bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessment of CCN2 Independent Modules Regenerative Capacity on Osteoarthritis and Further Selecting the Most Suitable Among them as a Potential Therapeutic Drug
Authors: Abdelkader Tarek , Aoyama Eriko , Nishida Takashi , Hattori Takako , Janune Danilo , Hara Emilio S. , Ono Mitsuaki , Tabata Yasuhiko , Kuboki Takuo , Kubota Satoshi , Takigawa Masaharu 
Published: November 2023

CCN family proteins (Connective tissue growth factor (CTGF)/CCN2, Cystein rich protein (Cyr61)/CCN1, and Nephroblastoma overexpressed gene (nov)/CCN3) play important roles in differentiation and regeneration of cartilaginous tissues. CCN2/CTGF is known to promote the regeneration of articular cartilage. It's comprised of 4 highly interactive independent modules, each of which was suggested to have its own biological activity. This study aims to assess the effects of these modules independently and their combinations on chondrocytic cells in vitro and damaged cartilage in vivo, especially in relation to full length CCN2.MethodsEffect of a single module, or their different combination, was evaluated with human chondrocytic HCS‐2/8 cells. The cellular phenotype was estimated by the gene expression of chondrocytic markers. Proteoglycan synthesis and proliferation were evaluated by [35S]‐sulfate and [3H]‐thymidine incorporation assays, respectively. Physical interaction of 2 modules was kinetically examined by a surface plasmon resonance (SPR) methodology. Cartilage regeneration in vivo was evaluated by using 2 rat models simulating osteoarthritis via surgical or chemical intervention. Angiogenic affinity of independent modules was assessed in vitro by tube formation assay using matrigel with human umbilical endothelial cells (HUVEC) and ex ovo by CAM (chorioallantoic membrane) assay with chick embryo.ResultsFunctional analysis in vitro revealed a biological activity even stronger than the full length CCN2 in a particular module. Interestingly, mixed application of all 4 modules almost reconstructed the bioactivity to the level of the full length. SPR analysis uncovered significant interaction of 2 modules and full‐length CCN2. The result of cartilage regeneration experiments in vivo was also consistent with the findings obtained in vitro. The results of the matrigel assay indicate that the same module has tube formation potential under special circumstances and a similar effect could be observed in the CAM assay ex ovo.ConclusionThese results indicate independent bioactivity of each module for cartilage regeneration and suggest unknown means of binding to construct a possible tetramodular complex that is functionally comparable to the full length CCN2. Also the effect of the proposed independent module as a regenerative drug on angiogenesis must be kept in mind and further analyzed as this characteristic might be beneficial for vascular regeneration in spite the fact that it's considered a side effect for a proposed OA treatment.
Visualization explorer for classification and functional enrichment of fecal microbiome to metabolites
Authors: Kumar Raina , Yang Ruoting , Gautam Aarti , Hammamieh Rasha , Mudunuri Uma , Jett‐Tilton Marti 
Published: November 2023

Advanced high throughput technologies such as Next Generation Sequencing have created opportunities for scientists to explore the human microbiome more intensively and effectively. This enables us to understand the impact of imbalances in normal microbial flora through determining changes in the microbial composition and investigating how these correspond with changes occurring at the molecular level and the physiological level in the human body. Here, we developed the visualization explorer component for our in‐house automated microbiome analysis pipeline. The explorer takes enriched microbial composition per sample as input and provides a unique way to classify and visualize the microbiome data, organized under tabs. Under the relative abundance tab is visualization of the microbial composition across different samples with taxonomic annotation using different charts such as bar and radial charts. The PCA/Diversity index tab allows one to perform and visualize the principal component analysis and microbial diversity index comparison between treatment and control categories. Under the functional enrichment tab, the metabolite enrichment for the present microbial composition per sample is conducted and provides the predicted significant pathways between two categories in a network view. This visualization explorer was built using R open source language and Shiny server API. PICRUST performs the metabolite prediction and pathway enrichment. For the network visualization, explorer uses Graphviz open source software. The other features of this explorer include several visualization options such as heatmaps, histograms and line charts to compare differentially abundant bacterial species between disease and control samples.Support or Funding InformationDHPRDTEGDF 374000GJ
Gene Expression Patterns in Trauma Patients: A Systems Biology Approach
Authors: Srinivasan Seshamalini , Donohue Duncan E. , Gautam Aarti , Conroy Amanda S. , Nelson Mary F. , Cohen Mitchell J. , Hammamieh Rasha , Jett Marti 
Published: November 2023

Coagulopathy in trauma patients is associated with a threefold to fivefold increase in mortality. An initial abnormal coagulation profile, as measured by routine clinical tests such as PT and PTT, increases the odds of dying by 35%–326%. (MacLeod et al. 2003). Yet, the current understanding of the mechanisms underlying the development of coagulopathy following severe injury is extremely limited making its prevention and treatment limited. The aim of this study is to use systems biology approaches to integrate multiple data types including clinical findings, various “‐omics” approaches from the onset of the disease to develop a universal picture of the progression of this hemostatic dysfunction. Adult trauma patients were recruited based on previously published inclusion/exclusion criteria. Blood collection was performed every 2–6 hours for the first 24 hours and daily up to 4 days. Comprehensive demographics, injury, treatment and outcome data was prospectively collected. A temporal change in gene expression was studied using whole genome microarrays which probe 50,000 biological features (Agilent Technologies, CA). 79 samples representing 25 patients were processed and analyzed. A comparison of samples from patients with ISS (Injury Severity Score) below the median of 19 (38 samples) vs. those at or above 19 (n=41) showed significantly higher expression (FDR adjusted moderated t‐test p<.05) for DHX58, which is involved in interferon signaling, and lower expression for MIAT, a known biomarker of myocardial infarction, in samples from high ISS patients. Work is ongoing to fully characterize mRNA expression changes in these patients to better understand the contribution of demographic, clinical, and treatment confounds towards identifying molecular targets with potential utility in informing treatment decisions.DISCLAIMERResearch was conducted in compliance with IRB approved human subjects protocol #H7462‐25307‐07A. The views, opinions, and/or findings contained in this report are those of the author(s) and should not be construed as official Department of the Army position, policy, or decision, unless so designated by other official documentation.
Mapping Tyrosine Phosphorylation Networks by Chemical Proteomics
Authors: Rogers William Eric , Ghassemian Majid 
Published: November 2023

Chemical Proteomics is an unbiased approach to cellular signaling, but it relies on the ability to chemically react a specific post‐translational modification for analysis. Previously, a Beta‐elimination/Michael's addition reaction has been developed to chemically probe phosphorylated Serine and Threonine residues. The stability provided to the phosphate in by attachment to the ring structure of Tyrosine makes this process impossible. Herein, I present a procedure for chemically labeling phospho‐Tyrosine (pY) through a modified Suzuki coupling on tryptic peptides. This is followed by a biotin‐streptavidin affinity based enrichment and mass spectrometric (MS) analysis. This procedure solves the two main problems of pY analysis. First, the “flyability” of the peptide is increased drastically by removing the negative charge that is present on the phosphate (pKa=1.76) at the standard pH (~2.0) used in MS. Second, and as importantly, the problem of rarity of phosphorylated peptides in a cellular system is also solved. With the addition of SILAC, ICAT, or other isotopic labeling techniques it is straightforward to map the propagation of signaling along pathways and possibly uncover novel effects by that signaling that can not be determined via conventional methods.Support or Funding InformationW.E.R is a recipient of an Arne N. Wick pre‐doctoral research fellowship from the California Metabolic Research Foundation.
Using Polymerase Chain Reaction Inhibition to Study Small Molecule Binding to DNA
Authors: Ghannam Ryan , Smith Sheila R , Benore Marilee Ann 
Published: November 2023

Detection of DNA‐molecule interaction can be challenging. Knowing that Polymerase Chain Reaction (PCR) can be inhibited by interfering agents, this interaction was exploited as a potential assay. Using whole plasmid and DNA fragments, the ability of small molecules to inhibit the PCR reaction was studied under diverse conditions and testing several potential inhibitors.Support or Funding InformationUniversity of MI Dearborn campus grants
Purification and Characterization of DNA from Fresh Versus Frozen Chicken Embryos
Authors: Byk Jennifer , Smith Sheila R , Benore Marilee Ann 
Published: November 2023

Purification of DNA from tissues can be carried out by several methods. The ease of purification, yield and functionality of the DNA isolated varies depending on the method used. Using both frozen and fresh chick embryos, we tested several kits and traditional methods. Comparative analysis of the yields, purity, and integrity of fresh tissue versus frozen tissue were measured.Support or Funding InformationUniversity of Michigan Dearborn Campus Grants
Evaluating the Extraction Efficiency of Food Borne Pathogens on Automated Homogenizer Platforms
Authors: Garrett Shari , Atwood James , Easparro Brandon 
Published: November 2023

There is an ever increasing demand for food and beverage manufacturers to adhere to rigorous food safety regulations. As part of the global need for safe food, food and beverage manufacturers test their products for the presence of food borne pathogens. While safety regulations describe what pathogens should be controlled through testing there is no predefined method of sample preparation or detection. Typically, the first step in a food borne pathogen detection study is the disaggregation of the food product or the washing of the sample to release bacteria from the food surface using a Stomacher. While the later method is great for large masses of food stuffs, some researchers may require small amounts of product for analysis. Bead mill homogenizers, as well as rotor‐stator homogenizers, can be used to aid in the sample preparation process. Processing can be completed in less than one minute while maintaining bacterial cell and analyte integrity. Depending on the pathogen of interest and the food matrix, the downstream results, including sensitivity can vary greatly based on the sample preparation method chosen.With this is mind, we analyzed two sample preparation techniques on commonly contaminated foods. Meat and spinach samples were inoculated with known levels of recombinant E.coli expressing Green Fluorescent Protein. The inoculated samples were processed on a bead mill homogenizer and an automated rotor‐stator homogenizer. The homogenates were grown on nutrient agar and the proportion of viable cell recovery was quantified. Limit of detections were determined by lysis of recovered homogenates, DNA purification and PCR amplification.It was determined that both homogenization techniques were able to process food samples for the extraction of live cells and plasmid DNA. The processing efficiency can deliver a 76% cell recovery with a PCR detection limit to 1×10−5 CFUs/ml. A linear relationship was demonstrated between the number of CFUs to intensity of DNA amplified via PCR. A detection limit of 10−5 CFUs is comparable to some food testing methods; however the vigorous processing of bead mill and rotor stator technology has the potential to eliminate the standard pre‐enrichment step needed for the detection of food pathogens.
Development of an easy, inexpensive, and precise method to identify Hemoglobin C for use in underdeveloped countries
Authors: Ohiri Chiemeziem D , Randolph Tim R 
Published: November 2023

Hemoglobin C is one of the most common structural hemoglobin variants in the human population, second only to HbS (sickle cell). Like HbS, the HbC mutation exists in places which have inadequate resources to detect the presence of Hemoglobin C. This study aims to develop an easy, inexpensive, yet precise method to detect the presence of Hemoglobin C and determine zygosity. The hypothesis is that Hemoglobin C crystal formation can be induced intracellularly when incubated in a hypertonic salt solution and visualized microscopically when stained with new methylene blue. Crystal formation was achieved by incubating de‐identified patient blood samples bearing the Hemoglobin SC mutation in sodium chloride solutions ranging in concentration from 2%–6% and in concentrated Dulbecco's phosphate buffered saline. Sodium chloride concentrations between 3% and 5% showed the highest crystal formation. With further study, we hope to optimize the method to detect Hemoglobin C and establish guidelines to determine zygosity of blood samples.Support or Funding InformationCO is supported by the DeNardo Education and Research Foundation Fellowship.
Comparison of Enzymatic Activities of Pokeweed Antiviral Protein (PAP) Isoforms toward RNA from the Tobacco Etch Virus
Authors: Aitbakieva Valentina R , Domashevskiy Artem V 
Published: November 2023

Plants produce ribosome‐inactivating proteins (RIPs) phytotoxins that serve an important role in plants’ defense mechanisms against pathogenic invaders. RIPs are RNA N‐glycosidases that inhibit protein synthesis of a host cell through depurination of highly conserved sarcin/ricin loop of large ribosomal RNA. After synthesized inside the cells, RIPs are exocytosed and stored within extracellular spaces. Once a pathogenic attack occurs, RIPs gain access into the cell, alongside with a pathogen. Lesions within depurinated ribosomal RNA, produced by enzymatic activity of RIPs, avert the translocation step of protein synthesis. As a result, ribosome stalls.. The common pokeweed plant, Phytolacca americana, produces pokeweed antiviral protein (PAP), a type 1 RIP. PAP had been interacts directly with viral RNAs, depurinates them directly, and does so without substantial damage to the host ribosoms. PAP lowers the infectivity of many plant and animal viruses, including HIV‐1, poliovirus, and influenza. Nine known isoforms of PAP are produced in different parts of the plant; these vary during seasonal development. The mechanism of PAP‐viral RNA selectivity is not well known, and antiviral activity among different PAP isoforms varies. The focus of this study is to gain a deeper understanding into PAP‐RNA selectivity mechanism, and examine which isoforms of PAP exhibit the greatest activity toward tobacco etch virus (TEV) RNA. This knowledge will further be used in the development of efficient antiviral treatments.Support or Funding InformationTitle V, HSI‐STEM and MSEIP programs within the U.S. Department of Education; the PAESMEM program through the National Science Foundation; and New York State's Graduate Research and Technology Initiative. Professional Stuff Congress of the City University of New York (PSC‐CUNY), Cycle 44 Award.
Spectroscopic and Electrochemical Characterization of a Unique Radical S‐Adenosylmethionine Methylase
Authors: Blaszczyk Anthony J. , Silakov Alexey , Zhang Bo , Maiocco Stephanie J. , Lanz Nicholas D. , Kelly Wendy L. , Elliott Sean J. , Krebs Carsten , Booker Squire J. 
Published: November 2023

TsrM catalyzes the methylation of carbon 2 of the indole ring of L‐tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group derives from S‐adenosylmethionine (SAM); however, the enzyme also requires cobalamin and iron‐sulfur cluster cofactors for turnover. In this work we demonstrate the ability to overproduce and purify TsrM and characterize its metallocofactors by UV‐visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mössbauer spectroscopies, in addition to protein film electrochemistry. The enzyme contains one equivalent of its cobalamin cofactor in its as‐isolated state, and can be reconstituted with iron and sulfide to contain one [4Fe–4S] cluster with a site‐differentiated Fe2+/Fe3+ pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five‐coordinate base‐off/His‐off conformation, whereby the dimethylbenzimidazole is replaced by a ligand lacking nuclear spin, likely a water molecule. Electrochemical analysis of the protein by protein‐film electrochemistry indicates a one‐electron redox wave that exhibits a midpoint potential of ‐550 mV, assigned to a [4Fe–4S]2+/[4Fe–4S]+ redox couple. Analysis of TsrM by Mössbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron ion of the cluster in the same manner as other radical SAM enzymes, yet still disturbs the electronic configuration of the Fe/S cluster. These biophysical studies suggest that TsrM is an atypical radical SAM enzyme, consistent with its reported inability to catalyze formation of a 5′‐deoxyadenosyl 5′‐radical.Support or Funding InformationNational Institutes of Health
Induction of Apoptosis in DLD‐1 Colorectal Cancer Cells using Water Soluble Compounds from Rumex Crispus
Authors: Bhandari Mukund , Lockwood Hannah , Clack Beatrice 
Published: November 2023

Colorectal cancer is the third leading cause of cancer death in the United States (American Cancer Society, 2015). Colorectal cancer cells can become resistant to current chemotherapy agents which prompts the need for new anticancer compounds (Crawford, 2014). In this study, water soluble compounds from R. crispus root and leaf were extracted, purified and separated using reversed phase High Performance Liquid Chromatography (HPLC). Cell viability assays were performed on human colorectal adenocarcinoma (DLD‐1 ATCC® CCL‐221TM) cells for each fraction and were further screened for caspase 3/7 activity to show that cell death was through apoptosis. R‐26, L‐26 and L‐19 fractions from HPLC exhibited cell viability reduced to 8.7 %, 10 % and 9.8%, respectively. The caspase‐3/7 fold change was observed to be 12.3, 3.5 and 26.9 fold increase for R‐26, L‐26 and L‐19 respectively, compared to untreated cells. mRNA from L‐19 treated cells was used to determine caspase 3,7,8, 9 and 10 levels of gene expression by qRT‐PCR. The ΔΔCt values for each caspase was determined using GAPDH for normalization. The ΔΔCt values for initiator caspases 8 and 10 were found to be 3.0 and 6.5 fold higher relative to untreated cells respectively. The initiator caspase‐9 showed no change. These results suggest the extrinsic pathway of apoptosis to be involved. Gas chromatography‐Mass Spectrometry (GC‐MS) of the HPLC fractions provided possible candidate compounds.Support or Funding InformationOffice of Research and Sponsored Projects, SFASU and Ed and Gwen Cole, Nacogdoches, TX
The Effect of Phytochemicals on a Chemoresistant, Epithelial‐Mesenchymal Transitioned, Colorectal Cancer Cell Line
Authors: Murden Kathryn , San KayKay , Martino Alice , Ezekiel Uthayashanker 
Published: November 2023

In 2012, colorectal cancer was the second most common cancer diagnosed in women and the third most common in men. In 2015, an estimated 132,700 new individuals will be diagnosed and 49,700 people will die from the disease. Colorectal cancer, though one of the most common cancers worldwide, is not distributed evenly among the world's population. It is concentrated mainly in countries with a Western culture, specifically the Western diet, such as the United States and Western Europe. A considerably smaller rate of incidence of disease is found in Africa and parts of Asia. This difference in colorectal cancer distribution may be attributed to the increased presence of certain phytochemicals in non‐Western diets. Our hypothesis is that phytochemicals inhibit proliferation of both metastatic and non‐metastatic cancer cell lines. Studies have shown that transdifferentiation into epithelial‐mesenchymal transition (EMT) by chemotherapy leads to the formation of resistant cells. EMT is a process in which epithelial cells acquire a mesenchymal phenotype, characterized by increased migratory and invasive properties. We developed a chemoresistance‐induced epithelial to mesenchymal transitioned colon cancer cell line (DLD‐1 OxR) by exposing the parental cell line (DLD‐1) to increasing concentrations of oxaliplatin (a drug used in the treatment of metastatic colon cancer). The DLD‐1 OxR cell line has undergone EMT and lost its epithelial properties to become a more motile mesenchymal cell type. We used a wound healing assay and transwell assay to assess the migration and invasion of the EMT‐chemoresistant cell line respectively. DLD‐1 and DLD‐1 OxR cells were exposed to curcumin and kaempferol. Curcumin is a phytochemical derived from turmeric, which has been shown to suppress inflammation and inhibit cell proliferation. Kaempferol, a phytochemical found in plants such as broccoli, kale, beans, and leeks, has also shown anti‐inflammatory and anti‐cancerous activity. Both phytochemicals inhibited proliferation of DLD‐1 and DLD‐1 OxR cell lines.Support or Funding InformationKM, KS, and AM are supported by the DeNardo Education and Research Foundation Fellowship.
Mechanism of SAMT Analog Inactivation of HIV‐1 Gag Polyprotein
Authors: Paine Elliott L. , George Rosenker Kara M. , Scerba Michael T. , Deshmukh Lalit , Appella Daniel H. , Miller Jenkins Lisa M. 
Published: November 2023

Although antiretroviral therapy has advanced, there remains a critical need for new therapeutics, especially those targeted at resistant strains of HIV. The Gag polyprotein, and in particular its constituent nucleocapsid protein, NCp7, represents a prime target for antiretroviral inhibition. NCp7 is composed of two highly conserved zinc‐binding domains and plays multiple roles throughout the virus replication cycle. We have developed a class of small molecule inhibitors of NCp7, based upon an S‐acyl‐2‐mercaptobenzamide thioester (SAMT) scaffold, that covalently modify the zinc‐binding domains of NCp7 in vitro and in cells. These compounds display potent antiviral activity without evidence of cytotoxicity in cell models, ex vivo cervical explants, and in animal models. We investigated the mechanism of viral inactivation of the Gag polyprotein both in vitro and in virus‐like particles (VLPs) released from cells. We utilized mass spectrometry and NMR spectroscopy to characterize the covalent SAMT reaction sites within the Gag polyprotein. Our results indicate that reaction occurs first within the NCp7 domain, but that sites within both matrix and capsid are also modified. These results represent an increased understanding of the mechanism of SAMT activity and will help to develop better inhibitors.Support or Funding InformationThis research was supported by the Intramural Research Programs of the NCI and the NIDDK, NIH.