in secretion are accompanied by increased protein levels in TBT‐exposed cells and this increase in protein production also utilized the ERK1/2 and p38 pathways. These data suggest that increased protein production accompanies the increases in secretion of IL‐1β seen in human immune cells exposed to TBT and these increases are dependent on MAPK pathway activation.Support or Funding InformationSupported by NIH grant U54CA163066.
Involvement of MAPK Signaling Pathways in Tributyltin‐induced Increases of Interleukin 1 beta and Interleukin 6 mRNA in Human Lymphocytes
Authors: Brown Shyretha D , Boules Mariam , Hamza Nafisa , Whalen Margaret 
Published: November 2023

IL‐1β and IL‐6 are pro‐inflammatory cytokines produced by monocytes, macrophages, and lymphocytes. Both are responsible for immune response regulation and cellular growth. Increased levels of these two cytokines are associated with several pathologies, including rheumatoid arthritis, multiple sclerosis, and specific cancers (such as breast, lung, and pancreatic). Tributyltin (TBT) is an environmental contaminant which is found in human blood and other tissues, and it has been shown to increase incidences of tumors in mammals. Previous ex vivo studies have shown that when TBT is introduced as a stress inducer to the cellular environment, the production (secretion and/or intracellular levels) of IL‐1β and IL‐6 was increased in human lymphocytes, and ERK1/2 and p38 MAPK pathways were shown to be necessary for this TBT effect. The current study examines whether this increased production (protein levels) of IL‐1β and IL‐6 is due to TBT‐induced increases in the transcription of IL‐1β and IL‐6 mRNA. Additionally, this study will examine the roles of MAPKs (ERK1/2 and p38) on any demonstrated TBT‐induced increases in expression of IL‐1β and IL‐6 mRNA in lymphocytes (monocyte‐depleted (MD) peripheral blood mononuclear cells (PBMCs)). MD‐PBMCs were exposed to 0–200 nM TBT followed by isolation of RNA. The IL‐1β and IL‐6 mRNA levels were monitored using semi‐quantitative RT‐PCR. mRNA levels of each cytokine were also monitored in the presence and absence of ERK1/2 (PD98059) and p38 (SB202190) pathway inhibitors. Results indicated that exposure to 50–200 nM TBT caused increased levels of both IL‐1β and IL‐6 mRNA. Initial results suggest that the MAPK pathways, although involved in the TBT‐induced increased in cytokine production, may not have a role in the TBT‐induced increases on mRNA expression. These data suggest that MAPK pathway activation is needed for TBT to cause increased production for both IL‐1β and IL‐6.Support or Funding InformationNIH grant U54CA163066
Characterizing of the Novel Gene At4g33666 in the Abiotic Stress Response in Arabidopsis
Authors: Clark Amber Elizabeth Miller , Wershoven Nicole , Palmer Christine 
Published: November 2023

Expanding populations and environmental changes can leave the plants we depend upon to meet our food and energy needs facing increasingly difficult growing conditions. Enhancing our understanding of the methods that plants naturally employ to overcome abiotic factors such as heat, drought, high salinity, or poor light will enable us to better select for plants that can succeed under challenging conditions. Here we examine At4g33666, a novel gene in Arabidopsis thaliana, which is thought to play a role in perceiving or overcoming abiotic stressors. In order to begin to understand the role of this gene in the stress response, we have examined expression at both an RNA and protein level. We extracted RNA from plants exposed to abiotic stress and performed quantitative PCR. To visualize phenotypic changes caused by the loss or overexpression of the gene, we measured the rate of germination of different genotypes under osmotic stress. This protein is hypothesized to be localized to the chloroplast, and to learn more about post translational modifications that might occur, we used immunoprecipitation to isolate protein for analysis via mass spectrometry. By studying the role of At4g33666, we can improve our understanding of plant stress responses and optimize success of the plants we depend upon.Support or Funding InformationThis research was supported by a Castleton University student research grant.
Controlled BMP2 Release from Keratin‐based Hydrogels Modulates Osteoinduction
Authors: Olson Lucas C. , Hyzy Sharon L. , Saul Justin M. , Cohen David J. , Kajan Illya , Schwartz Zvi , Boyan Barbara D. 
Published: November 2023

Keratin biomaterials have potential for tissue engineering applications. When processed keratin is naturally non‐immunogenic, contains cell attachment sites, and has a tunable degradation rate, resulting in an effective scaffolding to simulate extracellular matrix (ECM). Keratose (oxidatively derived keratin) degrades in a period of weeks whereas kerateine (reductively derived keratin) degrades on an order of months to years, due to higher cysteine cross linking in kerateine. Clinically type 1 collagen sponge is soaked in bone morphogenetic protein (BMP2), which is a potent osteoinductive factor, in order to promote bone healing in orthopedic and craniofacial sites. The sponge, however, does not modulate BMP2 release in vivo, resulting in supraphysiological levels of the osteoinductive factor. Harmful ectopic bone formation and inflammation can result. In contrast, keratin degradation and BMP2 release rate are correlated. Modulation of BMP2 release by keratin, coupled with its inherent properties as an ECM substitute, could result in a suitable construct for osteoinduction via controlled osteogenic signal release, inflammatory mitigation, and cellular infiltration. The aim of this study was to examine the ability of keratin‐derived materials containing BMP2 to induce bone formation in vivo.Ectopic bone formation from collagen sponge, keratose gel, kerateine gel, and keratose: kerateine blends (30:70, 50:50, 70:30) ± 0.67μg BMP2/mg material was examined in a well‐known mouse model. Samples were placed in gelatin capsules and bilaterally implanted into pockets created in the gastrocnemius of 7w‐old male athymic nude mice (n=8 implants/variable). After 5w, new bone formation was ascertained by histology, microCT and histomorphometry. (Statistical significance: ANOVA with post‐hoc Bonferroni‐corrected t‐test).All +BMP2 samples produced new bone formation on microCT and histology, while there was no new bone in‐BMP2. On microCT, the most new bone was seen in 50:50+BMP2. 30:70+BMP2 had more new bone volume than collagen+BMP2 or keratose+BMP2. Histologically, 30:70+BMP2 contained greater total bone area than collagen+BMP2 and keratose+BMP2. 70:30+BMP2 had more cortical‐type bone and bone marrow than keratose+BMP2, but 30:70+BMP2 had more material still present than collagen+BMP2, keratose+BMP2, or 50:50+BMP2. Keratose (±BMP2) degraded completely. In‐BMP2 samples, remaining material increased with increasing kerateine. In conclusion, keratose/kerateine carriers facilitate greater BMP2‐induced bone than standard collagen or either biomaterial separately, suggesting their suitability as slow‐release BMP2 carriers for clinical bone regeneration.Support or Funding InformationNIH 5R01AR061391