bluuebunny/crossref_metadata_2025_split huggingface dataset:
Shoc2 Mediates Hematopoietic Signals of the ERK1/2 Pathway
Authors: JANG HYEIN , Jang Eun Ryoung , Morris Ann , Forbes‐Osborne Marrie , Galperin Emilia 
Published: November 2023

Activity of the extracellular‐signal‐regulated kinase 1/2 (ERK1/2) pathway plays a critical role in normal human development. Aberrant ERK1/2 signaling caused by germline mutations in genes of the Ras‐mediated ERK1/2 pathway results in developmental disorders cumulatively called ‘RASopathies’. Shoc2, the scaffold protein in the ERK1/2 pathway, accelerates ERK1/2 activity by bringing the signaling components of the pathway, Ras and Raf‐1, to close proximity. Noonan‐like RASopathy causing Shoc2 mutations emphasize the significance of ERK1/2 signaling transmitted through the Shoc2 scaffolding module. However, the physiological role of Shoc2 and the specificity of the signals transduced through the Shoc2 scaffolding complex remain largely unknown.In our work, we took advantage of the vertebrate zebrafish model that is ideally suited for developmental genetic studies due to its optical transparency and ex vivo development. To delineate the developmental ERK1/2 signals mediated through the Shoc2 complex, a translation‐blocking shoc2 morpholino (MO) was injected into 1–2 cell stage zebrafish embryos. The outcomes of the Shoc2 depletion were examined using biochemical methods such as Western blot analysis, RT‐PCR and whole embryo staining/imaging. We found that Shoc2 depletion in zebrafish embryos results in decreased ERK1/2 activity. Shoc2 MO‐injected embryos also possessed a larger yolk, kinked tail, and an enlarged hindbrain. Importantly, Shoc2 morphants displayed dramatically reduced numbers of circulating blood cells and loss of erythropoietic and myeloid lineages. To further understand the role of Shoc2 in differentiation of hematopoietic stem cells, we utilized CRISPR/Cas9 gene editing to generate the Shoc2 zebrafish knockout. 12 germ‐line transmitted alleles were recovered from the mutagenesis. All alleles had lesions in similar locations, causing frame‐shifts around amino acid 225 and introducing aberrant stop codons (TAA or TAG) prior to termination.Our data demonstrate that Shoc2 depletion affects both waves of hematopoiesis: primitive and definitive. Shoc2 CRISPR zebrafish knockout will now be utilized to examine quantitative changes in the hematopoiesis‐specific ERK1/2 transcriptional program.Support or Funding InformationFunding sources: National Institute of General Medical Siences, American Cancer Society, American Heart Association
Phosphorylated Astrin regulated kinetochore function mediated by TRAIP in mitosis
Authors: Chung HeeJin , Park JiEun , Lee JooRak , Kim Hongtae 
Published: November 2023

Protein‐protein interaction post‐translational modification and protein degradation are important for the mitotic progression such as chromosome segregation to opposite spindle poles and the stable attachment of spindle microtubules (MTs) to kinetochores (KTs). Error‐free chromosome segregation required precisely timed search and capture of chromosomes by spindles in early mitosis. However, the molecular mechanism underlying conversions from labile to stable attachment are poorly understood. Here, I show that the sequential action of the kinase Cdk1 (Cyclin‐Dependent Kinase 1) or Plk1 (Polo‐Like Kinase 1), TRAIP (TRAF‐interacting protein), and the phosphatase CDC14A (Cell Division Cycle 14A) controls the spindle attachment of Astrin to KTs.In previously study, the mitotic spindle protein SPAG5/Astrin phosphorylated during mitosis. However, unfortunately there's no evidence which site of Astrin phosphorylated. This study contains that Astrin is transported into centrosomes by Kinastrin and I identified phosphorylation sites of Astrin at S135 and S249 using PhosphoSite program (http://www.phosphosite.org), and it's phosphorylated by Cdk1 or Plk1. Somehow, what protein loaded Astrin onto the spindle and targeted to KTs is unknown. I found TRAIP as a new Astrin interacting protein by using yeast two hybrid screening and confirm that TRAIP binds and co‐localizes with Astrin during early mitotic progression by immunoprecipitation and immunofluorescence assay. Depletion of TRAIP resulted in hyper‐stabilized and multipolar spindles, accompanied by aberrant chromosomal alignment measured by phosphor‐Histone H3 (p‐Histone H3) staining for Fluorescence‐Activated Cell Sorting (FACS), Live cell imaging observation for time‐lapse microscopy and Fluorescence Loss on Photobleaching (FLIP) experiment. Mechanistically, TRAIP regulates Astrin phosphorylation and targets phospho‐Astrin to KTs, leading to the attachment of spindles to KTs. I also demonstrate that CDC14A dephosphorylates Astrin and therefore, overexpression of CDC14A sequestrates Astrin in the centrosome and results in aberrant chromosome alignment. These comprehensive findings uncover a regulatory circuit for protein targeting to KTs that controls the change of KT composition for stable spindle attachment and chromosome integrity.In summary, this study reveals a new agent involved in search and capture, the signal messenger phospho‐Astrin, which is generated by Cdk1 and targeted to KTs by TRAIP through spindle MTs, and is involved in stable KT attachment to spindle. It elucidates a key mechanism in mitotic progression. This study provides additional information about both the mechanism underlying bi‐oriented spindle formation and chromosome instability in cancer cells.Support or Funding InformationThis work was supported by National Research Foundation of Korea grants funded by the Korean government (MSIP)(No. 2011‐0030043)
Arsenic and Line‐1 Disrupt Developmental Epithelial to Mesenchymal Transition: Implications for Cardiac Morphogenesis
Authors: Perrera Alec Bennett , Huang Tianfang , Bojang Pasano , Tavera‐Garcia Marco , Reyes‐Reyes Elsa M. , Ramos Kenneth S. , Camenisch Todd D. 
Published: November 2023

Chronic exposure to arsenic is linked to increased risk for cancers of the lung, kidney, bladder, and liver. Non‐cancerous disease endpoints such as atherosclerosis, birth defects, metabolic syndrome and hypertension are also associated with arsenic exposure. Many of the organ systems impacted by arsenic require epithelial to mesenchymal cell transition (EMT) to establish and maintain tissue homeostasis. For example, developmental EMT contributes to lung, neural tube and heart morphogenesis. As such, disruption of EMT programming by arsenic can result in birth defects and predispose to diseases later in life. The mechanisms by which arsenic disrupts EMT is not known. Long interspersed element 1 (LINE‐1 or L1) is a retrotransposon of the human and mouse genomes, and recently linked to conotruncal heart defects. Such defects arise from malformation of the outflow tract of the embryonic heart through altered EMT. In the present study, we discovered induction of Line‐1 by arsenic and investigated whether Line‐1 impacts cardiac developmental EMT. An increase in ORF‐1 and ORF‐2 Line‐1 mRNAs was detected, starting 2 hr after exposure to arsenic and peaking at 8 hr. Line‐1 protein is also increased following arsenic exposure with increased dose‐dependent localization of Line‐1 in the nuclear compartment. Furthermore, the transcriptional activity of Line‐1 was induced by arsenic in a time and dose dependent manner. Lastly, using a unique mouse genetic model we found that ectopic Line‐1 expression functionally attenuated cardiac EMT in an ex vivo collagen gel invasion assay. On the bases of these findings we conclude that arsenic and Line‐1 block developmental EMT.Support or Funding Information(NIH. ES04940; ES06694; SWEHSC P30ES006694; NIEHS R25ES025494)
Inhibition of microRNA‐17 Promotes the Progression of Thyroid Cancer in a Xenograft Murine Cancer Model
Authors: Yan Yan , Eliason Steve , Ries Ryan , Hong Liu , Cao Huojun , Amendt Brad A. 
Published: November 2023

MicroRNAs (miRs) are small non‐coding RNAs that bind to target mRNAs and negatively regulate their expression. The expression of miRs are altered in many cancers and cancer cell types. We previously generated a miR‐17‐92 knockdown mouse model using the Plasmid based MicroRNA Inhibitor System (PMIS). With this new PMIS system we found that thyroid development was abnormal or absence in these mice. Thus, thyroid development requires miR‐17 for normal development. We investigated the role of miR‐17 in SW579 thyroid cancer cells, which are derived from human thyroid tissue. PMIS‐miR‐17, specifically targeting miR‐17‐5p completely inhibited the activity of miR‐17‐5p in SW579 cells. However, inhibiting miR‐17‐5p activity in SW579 thyroid cancer cells also promotes tumor growth in a nude mouse model for thyroid cancer. The tumor formation was due to increase cell proliferation and aberrant gene expression. RNA‐seq data shows knocking down miR‐17 in thyroid cancer cells induces large‐scale changes in miR‐17‐5p target gene expression, including PDGFR and MMP2, which may play oncogenic roles in tumorigenesis. Bioinformatics analyses has identified several new gene expression networks regulated by miR‐17 in thyroid cancer. We have identified several new gene expression mechanisms for miR‐17 and more importantly regulation of miR‐17‐92 expression and processing of the miR‐17‐92 cluster during thyroid development and thyroid cancer.Support or Funding InformationIowa Institute for Oral Health Research, College of Dentistry, The University of Iowa, Iowa City, IA
Synthesis of a Mini‐Reporter Construct to Test Gene Transfer of RNA Therapeutics.
Authors: Muralidharan Koushik , Khan Arbaz M , Fuoco Kerriane , Patel Hemangi , Hicks Martin J 
Published: November 2023

Glioblastoma multiforme (GBM), a grade IV tumor of the central nervous system, is the most common malignant primary brain tumor in adults, with a median survival of only 14 months. This poor survival rate is due to a lack of efficacy in current therapies, including chemotherapy and radiation, which are limited by the blood‐brain barrier. GBM survival depends on tumor cells to generate new vasculature, which is essential for the exchange of wastes and nutrients. Endothelial cells connect with each other and form the walls of new blood vessels, bridging the gap between the growing tumor mass and the established vasculature of the circulatory system. The membrane receptor that activates this growth is vascular endothelial growth factor receptor 2 (VEGFR2). In our lab, we are developing a novel therapy to alter the VEGFR2 receptor so that it cannot form a bridge with its cognate ligand, vascular endothelial growth factor. Alteration in VEGFR2 so that it cannot form this bridge, blocks its activation and inhibits the development of new blood vessels. We have designed therapies to deliver the genetic sequences of anti‐sense RNA therapy molecules to alter the splicing pattern and expression of the VEGFR2 transcript, creating a soluble VEGFR2 decoy. We have designed and cloned a mini‐reporter‐system that contains the regulatory elements of VEGFR2 splicing. This system measures the efficacy of RNA anti‐sense therapeutics to alter the splicing of the VEGFR2 transcript. The visual marker, eukaryotic green fluorescent protein is used to mimic the natural splicing product, whereas the red fluorescent protein, mCherry detects changes in the the efficacy of our RNA anti‐sense therapy. We use a GBM tissue culture model to measure mini‐reporter and therapy expression. In this manner, the mini‐reporter construct provides a quick and visually measurable test to optimize RNA anti‐sense therapies directed against VEGFR2.Support or Funding InformationBristol‐Myers Squibb
Muscle Growth by Activin Type II Receptor Blocking Ameliorates Weakness in GNE Myopathy Mice
Authors: Noguchi Satoru , Yonekawa Takahiro , Malicdan May Christine V , Miyakawa Miho , Lach‐Trifilieff Estelle , Nonaka Ikuya , Nishino Ichizo 
Published: November 2023

BackgroundGNE myopathy is an autosomal recessive muscle disease clinically characterized by skeletal muscle atrophy and weakness. We have established that a key factor in the mechanism of disease is sialic acid deficiency, a target for recent and ongoing clinical trials. In GNE myopathy patients with existing severe muscle atrophy and degeneration, however, exploring additional therapeutic options may prove beneficial.ObjectiveIn this study, we demonstrate the efficacy of myostatin pathway blockade, which promotes muscle growth, on GNE myopathy model mice.MethodsCDD866, a murinized anti‐activin type II receptor antibody, were administered to GNE myopathy model mice once weekly with subcutaneous injections for 8 weeks. Physiological examination and pathological observation on the skeletal muscles of treated mice were performed.ResultsCDD866, not only promoted body weight gain and increase in muscle size, but also led to the recovery of specific tetanic force, which is a functional indicator of muscle contractile devices. In addition, we demonstrate that increased S‐nitrosylation of contractile and metabolic proteins leads to muscle weakness in GNE myopathy mice. More importantly, CDD866 treatment in mice reversed S‐nitrosylation of the muscle proteins along with the recovery of muscle function.DiscussionThis results suggest that protein synthesis via myostatin pathway blockade has a beneficial effect by providing newly‐synthesized fresh muscle proteins to chronic myopathic muscles in addition to muscle size increment. Our results also suggest that the myostatin pathway blockade may be a promising therapy on GNE myopathy in addition to sialic acid supplementation.Support or Funding InformationIntramural Research Grant (28‐6) for Neurological and Psychiatric Disorders of NCNP
Evaluating the activity of bacterial enzyme DXP synthase a potential target for newer antibiotics
Authors: Kessler Alicia M , Anand Sridhar , Callahan Kevin P 
Published: November 2023

An emerging medical crisis involving the development of antibiotic resistant bacteria in the human body is compromising treatment of infections, leaving patients in critical condition. Infections that are becoming unresponsive to current treatment are creating a need for new antibiotics. In order to develop new antibiotics, researchers need to identify new targets that have high specificity for certain bacterial pathways. The enzyme D‐xylulose 5‐phosphate (DXP) synthase catalyzes the first step in the MEP (methylerythritol phosphate) pathway in human pathogens, which produces the biological molecules necessary to regulate numerous cellular functions, including central metabolism. Inhibition of DXP synthase would prevent or heavily reduce the rate of DXP production, which can then prevent or delay the continuation of the MEP pathway and thus is an ideal candidate for antibiotic development. In order to test the effectiveness of potential inhibitors of DXP synthase it is first necessary to establish a system to monitor the activity of DXP. We have purified Histidine tagged versions of DXP synthase and 1‐Deoxy‐D‐Xylulose 5‐Phosphate Reductoisomerase (IspC). The activity of DXP synthase cannot be directly measured using spectroscopic techniques, however it can be coupled to the activity of IspC, which in the process of converting DXP into MEP oxidizes NADPH. The establishment of this in vitro system will enable us to test the efficacy of novel inhibitors of DXP that are currently being developed.Support or Funding InformationSupported by internal grants from St. John Fisher College School of the Arts and Wegmans school of Pharmacy
Exploring inhibitors of the periplasmic chaperone SurA using fluorescence anisotropy
Authors: Zheng Erica J , Bell Eric W , Ryno Lisa M 
Published: November 2023

A requirement for cell homeostasis is the correct functioning of chaperones, which inhibit the aggregation of other proteins in the cell. The chaperone SurA, present in gram‐negative bacteria, prevents the aggregation of outer membrane porins as they traverse the aqueous periplasm. Disruption of SurA activity renders the bacterial cell more sensitive to agents that would normally be kept out by outer membrane porins. Here we describe an in vitro screen to test potential small molecule inhibitors of SurA that could be used to decrease the virulence of bacterial cells. Using fluorescence anisotropy, we conduct competitive binding assays using fluorescently‐tagged peptides that bind SurA with high affinity and small molecules discovered using in silico screening.Support or Funding InformationThis work was supported by Oberlin College.
Diadenosine Polyphosphatases of the NUDIX Hydrolase Superfamily in M. tuberculosis
Authors: DiCola Andrew Michael , Knowles Alexander , O'Handley Suzanne 
Published: November 2023

M. tuberculosis contains 11 potential Nudix hydrolases, and we are characterizing these enzymes as potential novel antibiotic targets. The diadenosine polyphosphatases (ApnAases)/mRNA decapping enzymes are a family of enzymes within the Nudix hydrolase superfamily. In M. tuberculosis there is the primary Nudix ApnAase and the secondary Nudix ApnAase. The diadenosine polyphosphatases from Legionella pneumophilia and Bartonella bacilliformis have been found to be important in each pathogen's ability to invade its host cells. It is of interest to know whether these enzymes act in the same way in M. tuberculosis. If they are all found to be involved in invasiveness and thus in virulence, then these enzymes could be novel antibiotic targets. We have cloned and overexpressed each protein and have subcloned each into a HisTag vector to optimize purification. We have purified the secondary Nudix ApnAase from M. tuberculosis so that we can complete its characterization. Thus far we have determined its substrate specificity (Ap4A is the best substrate), ApnAase activity, optimal pH (7.8), and divalent metal ion requirements (1 mM Mn2+ yields optimal activity, however Mg2+, Co2+, and Zn2+ yield activity as well).Support or Funding InformationThis research has been supported by an NIH AREA grant and RIT COS and honors summer undergraduate research fellowships.
Design and Analysis of a Microbiome Mock Community: Understanding and Mitigating Methodological Biases
Authors: Mosby Suquoia , Kiflezghi Michael , Edwards David , Brooks Paul J , Rivera Maria 
Published: November 2023

The gut microbiome has been shown to influence immunity, to play a role in obesity, colorectal cancer and diabetes among others. Although much research has been done, there is still a lack of understanding of what factors in the gut microbiome are influencing the health and/or disease state of the host. Among the challenges facing microbiome research are the difficulties in accurately identifying and quantitating the components of a microbiome. A typical protocol for analyzing and comparing microbiomes is to conduct a taxonomic and phylogenetic survey or census of the members of the microbiome community. The microbiome survey is typically performed by high‐throughput sequencing of PCR amplification of the 16S rRNA gene from the community metagenome, followed by applying bioinformatics methodology to assign a taxonomic identification to each one of the sequenced fragments. These processing steps can introduce bias that can distort the true or original composition of the microbiome. The ultimate goal of the experiments presented here is to understand the type and magnitude of the bias introduced by the typical protocols used for microbiome characterization. In order to uncover potential biases affecting microbiome studies we are designing and analyzing a mock community or a synthetic microbiome. The mock community is designed by combining in vitro known proportions of a set of well‐characterized bacterial species. Processing the mock community with the same protocols used by microbiome experiments will allow us to generate a quality control for these types of experiments. We designed a low complexity mock community composed of 10 species of bacteria isolated from different hosts. These 10 bacterial species will be mixed at different ratios and the different mixes will be subjected to a full microbiome analysis. This analysis includes DNA extraction, PCR amplification, NGS sequencing and bioinformatics analysis. The goal of the experimental design is to determine our ability to recover the original input ratios. The ratios obtained by our experimental protocols will ultimately be used to develop mathematical models to predict the “true” composition of the microbiomes. In the initial phase of the project we grew each organism individually as a pure culture followed by PCR amplification and sequencing to assess the purity of the cultures. We analyzed the sequences by comparing them to the NCBI nucleotide database using BLASTN. The BLASTN results indicated we had to distinct cultures sharing only 95% sequenced identity; the sequence of one of the cultures showed a 99% sequence identity to Escherichia coli and the other showed 99% sequence identity to Shimwellia blattae. The same methodology will be used to analyze another 8 pure cultures.Support or Funding InformationInitiative for Maximizing Student Development R25GM090084
The Metatranscriptome of the Rhesus Macaque: Investigating Potential Causes of Idiopathic Chronic Diarrhea
Authors: Westreich Samuel T , Ardeshir Amir , Kable Mary E , Korf Ian , Lemay Danielle G 
Published: November 2023

The study of the gut microbiome—the collection of microbes within the intestinal tract and the genes they express—is growing in popularity as associations are found between diet, gut microbiome activity, and host health and disease. However, current metagenomic and ribosomal profiling approaches are unable to capture changes in activity within microbiome populations. In this environment, shifts in diet may not result in microbiome population changes, but may instead influence the activity of the existing microbial community. Metatranscriptomics, the study of microbial population activity based on RNA‐seq data, offers detailed data on the gene expression of all microbes within the sampled community. We have created a new bioinformatics pipeline, SAMSA (Simple Analysis of Metatranscriptome Sequence Annotations), designed specifically for metatranscriptome dataset analysis, with options for either in‐house or external server‐based computational processing. Designed for use by researchers with relatively little bioinformatics experience, SAMSA offers a breakdown of metatranscriptome activity by organism or transcript function, and is fully open source.We have applied SAMSA to examination of Idiopathic Chronic Diarrhea (ICD), a long‐term inflammatory bowel condition in rhesus macaques that leads to significant mortality in captive populations. Previous studies on ICD have failed to identify a causative pathogen, and the disease is believed to be the result of a wider shift in the microbiome across multiple taxa. Through metatranscriptomic examination of both organism and functional activity shifts, we have identified underlying shifts in the gut environment of ICD‐afflicted animals, and observed changes in the activity of affected microbial communities. These results will offer new approaches for not simply providing palliative treatment for these affected animals, but permanently eliminating the ICD condition from captive rhesus macaque communities. These results demonstrate the value of a metatranscriptome‐based approach for studying microbiome environments.Support or Funding InformationFunding provided by Signature Research in Genomics award.