bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generation of an Endogenous Dominant Negative Allele of chd5 Using a Novel Homologous Recombination Strategy to Reveal the Role of chd5 in Tumor Suppression and Neural Differentiation
Authors: Denning Ellen , Sorlien Erin , Ogas Joseph 
Published: June 2021

CHD5 is a vertebrate specific member of a family of ATP‐dependent chromatin remodeling proteins. Chromodomain/Helicase/DNA‐binding (CHD) proteins play a variety of roles in vertebrate development, and loss of CHD5 causes failure of proper expression in a cohort of genes involved in neural development. CHD5 plays a critical role tumor suppression in humans, and loss of CHD5 contributes to the formation or progression of numerous cancers, in particular neuroblastoma. To understand how CHD5 plays a role in tumor suppression, it is necessary to understand how CHD5 contributes to normal development and how its normal functions are misregulated during tumor progression. Our lab is establishing zebrafish as model system in which to study the role of chd5 dependent processes.We have engineered chd5 knockout fish using CRISPR‐Cas9 technology. The resulting fish do not exhibit any overt developmental phenotypes, and do not produce tumors during adulthood (N=165, aged >16 months). We hypothesize that the closely related remodelers CHD3/4 function redundantly with CHD5, and propose to use a novel knock‐in approach to generate zebrafish carrying a dominant negative mutation in the ATPase domain that has previously been shown to result in strong mutant phenotypes when generated in closely related remodelers.To engineer a dominant negative allele of chd5 we have engineered a homologous recombination (HR) strategy that uses piggyBac transposase technology, which has previously been used in Drosophila, mice, and human cells to engineer scarless genomic modifications. This design uses two‐steps to first, introduce a homologous donor carrying the mutation of interest and a fluorescent marker for easy identification. Second, the HR positive fish will be bred with piggyBac expressing fish lines to seamlessly excise the exogenous sequences, thus leaving only the desired genome modification.Once we have engineered this method into the fish, we will be able to use piggyBac to control when and where we express the dominant negative allele of chd5 so that we can score the resulting fish for developmental and tumor phenotypes. The screening of the dominant negative, knock‐in lines is in progress. The resulting dominant negative lines will give great insight into the role of chd5 in neural development and tumor suppression.Support or Funding InformationFunding made available by: Purdue University Center for Cancer Research, Purdue University Department of Biochemistry, Purdue University College of AgricultureThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genome Editing of a Secondary Alcohol Dehydrogenase Gene by CRISPR‐Cas9 Technology
Authors: Huang Jenq‐Kuen , Samassekou Kadidia , McClenahan Shawn , Seiver Jacob , Holt Scott , Wen Lisa 
Published: June 2021

Nocardia Cholesterolicum NRRL 5767 (NC NRRL5767) is well‐known for its ability to transform ~95% of added oleic acid, an abundant agricultural commodity, to value‐added product of 10‐hydroxystearic acid (10‐HSA). A small amount of unwanted 10‐ketostearic acid (10‐KSA) was also produced. The conversion of oleic acid to 10‐HSA and then to 10‐KSA is catalyzed by oleate hydratase and secondary alcohol dehydrogenase (2°‐ADH), respectively. The objective of this project was to knockout the 2°‐ADH gene in NC NRRL5767 so that the sole biotransformation product from oleic acid would be 10‐HSA. Here, we report construction of CRISPR/Cas9/sgRNA chimeric plasmids that specifically target two different loci in the coding region of the 2°‐ADH gene by golden gate assembly. The constructs were confirmed by DNA sequencing and transformed into NC NRRL 5767 via electroporation. The transformants were selected by apramycin resistance and screened for the presence of the target insert by PCR. The ability of the selected transformants to transform oleic acid to 10‐HSA was examined by TLC. Our results showed that both 10‐HSA and 10‐KSA were produced by the transformants from oleic acid. Apparently, either Cas9 was not expressed or it was expressed but did not target the 2°‐ADH gene. The expression of Cas9 in the cell‐free extracts of selected transformants was examined by Western blotting using Cas9 antibody. However, the Cas9 protein was undetectable in these transformants.Support or Funding InformationWestern Illinois University FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential nicking patterns of Cas12a variants
Authors: Murugan Karthik , Sashital Dipali G 
Published: June 2021

Cas12a is an endonuclease from the bacterial CRISPR‐Cas immune system that can be easily programmed for use in various biotechnological applications. Despite its prevalent use in genome editing, the cleavage specificity and activity of Cas12a remain to be fully determined. We employed high‐throughput in vitro cleavage assays to determine and compare the native specificities of different Cas12a variants. Surprisingly, we observe pervasive nicking of randomized target libraries. Cas12a has distinct nicking activity against targets with multiple mismatches, where it can linearize certain mismatch targets, but only nick others. Our results also demonstrate that Cas12a variants have differential in vitro cleavage activity and mismatch tolerance. Francisella novicida (Fn)Cas12a is less tolerant to mismatches than Lachnospiraceae bacterium (Lb)Cas12a and Acidaminococcus sp (As)Cas12a. The mismatch position plays a vital role in determining target specificity of Cas12a. Like Streptococcus pyogenes (Sp)Cas9, the mismatches in the PAM‐proximal “seed” region are especially deleterious to Cas12a target cleavage, however, the seed region is shorter (~ 6bp) when compared to SpCas9 (~ 8–10bp). Overall, we observe that Cas12a variants have sequence‐ and seed‐specific nicking and cleavage activities, with variable sensitivities to mismatch position.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Divulging the Enigma of Epitranscriptome in Plasmodium falciparum
Authors: Govindaraju Gayathri , Jabeena CA , sethumadavan Devadathan , Raja Nivethika , Rajavelu Arumugam 
Published: June 2021

In spite of prime posttranscriptional modifications, eukaryotic RNA is subjected to internal epitranscriptomic marks. Thus, these changes influence the rate of transcription which regulates the translational plasticity. However, RNA methylome and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, for the foremost a piece of our work demonstrated the cytosine methylation in the tRNA Asp at the C38 position of Plasmodium falciparum, which is accomplished by the tRNA aspartic acid methyl transferase1 (TRDMT1), a conserved homologue of DNA methyltransferase DNMT2. Through RNA bisulfite sequencing, we mapped the 5‐methylcytosines in endogenous tRNA Asp, followed by in‐vitro characterisation of TRDMT1. Accordingly, P. falciparum proteome divulged the significant role of polyaspartic acid repeat‐containing proteins and its abundance. Those proteins may play astonishing roles in host‐pathogen interactions. Further studies focus on the generation of TRDMT1 knock out parasites which may aid us to find out the critical role of TRDMT1 mediated C38 methylation of aspartic acid tRNA. In particular, translational regulation of essential proteins which could modulate the malaria parasite pathogenesis. On the other hand, the significant genes in P. falciparum show discrepancy in mRNA expression and its cognate protein, through which ‘time shift’ is displayed. Moreover, P. falciparum exhibits decelerated ring stage as a distinctive policy for drug resistance. In recent years, it is evidently proven that the epigenetic modifications are existed in eukaryotic mRNA, especially methylation at the nucleotide level and is the reason for time shift.In support, our preliminary data's on P. falciparum RNA implied the methylated nucleotides. We anticipate that parasites may modulate the translation process at the transcription level through methylation that could be installed by a multicomponent methyltransferase complex and follow‐up by the RNA binding proteins. Thus so, parasite might adopt the ‘just‐in‐time’ transcription and translation strategy for its emergence that are controlled by epigenetic machinery. Consequently, further mining of RNA methylome in parasite could be insightful about the host pathogen interaction. Hence, by cracking the epitrancriptome of P. falciparum, the underlying mechanisms of dynamic gene regulation of the parasite pathogenesis could be explored.Support or Funding InformationDepartment of science and technology, Government of India.Department of Biotechnology, Government of India.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cypermethrin Induced Liver Toxicity: Altered Gene Expression and DNA Methylation
Authors: Mahna Dolly , Puri Sanjeev , Sharma Shweta 
Published: June 2021

Cypermethrin, a type II pyrethroid, is a widely used insecticide worldwide. It is considered a safe insecticide to be used in pet shampoos and household cleaning agents thus ignoring the possible health hazards caused by it. Cypermethrin may contaminate food, water, fruits or get deposited on furniture, mats or floors leading to acute or chronic disorders in non‐target organisms. Very limited studies with low and non‐frequent dose mimicking the unintentional cypermethrin intake in non‐target organisms including human beings have been carried out. The present study was aimed at evaluating the toxic effect of cypermethrin in the liver of Balb/c mice exposed to a low and once in a week oral dose (1/10 LD50) for 28 days. Serum was extracted from blood after the dosing period and utilized for Liver Function Tests. The gene regulatory studies were performed by amplifying the cDNA, employing specific primers for p21 and p53 and other cell cycle regulatory genes through PCR (Polymerase Chain Reaction). DNA methylation studies were carried out by bisulfite treatment of DNA followed by Bisulphite specific PCR. The methylation status was found out by analyzing the gene promoter regions through sequencing.The changes in liver enzyme activities and histo‐architecture of liver cells show that sub‐chronic exposure to cypermethrin causes liver damage. It also induces a significant increase in hepatic markers enzymes (ALT, AST, ALP) along with necrosis, vacuolar degeneration, increased kupffer cells and a decrease in the number of hepatocytes. A significant change in the gene expression of p21 and p53 is seen in the test animals pointing towards the relation of liver injury by cypermethrin with cell cycle regulatory mechanisms. The gene promoter region of p53 gene show alteration in methylation status in the test animal sample as compared to the control.Sub‐chronic exposure to cypermethrin causes significant changes in liver enzyme activities, cell architecture, cell cycle regulatory gene expression and methylation status of the concerned genes. These genes along with the other cell cycle regulatory genes can be used as biomarkers for liver injury caused by low doses of cypermethrin.Support or Funding Information1. University Grant Commission, New Delhi, India.2. DST‐PURSE grant, New Delhi, India.3. Institute of Forensic Science and Criminology, Panjab University, India.4. Centre for Stem cell and Tissue Engineering, Panjab University, India.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Function and Mechanism of Hepatitis C Virus Non‐structural Protein 3 (HCV NS3) Unfolding of Viral G‐quadruplex RNA Structures
Authors: Belachew Binyam Ateka , Byrd Alicia , Gao Jun , Raney Kevin 
Published: June 2021

Hepatitis C virus (HCV) is the major cause of chronic liver disease and hepatocellular carcinoma. It is currently estimated that over 71 million people in the world are chronically infected with HCV. The current HCV drugs are extremely expensive, and thus they are not available for the majority of patients infected with the virus. Moreover, due to high rate of mutation of the HCV genome, it is likely that the virus develops resistance to these drugs. Therefore, it is important to develop a cost‐effective anti‐viral compound that targets conserved regions within the HCV genome and prevent replication of various HCV genotypes and subtypes.The HCV genome encodes for ten different viral proteins, one of which is Non‐structural protein 3 (NS3). NS3 has both protease and helicase domain. The helicase domain of NS3 is essential for viral replication. However, its mechanism of action is not well understood. Previous works have shown the presence of conserved guanine‐rich consensus sequences within the HCV genome. These guanine rich sequences form G‐quadruplex (G4) secondary structures through Hoogesteen hydrogen bonding. It has been reported that both replication and translation of HCV genome could be inhibited by the formation of G4 structures. However, it is still unknown how these HCV G4RNA structures are regulated. We hypothesized that the Helicase domain of NS3 facilitates HCV replication by unfolding conserved G4 structures within the HCV genome.To investigate whether NS3 helicase resolves HCV G4RNA structures, we purchased synthetic HCV guanine rich RNA sequences and performed circular dichroism spectroscopy to confirm G4 formation. In addition, fluorescence anisotropy NS3 binding and trap unfolding assays were conducted. We showed that NS3 binds and resolves HCV G4RNA structure in an ATP dependent manner. Understanding the mechanism by which NS3 resolves HCV G4RNA might allow us to find processes and factors that could be targeted to prevent HCV replication in a host cell.Support or Funding InformationThis work is supported by MIRA grant R35GM122601This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Tissue Preparation, Handling and Dissociation Methods for Total RNA
Authors: Easparro Brandon , Soldat Shelby , Proctor Caleb , Nash Rodney J , Atwood James 
Published: June 2021

The extraction and isolation of RNA is an integral part of downstream analyses such as RT‐PCR, RT‐qPCR, Northern blotting, and cDNA library construction. The importance of using pure, intact RNA for these processes is well documented and a critical part of downstream analysis success. It is well known that RNA is sensitive to degradation due to mechanical shear, temperature, storage conditions and freeze‐thawing. Furthermore, RNA is highly susceptible to RNAse degradation following the release of nucleases during the tissue disaggregation process. Thus, proper sample handling during the tissue preparation and homogenization processes is crucial when performing any RNA based assay.Herein, we evaluate the impact of multiple methods for tissue harvesting, storage, storage buffer and mechanical dissociation processes on purified RNA yield and integrity. Multiple, freshly harvested murine tissues, including kidney, liver, heart, and lung were snap frozen in liquid nitrogen and stored without buffer or in an RNA stabilizing reagent at −80°C. Tissues were then dissociated via cryomilling or ambient bead milling and RNA was purified using a silica spin column method. The process above was repeated following one, two and three freeze‐thaw cycles. After each purification, RNA was analyzed on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano kit chip, as per the manufacturers' protocol. Gel images, electrophoretograms and RNA integrity numbers (RIN's) were visualized and analyzed on the 2100 Bioanalyzer Expert software (Agilent).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying a Novel Role for Circular Tau RNAs in Neurodegenerative Diseases
Authors: Welden Justin Ralph , Dockins Stephen , Blackburn Jessica , Nelson Peter , Stamm Stefan 
Published: June 2021

The Tau protein, encoded by the Microtubule‐Associated Protein Tau (MAPT) gene, is the major component of the intracellular filamentous deposits that define many neurodegenerative diseases (Tauopathies) and is a critical component in the etiology of Alzheimer's disease. However, the essential regulation of the MAPT gene expression is still poorly understood. It is unclear what causes the formation of neurofibrillary tangles, but tauopathy conditions show changes in mRNA isoforms, generated through alternative pre‐mRNA processing. Furthermore, mutations changing alternative splicing of MAPT cause frontotemporal dementia, underscoring the importance of tau pre‐mRNA processing.We have identified human‐specific circular RNAs (circRNAs) formed by the tau‐encoding mRNA from the MAPT locus generated by backsplicing of exon 12 (Welden et al. PMID: 29729314) and are investigating a role these circular RNAs may have in neurodegeneration. CircRNAs formed by backsplicing events occurs when pre‐mRNA forms loops, which is aided by primate‐specific Alu‐elements in humans.The tau circRNAs containing exons 10–12 consist of 281 nucleotides (nt), which is divisible by 3, that would give rise to 96 amino acids and does not include a stop codon. Another tau circRNA containing exons 7–12, with the exclusion of the alternatively spliced exon 8, is also divisible by 3 comprising 681 nt and does not have a stop codon; however, it does have a start codon in exon 7 and would give rise to 227 amino acids. Thus, if translation initiates, they could form high molecular weight multimers containing parts of the tau protein. Using minigenes, we are testing the role of human‐specific Alu‐elements in deciphering which cis‐factors are crucial for circRNA formation.Transgenic zebrafish expressing the tau circRNAs suggest a physiological relevance of tau circRNAs. We generated transgenic fish expressing only the human tau circRNAs under a neuron‐specific promoter. Importantly, the introduction of an in‐frame start codon that causes frontotemporal dementia in humans in the tau circRNAs causes early neurodegeneration in fish, suggesting that possible translation of the tau circRNAs may be occurring.Since circRNAs do not contain a canonical ribosomal entry site, we are testing whether tau circRNAs are translated after N‐6 methyl adenosine methylation of the circular RNA in biochemical studies.In summary, we found evidence of human‐specific tau circular RNAs, suggesting that not all RNAs made from this gene have been discovered. Our finding of a phenotype in fish indicate that the circRNAs could contribute to tauopathies, using a new disease mechanism.Support or Funding InformationNIA ‐ 5P30AG028383‐13, University of Kentucky College of Medicine Excellence in Graduate Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Cancer‐Associated Mutations in SF3B1 on Yeast pre‐mRNA Splicing
Authors: Groubert Brent Joseph , Kaur Harpreet , Paulson Joshua C , Hoskins Aaron 
Published: June 2021

In eukaryotes, a mRNA maturation step called splicing removes non‐coding intronic regions from pre‐mRNAs. Splicing is conducted by an enzyme called the spliceosome which uses a variety of protein and snRNA components in order to perform its catalytic function. One of these highly conserved components is the U2 snRNP protein SF3B1. In humans, mutation of SF3B1 can lead to improper regulation of alternative splicing. Many human diseases such as myelodysplastic syndromes (MDS), endometrial cancer (UCEC), bladder carcinoma (BLCA), and acute myeloid leukemia (LAML) have been linked to SF3B1 mutation and dysregulation of alternative splicing.The goal of this study is to introduce SF3B1 mutations observed in UCEC, BLCA, or LAML cancer patients into the homologous yeast protein (HSH155) to gain further insight into the consequences of these mutations on splicing. I have used site‐directed mutagenesis to incorporate these mutations into the HSH155 gene on plasmids and then shuffled these plasmids into a budding yeast strain in which the HSH155 gene was deleted from the genome. I then used temperature‐dependent growth assays and an in vivo splicing reporter to measure phenotypic responses due to these point mutations. These experiments will identify how cancer‐linked mutations change conserved, fundamental steps carried out by the spliceosome and how perturbation of these steps can led to changes in alternative splicing.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.