bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Small Molecule Enhanced CRISPR/Cas9 Methodology for Genome Editing of Dog (Canis familiaris) induced Pluripotent Stem Cells (diPSC)
Authors: Federico Francesca , Federico Niksha , Sternik Gabriel , Federico Tessya , Esmaeli‐Azad Babak 
Published: November 2023

Bacterial CRISPR‐Cas9 system has emerged as an effective tool for sequence‐specific gene knock‐out through non‐homologous end joining (NHEJ), but it remains inefficient for precise editing of genome sequences (gene knock‐in) via homology‐directed repair (HDR). Because of this a long and tedious screening process via cell sorting or selection, expansion, and sequencing is often required to identify correctly edited cells. Improving the efficiency of precise CRISPR gene editing remains a major challenge for many applications, such as generating sufficient numbers of genome‐edited animals or genome edited patient reprogrammed cell lines for disease modeling. Using our patented technology to develop Artificial 3D Microenvironment Niche (US Patent # 904737), we have developed a reporter based screening approach for high‐throughput identification of chemical compounds that can modulate precise genome editing through HDR. In preliminary experiments, we have identified small molecule classes that enhance CRISPR mediated HDR efficiency more than 9 fold. Additionally, we have found that small molecules that inhibit HDR, enhance insertion and deletion mutations via NHEJ and “vice versa”. We plan to further test this refined genome editing methodology for growth hormone gene modifications in dog (Canis familiaris) induced pluripotent stem cells (diPSC), attempting to correct genetically inherited disorders in dogs and for the development of “designer pets” of the future. Our preliminary findings with this experimental system will be presented.Support or Funding InformationThis project was supported by Internal R&D funds from DNAmicroarray, Inc.
Elucidating the Role of Transcriptional Mutagenesis in the Acquisition of Adaptive Mutations
Authors: Saxowsky Tina T. , Wilson Gracie , Nixon Gavin , Iverson Jessika , Angkanaworakul Pannapat , Brock Chance 
Published: November 2023

For nearly a century, we have understood mutagenesis as a growth‐dependent process involving DNA replication. More recently, cells have been shown to accumulate adaptive mutations in response to selective pressure that induces prolonged growth arrest. While multiple mechanisms related to DNA damage and genome maintenance have been shown to contribute, the specific role of DNA base damage and base excision repair in the process has not been explored. Base damage can be bypassed by RNA polymerase, resulting in a pool of mutant mRNAs through transcriptional mutagenesis (TM), and potentially a population of mutant proteins that could alter the phenotype of the cell. In non‐dividing cells, such as those subject to drug pressure, the influence of persistent DNA damage on transcription could affect cellular outcomes. We hypothesize that TM could be an important first step in acquiring drug resistance, as the mutant protein generated could allow cells to resume DNA replication and convert this damage to a permanent advantageous mutation, a process we call retromutagenesis. Using Saccharomyces cerevisiae as a model organism, we have established an experimental system that examines adaptive mutagenesis in response to canavanine exposure. Within this system, we have demonstrated that prolonged exposure to canavanine yields an increase in intracellular reactive oxygen species, and that cells deficient in the repair of the oxidative lesion 8‐oxoguanine (ogg1 knockouts) show an increase in adaptive mutation frequency. Additionally, DNA sequencing of the target gene in adaptive mutants from this repair deficient cell line has revealed a strand bias for the DNA damage leading to canavanine resistance in the template strand related to transcription. These results support our hypothesis that mutagenic transcription past DNA damage in non‐dividing cells could contribute to the mechanism by which adaptive mutations arise.Support or Funding InformationCottrell College Science Award, Research Corporation for Science Advancement
The Yeast Genome Project: Characterizing APD1
Authors: Douds Catherine , Ben Jelloun Idriss Touimi , Keeney Jill B. 
Published: November 2023

Yeast is considered to be “man's oldest industrial microorganism” and has been used by humans for over 5,000 years. Today, its use has expanded and Saccharomyces cerevisiae has become an ideal eukaryotic model due to its quick growth and easily manipulated genome. After its genome was sequenced in 1996, scientists came together to create a full encyclopedia of systemized gene functions characterized according to the Gene Ontology Consortium (GO). However, nearly twenty years later, the functions of more than 10% of these genes still remain unknown. The ORFan gene project aims to aid the characterization of the S. cerevisiae genome by exploring the function of specific open reading frames (ORFs). In order to classify these unknown ORFs, a putative pipeline has been constructed beginning with the identification of an ORFan of interest through the Saccharomyces Genome Database (SGD). This project focuses on YBR151W or APD1. The selected ORF was fluorescently tagged at the C and N terminals to verify cell localization. Next, a comparison between the wild type and target gene knockout strains will be analyzed through RNAseq. This comparative analysis uses programs such as FastQC, FastX, RSEM, Bowtie2 and R, and will provide information about the impact of gene deletion on global gene expression levels. Further exploration into the putative function of APD1 has led to studies involving actin patch localization, as well as the protein's potential role in oxidative stress mechanisms. We have observed the effects of actin patch localization under various forms of oxidative stress such as HU and H2O2 between wildtype and Δapd1 strains.Support or Funding InformationHoward Hughes Medical Institute Undergraduate Grant Program, Funded by HHMI GCAT SEEK SNF grant #1248096, NIH Grant R15‐GM0587 to Jill B. Keeney
TFAM Mutants Support mtDNA Replication In Vivo
Authors: Alexeyev Mikhail , Kozhukhar Natalya 
Published: November 2023

ObjectiveTo evaluate ability of the transcription‐deficient TFAM mutants to support mtDNA replication in vivo.MethodsWe developed a GeneSwap approach that allows for the replacement of WT TFAM allele with a mutant in cultured cells. The approach consists of the three steps: 1) inactivation of the chromosomal TFAM alleles with CRISPR/Cas, 2) introduction of a floxed WT TFAM allele, 3) reintroduction of mtDNA and 4) co‐introduction of Cre recombinase and mutant TFAM allele.ResultsWe were able, for the first time, to cultivate cells lacking TFAM and thus demonstrated that lethality of the TFAM knockout is conditional. TFAM knockout (KO) cells lack mitochondrial DNA (mtDNA) and are defective in respiration. We further demonstrate that TFAM mutants with severe defects in mtDNA transcription and respiration can support mtDNA replication.Conclusioneven very low levels of mtDNA transcription may be sufficient to support mtDNA replication in vivo.Support or Funding InformationHL66299 and OD010944.
Genome wide methylation of RASSFs associated genes in Kidney Cancer
Authors: ALOKAIL MAJED S , ALENAD AMAL M 
Published: November 2023

The RASSF are tumor suppressor proteins that undergo loss of expression through promoter methylation in numerous types of cancers including kidney cancer. RASSF proteins have several functional domains that modulate associations with other proteins like Salvador‐RASSF‐Hippo (SARAH). RASSFs can associate via the SARAH domain with downstream kinases such as MST1 and MST2 and SAV1 in order to promote apoptosis. This study identifying differential methylation patterns that can distinguish between conventional clear cell renal cell carcinoma (ccRCC), chromophobe, oncocytoma and papillary RCC. Genome wide methylation using the Illumina Infinium Human Methylation 450 Bead Chip, RASSF1A was highly methylated in ccRCC, Papillary and Oncocytoma tested (95%, 100% and 45% respectively), while in chromophobe, methylation was lower at only 15% of samples. Analysis of the Hippo pathway genes indicates that methylation of this pathway may vary between the cancer types in particualar methylation of MST1, which was identified to be methylated in 80% ccRCC, 38.9% papillary RCC, 20% chromophobe and 13.3% oncocytomas. Methylation of the RASSF2 promoter was detected in 36.8% papillary RCC, 31.6% ccRCC, 16.7% chromophobe and 5.3% oncocytomas. In conclusion, the methylation levels varied across the promoters of Salvador‐RASSF‐Hippo genes associated with kidney cancer.Support or Funding InformationKing Saud University
Molecular Analysis of Adaptational Strategies under Heat Stress by Whole‐Genome Sequencing of Laboratory‐Evolved Saccharomyces cerevisiae
Authors: Satomura Atsushi , Miura Natsuko , Kuroda Kouichi , Ueda Mitsuyoshi 
Published: November 2023

Experimental evolution is an effective method to connect genotypes to phenotypes. The outcomes obtained from the method potentially facilitate the rational engineering of useful strains for bioproduction.In this study, we carried out experimental evolution of Saccharomyces cerevisiae under heat stress [1, 2]. In order to track the cell populations, intermediate strains obtained during the adaptational experiments have been preserved. Whole‐genome sequencing of 19 intermediate strains revealed that a total of 49 genetic mutations had emerged during the experimental evolution. A phylogenetic tree analysis showed that there were at least 5 events in which these strains had acquired mutations in a CDC25 gene. One of the CDC25 mutants successfully adapted to a 38°C‐environment. These mutations led to the downregulation of cAMP‐dependent protein kinase (PKA) signaling pathway, which controls variety of processes such as cell‐cycle progression and stress tolerance. In general, excessive downregulation of this pathway causes a trade‐off between induced thermotolerance and diminished growth rate. However, these strains have acquired thermotolerance without any growth defects. Our study demonstrates that experimental evolution leads to fine tuning of signaling pathways and engineering of beneficial cellular traits [3].Support or Funding InformationThis work was supported by a Grant‐in‐Aid for JSPS Fellows (No. 26‐1413) from Japan Society for the Promotion of Science, the commission for Development of Artificial Genes Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI), Japan, and, JST‐CREST.
The involvement of histones and post‐translational modifications during DNA repair in bdelloid rotifers
Authors: Jones Alexander C , Ebijoyeldhas Miracline , Boerma Marjan , Orr Lisa M , Byrum Stephanie D , Tackett Alan J , Schurko Andrew M 
Published: November 2023

Histones are architectural proteins that package eukaryotic DNA into nucleosomes and regulate the expression and replication of the genome. Four canonical histones (H2A, H2B, H3, and H4) are present in duplicates to form an octamer within a nucleosome. Histone N‐and C‐terminal tails are often targets for post‐translational modifications (PTMs). For example, C‐terminal phosphorylation of the histone variant H2AX serves as an epigenetic marker for DNA damage and links damaged chromatin to DNA repair proteins. Bdelloid rotifers are aquatic microinvertebrates that have the ability to effectively repair DNA double‐strand breaks (DSBs) induced by high doses (>1000 Gray) of ionizing radiation. The bdelloid Adineta vaga possesses five histone H2A variants, three of which (H2Abd, H2Abd1, H2Abd2) have C‐terminal tails that are significantly longer than those in other eukaryotes. These tails have the potential to act as targets for PTMs. The objective of this project was to investigate the role of bdelloid H2A variants in DNA repair by characterizing PTMs associated with these proteins and to identify their binding partners. We induced DSBs by irradiating three species of bdelloids with 280 Gray of ionizing radiation. Histones were then isolated at different time points post‐irradiation and high‐resolution mass spectrometry was used to characterize the histones present. We identified each of the five bdelloid‐specific H2A variants along with histones H2B, H3 and H4. Several PTMs (acetylations, methylations and phosphorylations) were identified on the N‐and C‐terminal tails of all histones. Of the identified PTMs, many were only present during DNA repair; among these, several were located in the long C‐terminal tails of H2A variants. To determine the significance of the H2A variants and associated PTMS in bdelloids, we want to identify binding partners to these proteins during DNA repair. To accomplish this, we produced 6×histidine‐tagged proteins representing each of the five variants in A. vaga. Western blots with custom polyclonal antibodies specific for each variant were used to verify the identity of the tagged proteins. Recombinant proteins and peptides (+/− PTMs) will be used in pulldown assays to identify binding partners of histones during DNA repair. In sum, we have identified several novel histone PTMs associated with DNA repair in bdelloid rotifers. These findings will improve our understanding of the role of histones and epigenetics in the specialized DNA repair system in bdelloids.Support or Funding InformationThis work was supported by grants from the NCRR (5P20RR016460) & NIGMS (8P20GM103429) from the NIH and from the Arkansas Science and Technology Authority.
Modifying Trypsin to Weaken Inhibitor Interaction
Authors: Nzessi Anthony , Huynh Hanh , Baird Teaster 
Published: November 2023

Serine proteases have been used as therapeutics to help with health maladies such as Christmas Disease, a blood coagulation disorder. They may potentially be administered as drugs in order to aid many other conditions, but their activity is limited by natural regulatory inhibitors which serve to regulate the action of proteolytic enzymes and prevent diseases such as pancreatitis. A number of these inhibitors bind to the hydroxyl group of Y39 in the S1′ pocket of trypsin‐fold serine proteases. We expect that substituting this tyrosine with a leucine will weaken inhibitor interaction, thus increasing the half‐life of serine proteases in the body. Using trypsin as a model serine protease, the variant Y39L was designed via PCR mutagenesis, expressed in P. Pastoris, and purified through hydrophobic interaction chromatography. Before determining if inhibitor interaction with Y39L was weaker than with wild‐type trypsin, we compared their kinetics. In this study, we show that Y39L has comparable activity (kcat = 2.20 × 103 min‐1) and binding capability to Z‐GPR‐pNA (KM = 6.89 ± 1.28 μM) as wild‐type trypsin. Using the commercial competitive inhibitor benzamidine, we also show that the active site of Y39L has not been compromised by this amino acid replacement (KI = 5.28 μM). This implies that if the inhibition of this variant with macromolecular inhibitors is not consistent with that of WT, any difference must be due to the change made in the S1’ site. These studies will be instrumental in the development of trypsin‐fold serine proteases that may potentially be used as therapeutic agents to aid in several biological processes.Support or Funding InformationNational Institute of Health ‐ MARCNational Science Foundation ‐ CSU‐LSAMP
Trypsin as a model for investigating serine protease‐inhibitor interaction
Authors: Diaz Parris , Zhuo Weichao , Sharma Abriti , Baird Teaster 
Published: November 2023

For years proteases have been drug targets, but recently, several have been used as therapeutic agents themselves. However, macromolecular inhibitors present in the human body that inactivate proteases have been a hindrance in their further development as therapeutic agents. In this study, we use trypsin as a model protease to understand protease‐inhibitor interaction. We have substituted amino acids in S1’ pocket and determined if these variants are resistant to inhibitors while also retaining catalytic activity. The S1’ pocket contains residues Tyr‐39, Phe‐41, and Lys‐60 and has been chosen as a target site due to it being a common location at which multiple inhibitors bind to trypsin. The large phenyl group of Phe‐41 likely locks its backbone carbonyl into a conformation where it participates in a conserved hydrogen bond with inhibitors. If this interaction were disrupted, the interaction between enzyme and inhibitor may be weakened. To test this hypothesis, we created the F41L variant of trypsin (F41L‐Tn). In our initial experiments, we have observed that F41L‐Tn has a high binding affinity for the substrate Z‐GPR‐pNA (Km = 14.1 ± 2.8 μM) along with catalytic capability comparable to wild‐type (kcat = 2.30 × 102 s‐1). In addition, F41L‐Tn has similar affinity for the inhibitor p‐aminobenzamidine (KI = 5.8 μM) when compared to WT (KI = 6 μM). These results suggest that the substitution has not affected the active site is still intact and can still interact with substrates such as Z‐GPR‐pNA as well as competitive inhibitors such as p‐aminobenzamidine. Consequently, any differences observed between trypsin and macromolecular inhibitors may be attributed to changes at the S1’ subsite.Support or Funding InformationNIH MARC: T34‐GM008574LSAMP: HRD‐0802628
Characterization of Ecm14, a Fungal Pseudopeptidase
Authors: Idowu Temitope A , Schott Matthew J , Lyons Peter J 
Published: November 2023

Many peptidase families contain inactive enzyme homologs due to the alteration of critical active site amino acids. The function of these inactive homologs is in many cases an open question, and their study made more difficult by their lack of activity. For example, the M14 family of metallocarboxypeptidases is a large family of peptidases made up of 23 members in humans; three of these are inactive enzyme homologs for which we have little functional information. Interestingly, the yeast Saccharomyces cerevisiae has only one M14 metallocarboxypeptidase homolog, Ecm14, and this sole homolog is predicted to be inactive. Our goal was to investigate the molecular and biological function of this pseudopeptidase. An analysis of available fungal amino acid sequences showed that the Ecm14 gene was present and conserved in ascomycete fungi only. In order to characterize the function of Ecm14, we expressed a C‐terminally histidine tagged version of Ecm14 using the Sf9/baculovirus system. The protein was secreted and subsequently purified by metal affinity chromatography. Incubation of Ecm14 with trypsin or chymotrypsin resulted in the specific removal of the Ecm14 prodomain. The mature form of Ecm14 lacked any detectable enzymatic activity, but was able to prevent the cleavage of standard substrates by an active metallocarboxypeptidase. Preliminary results from a screen of knockout fitness in response to chemical stressors suggest a biological function for Ecm14. We propose that Ecm14 may perform a regulatory function through binding to specific C‐terminal amino acids, thus blocking their cleavage by active carboxypeptidases.Support or Funding InformationSupported by Andrews University Faculty Research Grants to PJL
FRET studies examining the interaction of hepatitis C NS3 helicase and human DExD/H box helicases
Authors: Corby Josie , Biener Gabriel , Raicu Valerica , Frick David 
Published: November 2023

The innate immune response to viral infection is the intracellular first line of defense against invading pathogens. One strategy that viruses, such as hepatitis C virus (HCV), use to establish a chronic infection is to short circuit this innate immune response. HCV encodes a multi‐functional non‐structural protein 3 (NS3), which is a protease covalently tethered to a C‐terminal helicase. One role of the NS3 protease in innate immune response evasion is to cleave mitochondrial anti‐viral signaling protein (MAVS). The function of the NS3 helicase is essential for replication but it is unclear why it is covalently linked to the protease.We propose that HCV NS3 helicase works to evade the innate immune response by binding to pathogen associated molecular patterns (PAMPs) in the HCV genome and interacting with the RIG‐I like receptor LGP2, to localize the NS3 protease near MAVs for cleavage.In order to study the interaction between human and viral helicases we used a process called Förster resonant energy transfer (FRET) where an excited fluorescent molecule (donor) transfers its energy to a nearby fluorescent molecule of a different type (acceptor) which in turn emits a red shifted fluorescent light. This process occurs in distances smaller than 10 nm between the donor and acceptor which indicates obvious interaction.We generated two sets of plasmids encoding donor (mCFP) and acceptor (mYFP) fused to viral and human helicases respectively. The donor plasmid set encodes recombinant proteins in which all or part of HCV NS3 and its cofactor NS4A were fused to a mCFP fluorescent protein. The first expresses only the NS3 helicase domain (mCFP‐NS3h) fused to mCFP. In the second, mCFP was fused with full‐length NS3 (mCFP‐NS3). In the third, mCFP was fused to NS3 and only the NS3 cofactor domain of NS4A (lacking the membrane anchor and C‐terminus, i.e. mCFP‐NS3‐4Acd), and in the fourth, mCFP was fused to NS3 and full length NS4A (mCFP‐NS3‐NS4A). The acceptor plasmid set encodes recombinant mYFP fused to DDX1, DDX3, DDX5, and DHX 58 (LGP2). HEK293T cells were co‐transfected with each donor plasmid and acceptor plasmid. The live cells were imaged using an optical micro‐spectroscope (OptiMis) which consists of a two photon excitation scanning optical microscope with high spectral resolution. In a separate set of experiments each of the donor plasmids were co‐transfected with a plasmid expressing RFP fused to MAVs and a nuclear localization signal.The fluorescently labeled recombinant NS3 proteins were visualized in real‐time in HEK293T cells. The RFP MAVs re‐localized to the nucleus only when it was co‐transfected with plasmids that encoded NS4A, in addition to the NS3 protease. FRET was observed between all recombinant HCV NS3 proteins and LGP2, but not between any recombinant HCV NS3 proteins and closely related DDX 1, DDX3, and DDX5. Our data indicates that the NS3 fusion proteins are biologically active and that NS3 interacts with LGP2 via its helicase domain.