bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating the Effect of Chromatin Environment on Susceptibility to Double Strand Breaks and Choice of Repair Mechanism
Authors: Rodier Kendall Marie , Ani Danika Ngozika , Burgess Rebecca C 
Published: June 2021

Double strand breaks are an extremely dangerous type of DNA damage. When a cell detects them they undergo apoptosis if it isn't repaired. If repaired improperly, mutations resulting in disease states can accumulate, so understanding the susceptibility and mechanism of DSB repair is important. While it is known how DSBs are repaired, it is unknown how the surrounding chromatin environment affects the DSB susceptibility and the choice of repair mechanism. The goal of this study is to compare DSB frequency and repair in regions with different chromatin environments around the genome. To induce DSBs, we first compared Galactose‐inducible and Tetracycline (tet)‐inducible CRISPR systems for cutting frequency, and found that the tet‐inducible system produces DSBs at a higher frequency in the time course examined. In our study, we will use this tet‐inducible system to measure DSB frequency at different genome sites as well as probe repair pathway choice, first comparing three cut sites near active genes and three near repressed genes.We first measured DSB frequency using quantitative PCR across break sites. Preliminary data shows that the repressed genes have higher levels of DSBs after 3 h of DSB induction. This suggests that the specific chromatin environment that surrounds the cut site that may contribute to the increased susceptibility to DSBs, and correlation to histone modifications is underway. Alternatively, the increased amount of DSBs may be due to a reduced repair efficiency in the repressed gene regions compared to the active gene regions, thus more double strand breaks were present at each time point. This a topic of ongoing investigation.The survivor analysis tests the ability of cells to become resistant to continuous DSB induction due to imprecise repair of the cut site and destruction of the Cas9‐guide RNA recognition site. Calculating percentage of survivor colonies can give an estimate of the mutation frequency of a genomic locus. Preliminary results suggest that active chromatin locations might generally have fewer survivors, but more data and genomic loci must be examined. Interestingly, a few active loci appear to be hypermutable. The TDH2gene, for example, showed a multitude of inaccuracies when repairing after cutting, one to two orders of magnitude higher than other active genes, including its paralog, TDH3. Sequencing of the imprecisely joined regions is underway to investigate this further. In addition, we will be determining if there is a correlation between specific histone modifications and mutation frequency.This work will help us better understand which locations in the genome are more susceptible to DSBs and to imprecise repair, and subsequent mutation, thus advancing our understanding of how DSB repair can drive genomic change.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modification of Ser7 of the RNA Polymerase II C‐terminal domain (CTD) regulates CTD phosphorylation patterns and transcription
Authors: Burkholder Nathaniel Tate , Sipe Sarah , Kumar Mukesh , Escobar Edwin , Irani Seema , Matthews Wendy , Sim Kelly , Brodbelt Jennifer , Zhang Yan Jessie 
Published: June 2021

RNA Polymerase II subunit RPB1 in eukaryotes contains a conserved, repetitive C‐terminal Domain (CTD) consisting of predominantly Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 heptad repeats. While Ser2 and Ser5 are strongly conserved and serve as essential phosphorylation sites for recruiting specific transcription factors, the role of Ser7 is less understood as it is not well conserved nor seemingly required for general transcription. However, Ser7 is indeed phosphorylated across most genes in vivo suggesting that it is an important mark in transcription. We hypothesized that different mutations and modifications of the Ser7 site regulate the phosphorylation of other CTD residues which alter transcriptional outcomes. To test this hypothesis, we first generated GST‐CTD substrates with Ser7 mutated to residues with different charges like Glu and Lys to mimic specific chemical modifications or naturally occurring mutations. We then treated these substrates with different CTD kinases and used ultraviolet photodissociation mass spectrometry (UVPD‐MS) to analyze the resulting phosphorylation patterns. We found that the CTD kinase ABL1 preferentially targets Tyr1 adjacent to Glu7 while phosphorylating Tyr1 away from Lys7 or Arg7, showing that charge in the seventh CTD position can regulate CTD kinase activity. Furthermore, we observed increased ABL1 phosphorylation of Tyr1 adjacent to Gln7 which mimics Lys acetylation, suggesting that this CTD kinase regulation is reversible. To test how Ser7 modification can affect CTD phosphorylation and transcription in vivo, we used UVPD‐MS and ChIP‐Seq to analyze samples from cells expressing RPB1 with Ser7 mutated to Glu in every other repeat. Our analysis provides some of the first in vitro and in vivo evidence that Ser7 modification or mutation can control CTD phosphorylation patterns thereby regulating transcriptional outcome.Support or Funding InformationNIH 1R01GM125882‐01A1 The Welch FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Cloning of a Fluorescence‐Based Reporter Plasmid for Cell‐Free Protein Synthesis
Authors: Dumeng Jeriel , Ferre Mariana , Rodriguez Jessica M , Rodriguez‐Martinez José A 
Published: June 2021

Cell‐free protein expression, also known as in vitro protein synthesis is a convenient and fast method for protein production. It bypasses the need to culture living cells, which can be time and resource consuming. Among multiple cell‐free protein expression systems available, the wheat germ extract system has become a popular choice for producing eukaryotic proteins due to high yields and commercial availability. The goal of this work was to generate a fluorescence‐based reporter plasmid to be used in wheat germ based cell‐free protein expression system. Because of its unique optical properties, the Green Fluorescent Protein (deGFP) can be used as a positive control during high‐throughput synthesis of proteins by a cell‐free system. To construct the reporter plasmid, Gibson Assembly strategy was used to clone a deGFP gene in a wheat germ extract compatible plasmid. The deGFP gene was successfully cloned in two plasmids, bearing a hexahistidine tag on either the N‐, or C‐terminus of GFP. The clones were validated by Sanger DNA sequencing. After validation, protein expression was confirmed by western blotting using an anti‐His antibody. The reporter plasmid will be used as a positive control during high‐throughput production of eukaryotic transcription factors by wheat germ cell free protein synthesis.Support or Funding InformationResearch Initiative for Scientific Enhancement (RISE), GRANT 5R25GM061151‐17This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biophysical characterization of DNAJB1‐PRKACA in Fibrolamellar Hepatocellular Carcinoma
Authors: Le Quang Nguyen Trung 
Published: June 2021

Fibrolamellar Hepatocellular Carcinoma (FL‐HCC) has historically been described as a rare and usually fatal pediatric liver cancer that almost exclusively affects adolescents and young adults without a history of underlying liver diseases. Occurring on a healthy background, FL‐HCC's lack of readily recognizable symptoms impedes early diagnosis and timely medical intervention that can prevent tumor's progression to more advanced and metastatic stages. Unfortunately, FL‐HCC is refractory to typically applicable curative regimens in traditional liver cancer such as radiation or chemotherapies, leaving surgical removal the only therapeutic mainstay. However, this approach is still unable to mitigate a 50% reoccurrence rate within 10–33 months.A recent identification of a recurrent chimeric fusion between the catalytic subunit alpha (PRKACA) of protein kinase A, and the J‐domain of the heat shock protein 40 (DNAJB1) in virtually all patients marks an important milestone for the study of FL‐HCC's molecular pathogenesis. The DNAJB1‐PRKACA is central in several ongoing FL‐HCC studies as the primary tumorigenic driver.We are performing a biophysical characterization of the chimera versus the wild‐type protein kinase A. For example, Circular Dichroism is being used to monitor the secondary structure of the chimera in comparison to PRKACA. Differences in thermodynamics and kinase activity due to the J‐domain's fusion in DNAJB1‐PRKACA chimera may modify the protein's interaction with its downstream phosphorylation targets, therefore changing protein kinase A's signaling network implicated in FL‐HCC development. Analysis on changes in the chimera's phosphorylation network relative to the wild‐type protein will provide a crucial insight into FL‐HCC pathogenesis, promoting the development of highly efficacious chemotherapies in treating this disease.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for the Binding of Synthetic Cannabinoids to Human and Bovine Serum Albumin
Authors: Vanderpuye Oluseyi Adewale , Agu Chiagoziem Sylvester 
Published: June 2021

Albumin is the most abundant protein in blood and binds to a large variety of small molecules such as drugs, fatty acids, and other organic compounds. The distribution and overall concentrations of some drugs in blood have been proposed to be affected by albumin because otherwise poorly soluble compounds may circulate in larger amounts by being bound to albumin. Synthetic cannabinoids comprise over 200 compounds with low aqueous solubility that bind and activate integral membrane cannabinoid receptors in the brain and other tissues. Although synthetic cannabinoid use is global and has been associated with serious side effects there is limited information on factors affecting their toxicity. It is hypothesized that despite their diversity of structures, synthetic cannabinoids bind to serum albumin and this renders them more soluble in plasma. The goal of the current research was to test for the formation of complexes between synthetic cannabinoids and albumin by using electrophoresis, uv‐vis and fluorescence spectroscopy. In the presence but not in the absence of BSA, fluorescent bands at the position of albumin were seen for mixtures of human serum and bovine serum albumin with Bay 59–3074 and JWH‐015, Nile Red, timolol, coumarin and eosin. In the absence of these compounds, the albumin band was not fluorescent or colored. The colors of eosin, bromophenol blue and Nile red changed in the presence of albumin indicating an influence of albumin on the spectral properties of small compounds. The binding of compounds to albumin was further examined and demonstrated by by uv‐vis and fluorescence spectroscopy. It was concluded that the binding of synthetic cannabinoids to serum albumin was observed for the first time. It was also shown that agarose gel electrophoresis can be used in a novel fashion to detect the interaction of certain colored or fluorescent compounds with albumin.Support or Funding InformationCenter for Undergraduate Research, Albany State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Examination of HSF Activation in a Saccharomyces cerevisiae Model for TPI Deficiency
Authors: Skolnick Kathryn , Schlosser Lily Jean , Barnes Carol , Koziara Kortnee , Fisher Isaac , Hrizo Stacy 
Published: June 2021

Triose Phosphate Isomerase (TPI) is a glycolytic enzyme that catalyzes the inter‐conversion of DHAP to G3P. Mutations in TPI have been identified that are associated with a neurodegenerative disease in humans known as TPI deficiency. To study the cause of TPI deficiency pathogenesis, we constructed Saccharomyces cerevisiae yeast strains expressing six different TPI alleles that are associated with the development of TPI deficiency in humans and Drosophila melanogaster. Interestingly, not all of the TPI alleles exhibit similar characteristics in terms of stability, temperature sensitivity and activity. Yeast with the M80T allele exhibits significant changes in growth and metabolic activity at all temperatures. Yeast with the E104D allele exhibit an acute temperature sensitive phenotype for growth and metabolic activity at 37C. All of the alleles except C41Y demonstrate protein instability at 37C. This study further examines the role of protein folding in TPI deficiency disease pathogenesis. Yeast expressing the mutant TPI alleles were examined for activation of HSF, a cytoplasmic protein folding stress response transcription factor. Understanding the activation of cell stress pathways in TPI deficient yeast may lead to understanding of disease pathogenesis in TPI deficiency patients and provide potential pharmacological targets.Support or Funding InformationResearch is supported by an SRU Student Faculty Research GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A possible connection between plant longevity and the absence of protein fibrillation: basis for identifying aggregation inhibitors in plants
Authors: Otzen Daniel E , Ward Jane , Mohammad‐Beigi Hossein 
Published: June 2021

The ability of proteins to aggregate to form well‐organized b‐sheet rich amyloid fibrils is increasingly viewed as a general if regrettable property of the polypeptide chain. Aggregation leads to diseases such as amyloidosis and neurodegeneration in humans and various mammalian species but is also found in a functional variety in both animals and microbes. However, there are to our knowledge no reports of amyloid formation in plants. Plants are also the source of a large number of aggregation‐inhibiting compounds. We reasoned that the two phenomena are connected and that the longer‐lived the plant, the more efficiently it can suppress unwanted protein aggregation. In support of this, we show that while protein extracts from the sugar maple tree A. saccharum fibrillate readily on their own, this process is efficiently abolished by addition of small molecule extracts from the same plant. Further analysis of 44 plants showed a correlation between plant longevity and ability to inhibit protein aggregation. Extracts from the best performing plant, the sugar maple, were subjected to chromatographic fractionation, leading to the identification of a large number of compounds, many of which were shown to inhibit aggregation in vitro. We suggest that plants have been under evolutionary pressure to maintain an efficient collection of aggregation‐inhibiting metabolites; consequently, long‐lived plants may be particularly appropriate sources of new anti‐aggregation compounds with therapeutic potential.Support or Funding InformationD.E.O. was supported by a grant to D.E.O. from The Danish Council for Independent Research | Technology and Production Sciences (grant number: 12–126186), and Parkinson Foreningen (the Danish Parkinson Society). Rothamsted Research receives grant‐aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Recruitment and Regulation of RPN13 in the 26S Proteasome
Authors: Hemmis Casey W , VanderLinden Ryan T , Yao Tingting , Robinson Howard , Hill Christopher P 
Published: June 2021

The 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anti‐cancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain that is recognized by proteasome‐associated ubiquitin receptors. One of these receptors, RPN13, was identified as the most commonly overexpressed gene in solid tumors and has been directly implicated in cancers such as hepatocellular carcinoma, ovarian cancer, and leukemia.RPN13 is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2. To better understand the interactions between RPN13, RPN2, and its substrates, we mapped the RPN13‐binding epitope in RPN2, and using structural and biochemical methods, validated the RPN2‐binding epitope, quantified binding interactions, and revealed details of RPN13 substrate binding. Examination of our structure revealed that the primary binding determinants in RPN2 bind into a small, surface exposed groove of RPN13. These features suggest that the RPN13‐RPN2 interaction could be modulated by small molecule compounds, thus making it an attractive target for developing orthogonal approaches to proteasome inhibition.Our findings provide a detailed characterization of interactions that are important for proteasome function, and have major implications for the development of novel anti‐cancer therapeutics.Support or Funding InformationThis work was supported in part by National Institutes of Health Grants R01 GM059135 and R21 CA191929 (to C. P. H.) and American Cancer Society Postdoctoral Fellowship PF‐16‐100‐01 (to C.W.H.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epigenetic regulation of osteoblastogenesis by blackcurrant fruit extracts in vitro and in vivo
Authors: Mahady Gail B , Raut Nishikant , Lawal Temitope O , Patel Shital 
Published: June 2021

Osteoporosis is a major public health concern and is characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures. Osteoporosis and osteopenia affect over 45 million Americans annually with an estimated cost of > $20 billion USD. Over 50% of women and ~13% of men over the age of 50 will suffer an osteoporosis‐related fracture in their lifetime resulting in loss of independence, health and productivity, thus the research and discovery of compounds that can enhance bone formation would be clinically significant. Blackcurrants (Ribes nigrum) are native to Central and Eastern Europe and Northern Asia and are used in traditional medicine for the treatment of cardiovascular disease, bone and muscle disorders, diabetes, viral infections and inflammation. The berries contain four major anthocyanins: delphinidin‐3‐O‐glucoside, delphinidin‐3‐O‐rutinoside, cyanidin‐3‐O‐glucoside, cyanidin‐3‐O‐rutinoside. We have previously shown that blackcurrant extracts (BCE) increase myoblastogenesis, and in this work, we investigate its effects in cultured hFOB human osteoblasts and osterix/Sp7:mCherry transgenic medaka. hFOB human osteoblasts were obtained from ATCC and cultured in 1:1 mixture of Ham's F12 Medium Dulbecco's Modified Eagle's medium, with 2.5 mM L‐glutamine (without phenol red). Double transgenic medaka (dT; osterix/Sp7:mCherry were maintained at UIC under protocol #17‐166. Larvae at 9–12 dpf were staged and then treated for 5 days with the extracts and compounds (1–10 mg/ml). Osteoblast formation and distribution were analyzed by fluorescence microscopy, bone mineralization was visualized by calcein. The results of this investigation demonstrate that BCE and delphinidin‐3‐glucose increased osteoblast proliferation and reduced apoptosis in cultured human osteoblasts (hFOB) by reducing Bax and p53 expression and altering HDAC1, HDAC3, SIRT 3 and PGC1amRNA expression. In osterix/Sp7:mCherry medaka, BCE treatment (10 mg/ml) increased osteoblast proliferation by increasing osterix/Sp7 expression. These data suggest that BCE and D3G increase osteoblastogenesis through epigenetic regulation of HDACs and mitochondrial biogenesis.Support or Funding InformationNCCIH at the National Institutes of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and Evaluation of a Photoswitchable ABL Kinase Inhibitor as a Biochemical Tool
Authors: Swartz Brock , Streu Craig 
Published: June 2021

With so much to learn about the various biological pathways that allow complex organisms to function, researchers are constantly working to expand their biochemical toolkit. A common way to study the role of an enzyme is to inhibit the protein with a drug and monitor effects such as metabolite buildup or increased activity in other enzymes. To assist these processes, the use of photoswitch molecules is beneficial. The electronics of these molecules allow them to change conformation in response to specific wavelengths of light. With the shape of a drug being so crucial to its binding affinity, molecules capable of changing their shape may be active in one conformation but inactive in the other. This allows these drugs to be activated and inactivated at will, allowing for more in‐depth studies of the spatial and temporal role of proteins. Azo‐stilbenes are among the most well established photoswitches. The azo‐stilbene structure, a chromophore commonly present in industrial dyes due to its vibrant colors, consists of two aromatic rings connected by an azo (N=N) bond. When irradiated with blue light (wavelength of 450–490 nm) these molecules undergo a trans‐to‐cis isomerization about the azo bond. Exposure to heat or red light (wavelength 620–750 nm) incites these molecules to relax back into their more thermodynamically favorable trans state. This poster will outline the synthesis and photophysical characterization of a photoswitchable inhibitor of ABL, a tyrosine kinase. A key component of the synthesis is the use of the Mills reaction, a condensation process that forms an azo bond between an amine and a nitroso compound. UV/Vis absorption spectroscopy was used to characterize properties such as the lambda max values for the cis and trans conformations as well as the thermal relaxation time. This molecule may also have potential for more targeted cancer treatments since the BCR‐ABL fusion gene causes chronic myeloid leukemia. By administering the drug in its inactive conformation and then irradiating sites to induce isomerization where cancer cells are present, unnecessary cell death may be avoided. Treatment of this sort could reduce the side effects of current CML treatments, namely low blood cell counts, which may result in infection and/or anemia.Support or Funding InformationAlbion College Foundation for Undergraduate Research, Scholarship, and Creative ActivitiesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.